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001 ENDOTHELIAL DYSFUNCTION AND MYOCARDIAL REMODELLING IN PATIENTS WITH ANKYLOSING SPONDYLITIS

D. A. Poddubnyy, A. P. Rebrov.Hospital Therapy Department, Saratov State Medical University, Saratov, Russian Federation

Background: Endothelial dysfunction is an imbalance between production of vasodilating, anticoagulant, anti-inflammatory factors (NO, prostacyclin, natriuretic factor etc.) and vasoconstrictive, procoagulant, proinflammatory factors (endothelin-1, thromboxane A2, von Willebrand factor, etc). Decreased NO production and increased endothelin-1 production are the main non-hemodynamic factors of left ventricle hypertrophy progression.45 Furthermore endothelial dysfunction (particularly NO deficiency) plays an important role in progression of pulmonary hypertension.2 Chronic systemic inflammation can affect endothelium and leads to endothelial dysfunction. This phenomenon is well described in rheumatoid arthritis and systemic lupus erythematosus13 and there is some evidence of endothelial dysfunction presence in ankylosing spondylitis (AS).

Objective: Assessment of endothelial dysfunction influence on myocardial remodeling in patients with AS.

Materials and methods: 86 patients (84 men and 2 women) with reliable AS due to modified New York criteria diagnosis of AS were included. Exclusion criteria were presence of ischemic heart disease, diabetes mellitus, renal amyloidosis with chronic renal failure. The mean age was 38.7±9.3 years and the mean duration of disease was 12.3±7.7 years. Screening of cardiovascular risk factors (arterial hypertension, smoking, heredity, body overweight, hypercholesterolemia), coronary disease risk and fatal cardiovascular risk assessment, echocardiography and spirography were performed in all patients. Diseases activity assessment was performed on the basis of Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and acute phase reactants, including erythrocyte sedimentation rate (ESR) and C reactive protein (CRP). Endothelial function was assessed during Dopplerography of brachial artery in tests with reactive hyperemia (endothelium-dependent dilatation). According to level of endothelium-dependent dilatation all patients were divided into two groups: I group—49 patients with normal endothelium-dependent dilatation (⩾10%), II group—37 patients with signs of endothelial dysfunction (endothelium-dependent dilatation <10%).

Results: Groups were comparable in age of patients, duration of disease, diseases activity, cardiovascular risk factors, coronary disease risk and fatal cardiovascular risk, spirography data. Arterial hypertension was noted in 30.6% of patients in I group and in 40.5% in II group (p>0.05). However patients with signs of endothelial dysfunction had significantly higher incidence of left ventricle hypertrophy (59.5% and 25.5% respectively, p<0.01), higher left ventricle mass (260.5±57.8 g and 218.5±48.0 g respectively, p<0.01), higher left ventricle mass index (139.9±26.5 g/m2 and 120.2±23.1 g/m2 respectively, p<0.01), higher left ventricular end-diastolic dimension (5.20±0.31 cm and 4.99±0.41 cm respectively, p<0.01) in comparison with patient with normal endothelium-dependent dilatation. Also we have revealed higher level of pulmonary arterial pressure (34.7±4.7 and 32.4±3.7 mm Hg respectively, p<0.05), higher incidence of right ventricle hypertrophy (48.7% and 24.5% respectively, p<0.05) and higher right ventricular end-diastolic dimension (2.74±0.28 cm and 2.60±0.26 cm respectively, p<0.05) in patients with lowered endothelium-dependent dilatation in comparison with patient with normal endothelium-dependent dilatation.

Conclusion: Lowered endothelium-dependent dilatation is the evidence of decreased production of vasodilating factors, mainly NO. We suppose that NO production deficiency in patient with AS is very important factor of myocardial remodeling (ventricles hypertrophy and dilatations) and pulmonary hypertension development.

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002 CONCENTRATION OF MARROW STEM CELLS: ROLE IN BONE HEALING

S. Muthian, V. Lee.Christian Medical College & Hospital, Vellore, India

Aim: To study the effect of marrow stem cell injection and the role of concentration of marrow stem cells in bone healing.

Study design: Randomised interventional trial.

Methods: This study included 55 patients who had either tibial or femoral shaft fractures of more than 3 months duration with inadequate callus formation. They were divided into 2 groups, centrifuged and uncentrifuged and were injected with marrow stem cells accordingly. After injection, they were followed up at 6 weekly intervals. They were followed up till 36 weeks.

Results: 48 out of 55 patients (87.27%) united at an average of 18 weeks (6–36 weeks). There were 24 patients in each group.

Conclusion: Injection of marrow stem cells is a highly effective method of augmentation of bone healing. In our study we found no improvement of bone healing when we concentrated the marrow before injection.

003 CONDITIONAL MUTAGENESIS REVEALS THAT NFATC1 IS REQUIRED FOR OSTEOCLAST DIFFERENTIATION IN VIVO

A. O. Aliprantis1, N. C. Walsh2, K. P. McHugh3, E. Gravallese2, L. H. Glimcher1.1Harvard School of Public Health, Department of Immunology and Infectious Diseases, Boston, MA, USA; 2University of Massachusetts Medical Center, Department of Rheumatology, Worcester, MA, USA; 3Beth Israel Deaconess Medical Center, Department of Rheumatology, Boston, MA, USA

Purpose: To study the role of the transcription factor Nuclear Factor of Activated T-cells c1 (NFATc1) in osteoclast function in vivo and in vitro.

Methods: NFATc1 deficient mice die in utero of heart failure, making an analysis of the adult skeletal system in these mice impossible. A conditional knockout allele of NFATc1 (NFATc1fl) was generated by flanking exon 3 with loxP sites. NFATc1fl/fl mice were intercrossed with Mx1-Cre transgenic mice that express the Cre recombinase from a type I Interferon (IFN) inducible promoter. The Mx1-Cre transgene was activated by intraperitoneal injection of polyinosinic-polycytidylic acid (poly I:C) to generate NFATc1 deleted mice (NFATc1Δ). Standard radiographic and histologic techniques were used to probe the phenotype of these mice. Littermates without the Mx1-Cre transgene served as wild-type controls.

Results: Injection of poly I:C resulted in complete and durable Cre mediated deletion of the “floxed” allele in bone marrow, as confirmed by Southern blotting and quantitative RT-PCR. Compared to Mx1-Cre negative littermate controls, NFATc1Δ mice displayed reduced tooth eruption, shortened long bones and markedly increased metaphyseal bone density on plain radiographs and quantitative micro-CT. This was associated with an absolute defect in the number of osteoclasts at the growth plate and a reduction in serum TRAP5b levels. Histomorphometric analysis confirmed reduced osteoclast numbers in NFATc1Δ mice and revealed massive amounts of calcified cartilage adjacent to the hypertrophic zone of the growth plate consistent with a defect in resorption of the primary spongiosa. The growth plates of NFATc1Δ mice demonstrated chondrodysplasia, with increased thickness, acellular areas and retention of hypertrophic chondrocytes beyond the chondroosseus junction. In vitro, bone marrow cells from NFATc1Δ mice failed to generate TRAP positive, multinucleated giant cells in the presence of recombinant M-CSF and RANKL, or in a co-culture system with wild-type osteoblasts. This in vitro osteoclast differentiation defect was accompanied by a reduction in bone resorption, TRAP secretion and a failure to upregulate genes associated with osteoclast differentiation including Cathepsin K, Beta-3 Integrin and the Calcitonin receptor. Interestingly, FACS analysis reveals normal numbers of osteoclast progenitors in the bone marrow of NFATc1Δ mice compared to littermate controls.

Conclusions: NFATc1 deleted mice display an osteopetrotic phenotype and a defect in osteoclastogenesis in vitro and in vivo. These mice confirm that NFATc1 is required for osteoclast differentiation in vivo and represent an inducible system to probe the role of the osteoclast in physiologic and pathologic circumstances.

004 A NON-CELL AUTONOMOUS REQUIREMENT FOR THE LEUKOTRIENE B4 RECEPTOR BLT1 ON NEUTROPHILS IN INFLAMMATORY ARTHRITIS

N. D. Kim, R. C. Chou, A. M. Tager, A. D. Luster.Division of Rheumatology, Allergy, and Immunology, Center for Immunology and Inflammatory Disease, Massachusetts General Hospital, Boston, Massachusetts, USA

Neutrophil recruitment into tissue plays an important role in host defense and disease pathogenesis, including the inflammatory arthritides. A multitude of diverse chemoattractants have been implicated in neutrophil recruitment, suggesting that they have overlapping functions in mediating this critical biological response. However, we have found a unique, non-redundant role for the leukotriene (LT) B4 receptor BLT1 in mediating neutrophil recruitment into the joint in the K/BxN mouse model of inflammatory arthritis. Despite increased expression of multiple chemokines active on neutrophils, we demonstrate that neutrophil expression of BLT1 is absolutely required for arthritis generation and chemokine production in this model, and that specific BLT1 inhibition reverses established disease. Adoptive transfer of WT neutrophils restores arthritis and chemokine production in BLT1-/- mice, but surprisingly, the primary effect of the transferred WT neutrophils into BLT1-/- mice is to promote the entry of endogenous BLT1-/- neutrophils into the joints of these mice. However, continued joint inflammation is dependent upon the presence of WT neutrophils, indicating an ongoing specific requirement for BLT1-activated neutrophils in mediating BLT1-/- neutrophil recruitment by other chemoattractants. These experiments demonstrate a novel non-cell autonomous role for BLT1 in neutrophil recruitment, which is required for subsequent amplification of inflammation in autoantibody-induced arthritis. We hypothesize that BLT1-activated neutrophils produce a specific factor(s) that then enables the migration of BLT1-negative neutrophils out of the vasculature and into the joint.

005 AUTOANTIBODIES TO TRANSCRIPTIONAL INTERMEDIARY FACTOR 1-GAMMA IN DERMATOMYOSITIS

I. N. Targoff, E. P. Trieu, M. Levy, T. Prasertsuntarasai, F. Miller.University of Oklahoma HSC, VAMC, Oklahoma Medical Research Foundation, and the NIEHS, NIH, DHHS

Purpose: Several autoantibodies (autoabs) have been associated with polymyositis (PM) and dermatomyositis (DM), some having relative specificity for these conditions. Anti-Mi-2 is the only established myositis-specific autoab found predominantly in DM. Preliminary reports, however, described anti-p155 in 29% of adult and juvenile DM, but rarely in PM or other diseases. Anti-p155, identified by immunoprecipitation (IPP), was known to react with a 155 kd protein and to show a nuclear pattern by indirect immunofluorescence, but the nature of the p155 antigen was unknown. The aim of this study was to further characterize p155 and develop better detection methods.

Methods: Anti-p155 autoab in sera was confirmed by IPP-blots (IPP-WB) using K562 cell extract. Some anti-p155-positive sera were known to react by IPP but not by WB. Patient serum was therefore used for the IPP step, and anti-p155 reference serum (p155-1) and rabbit antiserum or controls were used for WB. P155 antigen was prepared by affinity-purification and SDS-PAGE. The gel band was digested with trypsin followed by HPLC of peptides and mass spectrometry with MS/MS analysis. Rabbits were immunized and boosted with a 19-mer, 4 component MAP peptide synthesized based on a TIF-1 g sequence. Rabbit antibody (anti-TIFp), affinity-purified with the MAP, was used to coat wells for capture-ELISA. Wells were incubated with K562 extract, then patient ab, then rabbit anti-human IgG conjugate.

Results: Mascot analysis of the mass spectrometry gave a score of 81 for human TIF-1 g, with scores >47 considered significant. Other significant scores were only seen with related proteins and trypsin. Purification was repeated, and mass spectrometry of new sample also showed a significant Mascot score for human TIF-1 g (500). By IPP-WB, rabbit anti-TIFp blotted a 155 kd protein band in IPPs prepared with p155-1 serum, similar to the band blotted by the patient serum. Similarly, p155-1 serum, but not normal serum, blotted a 155 kd protein in IPPs prepared with rabbit anti-TIFp. Sera from 8 other myositis patients that were found to be positive for anti-p155 by IPP-WB in the previous study using p155-1 for WB, were retested here by IPP-WB using rabbit anti-TIFp or controls for WB, and all 8 were strongly reactive (3–4+). 19 myositis sera that were previously negative for anti-p155 using p155-1, were retested using rabbit anti-TIFp for WB. Only 4 of 19 were positive (3 at 1+, 1 at 2+). The capture ELISA using anti-TIFp also showed significant reaction with p155-1 and 6 previously positive anti-p155 sera, and not control.

Conclusion: These studies identify a target of anti-p155 autoabs as TIF-1 g, a nuclear protein found to be involved in regulating hematopoesis. Mi-2 is also a nuclear protein involved in transcriptional regulation. In contrast, aminoacyl-tRNA synthetases, which are cytoplastic proteins involved in translation, are often antigens in PM.

006 DIFFERENCES IN INITIAL PRESENTATION OF YOUNG AND OLDER SYSTEMIC SCLERODERMA PATIENTS

H. Weng, V. K. Ranganath, M. Shin, G. S. Park, K. M. Setoodeh, D. Khanna, P. J. Clements, J. R. Seibold, D. E. Furst.UCLA, Los Angeles, CA, USA

Purpose: Little is known about the differences in initial presentations of systemic scleroderma (SSc) in the young versus the older. In our study, we propose to compare the clinical and biochemical characteristics in younger versus older SSc patients.

Methods: Subjects were 264 diffuse SSc patients aged <39 or >53 from three different treatment clinical trials. All trials were double-blind, randomized, placebo controlled trials, conducted between 1991 and 2005. These age cutoffs represent the lowest and highest age quartiles from our pool of subjects. Student’s t-tests (without Bonferroni adjustment), chi-square tests, or Wilcoxon Rank sum test were used to compare the two groups for continuous and categorical variables. To evaluate for any differences in the two age groups, generalized linear models were created to evaluate whether the outcome variables were significantly different in the two age groups after adjustment for duration of disease, race, gender, BMI and medications. Normal reference values from the literature were used to adjust for any outcome variables there were significant different between the two age groups.

Results: Older SSc patients have a significantly higher proportion of Caucasians as compared to younger patients. Older patients are more likely to be on aspirin, steroids, and either ACE-inhibitor or angiotensin receptor blocker. There are no significant differences in gender distribution, disease duration, total skin score, oral aperature, %pred DLCO, Hgb, and patient global assessment in younger (age<39) vs older SSc (age>53). However, older subjects had significantly higher body mass index (BMI), systolic and diastolic blood pressures (SBP, DBP), creatinine, and HAQ-DI scores. Older subjects also had significantly lower albumin, and SF-36 physical summary score (PCS). After adjustment for gender, race, BMI, duration of disease and patient medications, there were significant differences in SBP, DBP, HAQ, SF-36 PCS, albumin, creatinine clearance between younger and older SSc patients. Adjustment for normal reference values from the literature revealed persistent differences in SBP, CPK, alkaline phosphatase, and SF-36 PCS. Older SSc patients have significantly higher SBP and lower SF-36 PCS.

Conclusion: There are baseline differences in BMI, systolic and diastolic blood pressure, albumin, creatinine, CPK, FVC, HAQ, and SF-36 PCS between younger and older patients with SSc. After adjustment for age and medications, differences in SBP, CPK, alkaline phosphatase, and SF-36 PCS remain. Younger patients with SSc score higher on SF-36 PCS compared to older patients after age adjustment. This suggests that, at enrollment into clinical trials, there may be blood pressure and functional differences between younger and older SSc pts. These data will have to be further confirmed.

007 THE HUMAN AUTOANTIGEN HNRNP-A2 (RA33) IS A MAJOR STIMULATOR OF AUTOIMMUNITY IN RATS WITH PRISTANE-INDUCED ARTHRITIS

M. H. Hoffmann1,2, J. Tuncel3, K. Skriner4, M. Tohidast-Akrad5, B. Türk1, S. Pinol-Roma6, G. Serre7, G. Schett*1, J. S. Smolen1,2,5, R. Holmdahl3, G. Steiner1,2.1Department of Rheumatology, Medical University of Vienna, Vienna, Austria; 2Center of Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; 3Department of Cell and Molecular Biology, Section for Medical Inflammation Research, Lund, Sweden; 4Department of Rheumatology, Charité University Hospital, Berlin, Germany; 5Ludwig Boltzmann Institut for Rheumatology and Balneology, Second Department of Medicine, Hietzing Hospital, Vienna, Austria; 6Department of Cell Biology and Anatomical Sciences, Sophie Davis School of Biomedical Education/CUNY Medical School, New York, USA; 7Laboratory of Epidermis Differentiation and Rheumatoid Autoimmunity, CNRS-Toulouse III University, Toulouse Cedex, France*Present address of Georg Schett: Clinic for Internal Medicine 3, University of Erlangen- Nurnberg, Erlangen, Germany

A single intradermal injection of the mineral oil pristane in susceptible DA.1F rats induces erosive arthritis closely mimicking rheumatoid arthritis (RA). Pristane-induced arthritis (PIA) disease is driven by autoreactive T cells but no autoantigen has been identified so far. We therefore analyzed B and T cell responses in rats with this disease to autoantigens potentially involved in the pathogenesis of RA, including immunoglobulin G, citrullinated proteins, stress proteins, glucose-6-phosphate isomerase, and heterogeneous nuclear ribonucleoprotein (hnRNP)-A2. Autoantibodies to hnRNP-A2 were detectable in sera of pristane-primed rats already one week before disease onset, reaching maximum levels during acute phase. Apart from rheumatoid factor autoantibodies to other antigens were not observed. CD4+ lymph node cells isolated 10 days after pristane injection produced interferon-gamma but not interleukin-4 in response to stimulation with hnRNP-A2, whereas none of the other candidate antigens elicited cytokine secretion. Surprisingly, hnRNP-A2 also stimulated non-primed lymph node cells to produce inflammatory cytokines in a MyD88-dependent manner. Furthermore, hnRNP-A2 was highly overexpressed in joints of rats with PIA as revealed by immunohistochemistry and Western blotting. Taken together, these data demonstrate hnRNP-A2 to be among the primary inducers of autoimmunity in PIA. Therefore, this antigen might play a pivotal role in the pathogenesis of PIA and possibly also human RA.

008 RHEUMATOID ARTHRITIS SUBTYPES IDENTIFIED BY GENOMIC PROFILING OF PERIPHERAL BLOOD CELLS: ASSIGNMENT OF A TYPE I INTERFERON SIGNATURE IN A SUBPOPULATION OF PATIENTS

T. C. T. M. van der Pouw Kraan1, C. A. Wijbrandts2, L. G. van Baarsen1, A. E. Voskuyl3, F. Rustenburg1, J. M. Baggen1, M. Fero4, B. A. C. Dijkmans3, P. P. Tak2, C. L. Verweij1.1VU medical center, Dept. of Molecular Cell Biology & Immunology, Amsterdam, the Netherlands; 2Academic Medical Center/University of Amsterdam, Div. of Clinical Immunology and Rheumatology, Amsterdam, the Netherlands; 3VU medical center, Dept. of Rheumatology, The Netherlands; 4 Stanford University, Stanford Functional Genomics Facility, Stanford, CA, USA

Aim: Rheumatoid arthritis (RA) is a heterogeneous disease with unknown aetiology. Here we aimed to identify peripheral blood gene expression profiles that may distinguish RA subtypes.

Material and methods: Large-scale expression profiling by cDNA microarrays was performed on peripheral blood from 35 patients and 15 healthy individuals. Differential gene expression was analyzed by Significance Analysis of Microarrays (SAM), followed by Gene Ontology analysis of the significant genes. Gene Set Enrichment Analysis (GSEA) was applied to identify pathways relevant to disease.

Results: We found a remarkably elevated expression of a spectrum of genes involved in immune defense in the peripheral blood of RA patients compared to healthy individuals. SAM analysis revealed a highly significant elevated expression of interferon (IFN) type I regulated genes in RA compared to healthy individuals, which was confirmed by Gene Ontology and Pathway analysis, suggesting that this pathway was activated systemically in RA. A quantitative analysis revealed that increased expression of IFN-response genes was characteristic of approximately half of the patients (IFNhigh patients). Application of pathway analysis revealed that the IFNhigh group was largely different from the controls, with evidence for upregulated pathways involved in coagulation and complement cascades, and fatty acid metabolism, while the IFNlow group was similar to the controls.

Conclusion: The IFN type I signature defines a subgroup of RA patients, with a distinct biomolecular phenotype, characterized by increased activity of the innate defense system.

009 MESENCHYMAL STEM CELLS REPAIR CAPABILITIES ARE DEFECTIVES IN RA IN RELATION WITH IN VIVO EXPOSURE TO INFLAMMATION

E. Jones, S. Field, A. English, R. Reece, P. Emery, D. McGonagle, F. Ponchel.LIMM, Section rheumatology, University of Leeds, U.K

Background: The potential value of Autologous Mesenchymal Stem Cells (MSCs) derived from patient’s joint is now well recognized in OA and RA for the repair of bone and cartilage damage. However, the fact that MSCs have been directly exposed to inflammation in the joint has not been given sufficient attention. We have shown recently that chondrogenesis was qualitatively diminished in MSCs derived from the synovial fluid of RA patients compared to OA (Jones et al. A&R, 2004. 48: 817–827). Here we investigate the effect of the joint microenvironment on MSC’s differentiation capability and the possible molecular mechanism by which cells become deficient.

Results: Synovial tissue (ST) was obtained during arthroscopy from 15 patients with RA and OA, fully documented with VAS scores. In vitro differentiation assays were performed towards bone and cartilage. We observed an inverse relationship between levels of inflammation in the joint (VAS) and the chondrogenic potential (R = −0.750, P<0.01). Such relationship was not observed for bone differentiation. The expression of two transcription factors (TF) is necessary for differentiation to take place: Sox9 for chondrogenesis and Runx2 for osteogenesis. We confirmed their induction during bone and cartilage differentiation of healthy bone marrow MSC. We measured the levels of expression of these TF in MSCs isolated from 9 synovial fluids (SF) from RA and OA patients and compared them with MSC isolated from healthy bone marrow (BM) donors (n = 6) and skin fibroblasts (negative control, n = 4). Sox9 expression was higher in OA SF-MSC that in BM-MSC but reduced to negative control levels in RA. Runx2 expression was reduced in both RA and OA. Sox9 and Runx2 expression was also investigated in ST-MSC from 6 RA patients. Sox9 and Runx2 expression were inversely correlated with VAS scores of inflammation (R = −0.737 P<0.01 and R = −0.843 P<0.01 respectively). To investigate the effect of inflammation on the ability of MSCs to differentiate we quantified the expression of Sox9 and Runx2 following treatment with TNF-a, TGF-b3, and IFN-g comparing BM-MSCs (n = 4) and RA ST-MSC (n = 6). TGF-b3 is a trigger of chondrogenesis and accordingly increased the expression of Sox9 and Runx2 in BM-MSC. TNF-a and INF-g reduced the expression of Sox9 and Runx2. In contrast in RA, TGF-b3 had lost the ability to induce the expression of Sox9 and Runx2 whereas TNF-a and INF-g increased it. The cytokine effects were all directly correlated with previous exposure to inflammation (all significant correlation with VAS).

Conclusion: Altogether, these data demonstrate that exposure to inflammation has a profound effect on the ability of MSCs to respond to differentiation triggers. Our data also suggest that cytokine(s) are likely to be involved in the acquired deficit in chondrogenesis in RA. Controlling joint inflammation with anti-TNF is associated with the down-regulation of inflammation-related parameters (including bone damage—Klareskog et al. Lancet, 2004. 363. 675–681) and would therefore be necessary, but may not be sufficient to allow MSCs to undertake cartilage repair in RA.

010 GENOMICS ANALYSIS OF FRESHLY-PURIFIED HUMAN MARROW STROMAL CELLS (MSCS): COMPARISON WITH HEMATOPOIETIC LINEAGE CELLS AND CULTURE-EXPANDED MSCS

E. Jones, S. Kinsey, A. English, S. Field, R. Jones, A. Rawstron, L. Strazsynski, S. Rundlett, S. Jain, R. Deans, P. Emery, D. McGonagle, F. Ponchel.Leeds Institute of Molecular Medicine, Paediatric Haematology and HMDS, Leeds University/Teaching Hospital and Athersys Inc, Stem Cell Institute, Minneapolis, USA

Background: Genomics and cell surface antigen expression profiles of bone marrow MSCs following in vitro culture have been thoroughly investigated, but whether the same characteristics can be extrapolated to MSCs in vivo remain poorly evaluated. In this study we used microarrays to compare MSCs freshly-purified from human bone marrow aspirates (fresh-MSCs) with donor-matched hematopoietic lineage cells (HLCs) and with standard culture-expanded MSCs (exp-MSCs).

Method: Following enrichment with Anti-Fibroblast MicroBeads, fresh-MSCs were further purified by cell sorting as CD45lowCD235a-LNGFR+ [Jones et al. 2002 A&R] and HLCs as CD45+CD235a+LNGFR-. Passage 2 exp-MSCs were used without further manipulation. RNA was extracted, amplified and hybridized to an Affimetrix U133 Plus 2.0 chip. Raw fluorescence data were normalised to GAPDH for direct validation by qPCR.

Results: The microarray data in fresh-MSCs highlighted the expression of several mesenchymal lineage transcription factors (TFs) (Sox9, Runx2 and PPARf×) and surface markers known to be present on exp-MSCs (CD73, CD90, and others), confirming the MSC nature of the CD45lowCD235a-LNGFR+ cells. All genes (cited above and below) were validated by qPCR (5 donors). Novel MSC surface markers were identified: Junctional Adhesion Molecule 2, Leptin receptor, CD81 and CD39, all confirmed at the protein level by 6-colour flow cytometry. A custom-made, Perl-based program was designed to search expression data for gene ontology, with particular focus on molecules involved in extra-cellular matrix (ECM) turnover, support for haemopoiesis and in osteogenic and adipogenic differentiation programs. Compared to HLCs, fresh-MSCs over-expressed genes encoding various ECM components, MMPs and TIMPs (a total of 45 genes). Molecules implicated in haemopoiesis-supportive function or reticular stromal topography were also detected in fresh-MSCs (including SDF-1, IL-7 and Jagged 1). Many components of the TGFb/BMP and Wnt pathways involved in osteogenic differentiation and several bone-related genes (osteocalcin and alkaline phosphatase) were also over-expressed in fresh-MSCs. Several mature fat tissue products were also detected in fresh-MSCs (FABP4, adiponectin and lipoprotein lipase) suggesting the presence of cells committed towards adipogenesis. The expression of ∼60% of molecules related to stroma and adipogenesis and specifically expressed in fresh-MSCs was significantly reduced in exp-MSCs (5–100 fold). In contrast, over 60% of genes related to ECM and osteogenesis remained stable.

Conclusion: Altogether, this work revealed the unique gene expression profile of fresh-MSC and described new surface molecules. It highlights the fact that gene expression data in exp-MSC cannot be directly extrapolated to fresh-MSC. Most importantly, the BMP2 autocrine loop maintaining the osteogenic readiness of fresh-MSC is lost in exp-MCS and may explain the lower bone formation capability of these cells in vivo (Banfi et al. 2000 Exp. Hem). Altogether, these data have broad implications for our understanding of the role of bone marrow stromal cells in skeletal remodelling and for the development of MSC-based therapies for bone repair.

011 CIRCULATING LEVELS OF INTERLEUKIN-7 IN RA

S. Churchman, S. Field, C. Burgoyne, A. Brown, N. ElSayed, P. Emery, F. Ponchel.Leeds Institute of Molecular Medicine, Section Rheumatology, University of Leeds, Leeds, UK

Background: We have previously demonstrated reduced circulating levels of interleukin-7 (IL-7) in active RA compared to health.1 IL-7 was shown not to correlate with CRP in active disease or with other disease activity markers (DAS28, ESR, joint counts, shared epitope, extra articular features and RF presence). Normal IL-7 levels were, however, found in 50% of patients in clinical remission and correlated with the recovery of thymic activity.1 Patients in clinical remission may represent a heterogeneous group and the aim of this work was to identify predictors of IL-7 recovery in demographic, clinical, imaging and functional data.

Methods: Patients deemed to be in clinical remission were recruited. These were outpatients, with stable rheumatoid arthritis (RA), with previous disease duration of at least 12 months, no clinically significant synovitis, no disease flare within preceding 6 months, no evidence of active inflammatory disease and CRP below 15 mg/l for the preceding 6 months. Blood and serum were collected from these patients (n = 106). Clinical data and imaging data were gathered at the time of sampling. High sensitivity ELISA was used for cytokine analysis, proliferation assays for T-cell function (mitogen, TCR stimulation, IL-2, recall antigens); real time PCR to quantify T-bet and GATA3 expression, and DNA sequencing was carried out to investigate 2 genetic polymorphisms in the IL-7 gene.

Results: Several studies in healthy controls indicate that normal levels of circulating IL-7 are between 10 and 25 pg/ml. In remission circulating levels of IL-7 vary between 2.47 and 23.85 pg/ml. Recovery of T-cell function was directly related to levels of circulating IL-7 in vivo (P<0.001, R = 0.873 for PHA n = 11, P<0.001, R = 0.786 for TCR stimulation n = 9 and P<0.001, R = 0.821 for recall antigen n = 9). Age and sex had no effect on IL-7. Combining active (n = 35) and remission patients (n = 106) showed no indication of a relationship between IL-7 and routine measures of disease activity (CRP, DAS28, ESR, PV and joint counts) suggesting that IL-7 is not an indicator of remission. To confirm this observation, we used imaging data (MRI and US assessment of hand and wrist) to address whether subclinical disease could predict lack of IL-7 recovery. Evidence of sub-clinical synovitis was found in 96% of patients in remission2 despite no evidence of clinically significant synovitis and there was no relationship between IL-7 and imaging scores. However, we found that levels of IL-7 in remission and the age of the patient at disease onset correlate (P<0.001, R = 0.498, n = 86). A family history of RA and smoking at the time of onset (both self reported) were also strongly associated with lower levels of IL-7 (both P<0.001, n = 66). We analysed 2 polymorphisms in the promoter and enhancer regions of the IL-7 gene. We found no allele frequency difference between RA patients and healthy controls, remission patients with or without family history, or in relation to IL-7 levels. Finally we analysed circulating cytokines known to regulate IL-7 expression (increase by IFN-g, decrease by TGF-b1 and found a strong correlation between IL-7 and IFN-g (P<0.03, R = 0.650, n = 10). Since IL-7 is a co-activator of Th1 polarisation, we analysed the expression of T-bet and found a direct relationship between the two (P<0.01, R = 0.601, n = 15) but not with GATA3 which is a regulator of Th2 polarisation.

Conclusions: Recent evidence suggests an important role for IL-7 in the pathogenesis of RA.3 Our data demonstrate that circulating IL-7 is not an indicator of disease activity. Despite the relationship with age at onset (genetic anticipation) and the family history association, levels of IL-7 in the circulation are apparently not driven by genetic alterations. Our results suggest that Th1 polarisation and IL-7 are related and that the mechanism by which IL-7 is recovered in remission may result from the recovery of Th1 polarisation.

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012 THE REGULATION OF IL-7 PRODUCTION IN SYNOVIAL STROMAL CELLS IN RA IS PROMISCUOUS

S. Field, E. Jones, A. English, C. Burgoyne, S. Churchman, R. Reece, P. Emery, F. Ponchel.Leeds Institute of Molecular Medicine, Section Rheumatology, Leeds University, UK

Background: Recent evidence suggests an important role for IL-7 in the pathogenesis of RA and a therapeutic potential for IL-7 blockade (Hartgring, 2006. ARD 65:69). IL-7 is a cytokine produced by synovial stromal cells (StrC) in RA but barely detectable in OA (Harada, 1999 A&R 42:1508). In contrast, our published data showed reduced production of IL-7 by bone marrow (BM) StrC and reduced levels of circulating IL-7 in RA compared to health (Ponchel, 2005. ART 7:82). These conflicting observations suggest that the regulation of IL-7 expression is tightly controlled at the level of tissue specificity. To support this hypothesis, we showed that IL-7 is induced by IFN-gamma in StrC from the bone marrow and in epithelial cells from the liver and the gut (using real time PCR and confirming the data with high sensitivity ELISA). In contrast, IL-1 and IL-10 can only induce IL-7 in liver cells. This work aims at identifying the factors that regulate IL-7 production in StrC isolated from synovial tissue of RA patients and healthy BM donors.

Results: RA synovial tissue StrC expressed IL-7 (detected by immunohistochemistry) in active disease but levels were below detection in clinical remission. Synovial fluid and tissue biopsy were obtained from RA (n = 6) and OA (n = 4) patients (documented with inflammation scores, VAS) and BM aspirates from healthy donors (n = 6). IL-7 expression (detected by real time PCR) was higher in synovial fluid StrC isolated from RA compared to OA (3 fold). We quantified IL-7 in freshly isolated StC (using advanced cell sorting strategy—Jones, 2006. Clin Cytometry. 70B-391) from 5 healthy BM and confirmed that the expression of IL-7 is 20 folds higher compared to hematopoietic cells. However expression declined rapidly in tissue culture (20 fold by passage 2) suggesting that environmental triggers are required to sustain IL-7 expression. We then compared IL-7 expression in expanded healthy BM and RA synovial tissue. At baseline IL-7 expression was 3 fold less in RA but directly correlated with a VAS score of inflammation performed during arthroscopy (rho = 0.930, P<0.001). IFN-gamma (10 ng/ml) induced IL-7 by 9 fold in health but this regulation is lost in RA in relationship (rho = 0.842, P<0.001) with exposure to inflammation (VAS score). In contrast TNF-alpha (25 ng/ml) and IL-1-beta (25 ng/ml) induced IL-7 by an average of 3 and 15 fold respectively in RA but had no effect in health. The effect of the cytokines was again correlated with exposure to inflammation (rho = 0.883, P<0.001 for TNF-alpha and rho = 0.964, P<0.001 for IL-1-beta). TGF-beta 1 (20 ng/ml) reduced IL-7 expression in health (5 fold) but increased it in RA (2 fold). Therefore, every cytokine appears to increase IL-7 expression in RA synovial StrC whereas in healthy BM, the tissue specificity is maintained.

Conclusions: The expression of IL-7 is tightly controlled in health, the accessibility of the IL-7 gene to regulation probably being restricted by tissue specificity. In contrast, in RA tissue specificity is abrogated, and every cytokine is able to induce IL-7. This last observation suggests promiscuity on the promoter of the IL-7 gene. This may be resulting from an alteration of the epigenetic regulation of the IL-7 promoter possibly through the effect of Line 1 mediated hypo-methylation of chromatin (Neidhart, 2000. A&R 43:2634).

013 EARLY EFFECTS OF RITUXIMAB ON THE SYNOVIAL CELL INFILTRATE IN PATIENTS WITH RHEUMATOID ARTHRITIS

K. Vos1, R. M. Thurlings1, C. A. Wijbrandts1, D. van Schaardenburg2, D. M. Gerlag1, P. P. Tak1.1Division of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands; 2Jan van Breemen instituut, Amsterdam

Objective: To study the specific effects of rituximab treatment on the synovium in patients with rheumatoid arthritis (RA) early after initiation of treatment.

Methods: Seventeen RA patients underwent an arthroscopic synovial biopsy procedure directly before and one month after two infusions of the chimeric anti-CD20 monoclonal antibody rituximab (1000 mg on day 1 and 15; at both time points without pre-medication with methylprednisolone). Immunohistochemical analysis was performed to characterize the cell infiltrate. Stained tissue sections were analyzed by digital image analysis. Statistical analysis was performed using the Wilcoxon signed ranks test.

Results: No significant change in the disease activity score (DAS28) was found 4 weeks after the first rituximab infusion. Two and 4 weeks after infusion B cells in peripheral blood were almost completely depleted. In the synovium most B cells were found in large lymphocyte aggregates. Interestingly, a significant reduction in B cell numbers at the site of inflammation was observed 4 weeks after treatment (before treatment: 26 cells/mm2±75; after treatment 11 cells/mm2±68; P<0.02). In 3 patients B cells disappeared completely, whereas there was partial depletion in 11 patients. In 3 patients no B cells were present in either pre- or post-treatment biopsies. No reduction in other synovial cell populations was observed at 4 weeks.

Conclusion: Rituximab treatment leads to a rapid and significant decrease in synovial B cell numbers, but not in all patients. Whether the variable tissue response is related to clinical response over time remains to be clarified.

014 A PHASE IB STUDY TO INVESTIGATE ATACICEPT (TACI-IG) IN PATIENTS WITH RHEUMATOID ARTHRITIS

P. P. Tak1, R. M. Thurlings1, A. Dimic2, V. Mircetic3, M. Rischmueller4, E. Nasanov5, E. Shmidt6, P. Emery7, C. Rossier8, I. Nesterov9, J. Hill9, A. Munafo8.1AMC/University of Amsterdam, Amsterdam, The Netherlands; 2Institute for Rheumatic and Cardiovascular Diseases, Niska Banja, Serbia and Montenegro; 3Institute for Rheumatology, Belgrade, Serbia and Montenegro; 4Queen Elisabeth Hospital, Woodville, Australia; 5GU Institute for Rheumatology, Moscow, Russian Federation; 6City Clinic Hospital, Moscow, Russian Federation; 7Leeds General Infirmary, Leeds, United Kingdom; 8Serono International, Geneva, Switzerland; 9ZymoGenetics Inc., Seattle, WA, USA

Purpose: To evaluate the tolerability of single and repeated doses of atacicept in patients with rheumatoid arthritis (RA). Atacicept (formerly known as TACI-Ig) is comprised of the extracellular portion of the TACI receptor fused to an Fc domain of human IgG. Atacicept is an antagonist of BLyS and APRIL, regulators of B cell function and survival that also play important roles in autoimmune disease.

Methods: A double blind, placebo-controlled phase Ib study was conducted in patients with active moderate to severe RA (n = 73). Six cohorts of patients received escalating doses of atacicept, either as a single subcutaneous dose, or as repeated doses. Standard safety assessments were performed; in addition a comparison of vaccine immunization status was assessed before and after treatment. Binding antibodies to atacicept were measured. PK was determined for serum concentrations of atacicept and of atacicept.BLyS complex. Lymphocytes, monocytes and NK cells were evaluated and levels of circulating Ig, anti-citrullinated peptides (anti-CCP), and rheumatoid factor were determined. ACR20 and DAS28 scores were assessed as measures of disease activity.

Results: No significant treatment-related adverse events were observed. No patient developed antibodies to atacicept. Vaccine immunization status to tetanus and diphtheria were not affected. Atacicept PK are nonlinear and consistent across the dose range studied, displaying a terminal half-life of 25 to 63 days. Atacicept was found to penetrate into inflamed joints. Biological markers decreased in accordance with atacicept’s MOA: after 3-months repeated dosing, serum IgM, IgA and IgG were reduced by 54%, 37%, and 21% respectively; there was a 41-44% reduction for all Ig-subclasses of RF, a 25% decrease in anti-CCP and a 40–50% decrease in mature and total B cells. T cells, NK cells and monocytes were not affected. Positive trends in ACR20 and DAS28 were also noted.

Conclusion: Atacicept was well tolerated in patients with moderate to severe RA. The observed reductions in circulating Igs and peripheral blood B cells were consistent with the predicted MOA for atacicept. Reductions in RF and in anti-CCP were observed and positive trends in ACR20 and DAS28 indicate atacicept’s potential to improve disease outcomes.

015 ENHANCED IL-7 EXPRESSION IN INFLAMED JOINTS CAN RESULT IN DIMINISHED SUPPRESSIVE TREG FUNCTION

S. A. Y. Hartgring, L. S. Taams, J. M. R. van Amelsfort, M. J. G. Wenting, J. W. J. Bijlsma, F. P. J. G. Lafeber, J. A. G. van Roon.UMC Utrecht, Dept Rheumatology & Clinical Immunology, Utrecht, The Netherlands

Introduction: IL-7, a member of the IL-2 family, is a potent immunoregulatory cytokine. Increased IL-7 levels are found in several inflammatory diseases including rheumatoid arthritis (RA). IL-7 effects are mediated through the high affinity IL-7receptor-alpha chain (IL-7Ralpha) in conjunction with the common gamma chain. IL-7 stimulates proliferation, survival and differentiation of T-cells and induces T-cell dependent monocyte, B-cell and osteoclast formation. We studied IL-7 and IL-7Ralpha expression levels in arthritic conditions and tested effects of IL-7 on CD25+Treg-mediated suppression.

Methods: IL-7Ralpha and IL-7 expression were determined by immunohistochemistry in synovial tissue of 24 RA patients, 15 osteoarthritis patients (OA, disease control) and 27 patients with undifferentiated (mono) arthritis (UA). IL-7Ralpha expression on CD4, CD8, NK T-cells, CD19 B-cells and CD14 monocyte/macrophages from RA synovial fluid (SF) and RA peripheral blood (PB) was assessed. Furthermore, IL-7Ralpha expression on CD25- vs. CD25+ cells was studied in addition to the effects of IL-7 on CD25+Treg mediated suppression.

Results: IL-7Ralpha and IL-7 expression in RA and UA synovial tissue was significantly higher compared to OA tissue. Co localisation of IL-7 and IL-7Ralpha expression was found at many sites and the expression of IL-7Ralpha and IL-7 significantly correlated. RA SF CD4+, CD8+ and NK T-cells strongly expressed IL-7Ralpha. Remarkably, compared to PB, SF B-cell and monocyte subpopulations also showed IL-7Ralpha expression, although this expression was considerably lower than the expression levels on T-cells. Interestingly, we found that CD25+ T-cells did not express IL-7Ralpha, in contrast to CD25- T-cells. IL-7 abrogated CD25+ Treg mediated suppression of CD25- T-cells, apparently mediated by IL-7-induced activation of IL-7Ralpha+CD25- effector T-cells.

Conclusion: Our data suggest that enhanced IL-7 expression in RA patients could significantly contribute to arthritis by activation of arthritogenic CD25- T-cells, overriding the suppressive effect of Tregs that lack IL-7Ralpha expression.

Drs Hartgring and Dr van Roon were supported by the Dutch Arthritis Association.

016 GSK3 REGULATION OF THE INFLAMMATORY RESPONSE TO TLR LIGANDS

A. Triantafyllopoulou1, X. Hu1, L. Wang1, A. Davidson2, L. Ivashkiv1.1Cytokine and Inflammation Laboratory, Hospital for Special Surgery, New York; 2Department of Medicine, Columbia University Medical Center, New York, USA

Glycogen synthase kinase-3 (GSK3) has recently emerged as an important regulator of the pro- and anti-inflammatory cytokine production.12 Administration of GSK3 inhibitors suppressed the proinflammatory response in mice receiving LPS, protecting them from endotoxic shock1 and ameliorated the clinical signs, radiographic and histologic findings of type II collagen induced arthritis in mice.3 IL-10 and TNF-a have been found to be induced early in the course of nephritis in NZB/W mice and thus potentially play a pathogenetic role in lupus nephritis. We sought to study the effect of GSK3 inhibition on the expression of inflammatory cytokines by CD14+ monocytes, following stimulation with a synthetic TLR7/8 ligand or LPS. Real-time PCR showed that GSK3 inhibition increased the relative expression of IL-10 in response to TLR7/8 ligands and LPS while the effect of GSK3 inhibition on the relative expression of TNFa varied. Further, GSK3 inhibition differentially regulated the induction of IFNβ by TLR7/8 ligands and LPS. These findings may have important implications for lupus nephritis where end organ damage seems to be mediated by inflammatory cytokines produced by effector cells which are activated in response to RNA and DNA containing immune complexes that bind to TLR7/8 and TLR9 receptors.

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017 MODULATION OF SERUM AMYLOID A ACTIVATION OF HUMAN ENDOTHELIAL CELLS BY TRANSFORMING GROWTH FACTOR-BETA1

K. Lakota, K. Mrak-Poljsak, B. Rozman, T. Kveder, M. Tomsic, S. Sodin-Semrl.University Medical Centre, Department of Rheumatology, Ljubljana, Slovenia

Background: Inflammation is considered to be the driving force leading to pro-atherogenic and pro-atherosclerotic mechanisms. Acute phase Serum Amyloid A (SAA) is highly elevated in patients due to inflammation and those at risk for future coronary occlusion. SAA’s increased levels predict the risk of coronary artery disease and even mortality from cardiovascular disease in humans. Recent animal studies have indicated that SAA plays a causal role in atherogenesis. Transforming growth factor beta1 (TGF-beta1) has been shown to be produced in the vascular endothelium where it can suppress pro-inflammatory cytokine and adhesion molecule expression.

Aim of study: The objectives of this study were to understand the role of SAA in activating possible atherogenic inflammatory responses in endothelial cells and to determine the effect of TGF-beta1 on this activation.

Materials and methods: Human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) were grown in order to analyze the effects of SAA on endothelial expression of pro-inflammatory cytokines, such as IL-6, chemokines, such as IL-8 and adhesion molecules by reverse transcription-PCR and ELISAs. TGF-beta1 was used as a potential inhibitor/modulator of these events.

Results: We compared the dose responses of SAA between HUVEC and HCAEC. SAA activated HUVEC in a dose dependent manner. In comparison, HCAEC showed greater sensitivity to SAA, with a higher level of expression of all pro-inflammatory markers at much lower concentrations of SAA and their greater stimulation at higher SAA concentrations. SAA also generated a dose dependent positive feedback response on its own mRNA expression in HCAEC as compared to HUVEC, where SAA did not generate any positive feedback. Incubations of both HUVEC and HCAEC with increasing doses of TGF-beta1 showed a tendency for significant inhibition of IL-6 and adhesion molecules, especially at the highest concentration used (2000 pM). SAA-stimulated IL-6 mRNA expression levels in presence of TGF-beta1 decreased in HUVEC as well as HCAEC. However, IL-6 protein levels were less affected by the addition of TGF-beta1 to SAA induction.

Conclusions: In summary, there are distinct significant differences in levels of inflammatory markers and adhesion molecules between HUVEC and HCAEC SAA dose responses that could account for HCAEC greater susceptibility to inflammation and atherogenesis. Treatment with TGF-beta1 alone at a concentration of 2000pM significantly inhibits background levels of IL-6 and adhesion molecule mRNA expression and protein levels in both HUVEC and HCAEC. When TGF-beta1 is used in combination with SAA, inhibition is more prominent at the mRNA expression levels than at the protein levels of all inflammatory and adhesion molecules tested.

018 HMGB1-CONTAINING NUCLEOSOMES RELEASED FROM SECONDARY NECROTIC CELLS CAUSE ACTIVATION AND CYTOKINE RELEASE FROM MONOCYTES/MACROPHAGES AND DENDRITIC CELLS—IMPLICATIONS FOR THE IMMUNOPATHOGENESIS OF SLE

V. Urbonaviciute1, B. Fuernrohr1, C. Weber1, P. Heyder1, J. R. Kalden2, M. Herrmann2, R. E. Voll1 2.1IZKF Research Group 2, Nikolaus-Fiebiger Center, University of Erlangen; 2Department of Internal Medicine 3, University Hospital of Erlangen

Background/aim: Autoantibodies against dsDNA and nucleosomes are hallmarks of systemic lupus erythematosus (SLE). The mechanisms and factors which are involved in breaking immunological tolerance towards nuclear autoantigens are not yet fully understood. High mobility group box protein 1 (HMGB1), an architectural nuclear DNA-binding protein and, in addition, a secreted pro-inflammatory mediator, could represent such a signal. In apoptotic cells, HMGB1 is tightly attached to the chromatin and, hence, not released during apoptosis. In conditions of defective clearance of apoptotic cells, which is observed in a subset of patients with SLE, non-eliminated apoptotic cells may undergo secondary necrosis, thereby releasing HMGB1 tightly attached to nucleosomes. To test this hypothesis we investigated if nucleosomes released from secondary necrotic cells in fact contain HMGB1 and can lead to activation of monocytes/macrophages and dendritic cells. In addition, we analyzed if circulating immunocomplexes and nucleosomes in the blood of SLE patients contain HMGB1.

Materials and methods: Nucleosomes were prepared from living, apoptotic, and necrotic cells by isolation of nuclei, digestion with DNase I, and subsequent purification of mononucleosomes via sucrose gradient centrifugation. Fractions were tested for the presence of mononucleosomes by agarose gel electrophoresis and for the presence of HMGB1 by immunoblot analysis. Human monocytes-derived macrophages and dendritic cells were incubated with mononucleosome fractions from living, necrotic and apoptotic cells. Cytokine secretion pattern was assessed by ELISA. Surface marker expression was analyzed by flow cytometry. HMGB1 was detected in the serum of patients by co-immunoprecipitations using either anti-nucleosome or anti-dsDNA antibodies covalently coupled to sepharose beads and subsequent immunoblot analysis for HMGB1. Circulating immunocomplexes from patients with SLE and other rheumatic and infectious diseases were precipitated from the serum using 2 % polyethylene glycol and analyzed by immunoblotting.

Results: Substantial amounts of HMGB1 were detected within immunocomplexes, precipitated by polyethylene glycol in sera of patients with SLE, but not in sera from healthy controls and non-SLE patients. Co-immunoprecipitations revealed a physical association of HMGB1 with nucleosomes. Importantly, incubation of human macrophages and dendritic cells with HMGB1-containing nucleosomes isolated from apoptotic cells potently induced the release of TNFalpha and IL-10, whereas NCs purified from living cells did not contain HMGB1 and failed to induce cytokine release. Also, surface expression of MCH class II molecules and costimulatory molecules such as B7 family members were predominantly induced by HMGB1-containing nucleosomes.

Conclusions: Our results suggest that HMGB1 attached to nucleosomes released from apoptotic cells may act as an “endogenous adjuvant”, thereby, increasing the low immunogenicity of nucleosomes/chromatin. Hence, impaired phagocytosis of apoptotic cells with consecutive release of HMGB1-nucleosome complexes may crucially contribute to the immunopathogenesis of SLE.

019 ANGIOTENSIN II PROTECTS FIBROBLAST-LIKE SYNOVIOCYTES FROM APOPTOSIS VIA THE AT1-NF-KB PATHWAY

L. Pattacini, B. Casali, L. Boiardi1, N. Pipitone1, C. Salvarani1.Molecular Biology Lab, Ospedale Santa Maria Nuova, Reggio Emilia, Italy; 1Rheumatology Unit

Aim of the study: To evaluate the effects of Angiotensin II (Ang II) treatment on apoptosis of fibroblast-like synoviocytes (FLS) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA).

Materials and methods: FLS were obtained from synovial fluids of OA and RA patients. AT1 receptor expression was detected by Western blot and flow cytometry. Apoptosis was induced by nitric oxide exposure and serum starvation and determined by nucleosome ELISA and TUNEL assay. Cell proliferation was determined by BrdU incorporation. AT1 function was antagonized by using Losartan; the expression of p65 subunit of NF-kB was switched off by RNA silencing, whereas caspase activity was inhibited by z-VAD-fmk. Caspase 3 activation was evaluated by western blot and by a colorimetric assay.

Results: Comparable levels of AT1 were expressed by FLS from OA and RA patients. Ang II pretreatment reduced FLS response to the above mentioned apoptotic stimuli. In fact, the absorbance values (mean± SD) obtained by ELISA in the absence or presence of AngII pre-treatment were 0.48±0.09 and 0.30±0.11 respectively for serum deprived OA samples and 0.80±0.20 and 0.44±0.18 (p<0.05) for nitric oxide exposure. In RA samples, the absorbance values were 0.37±0.09 and 0.15±0.02 for starvation and 0.32±0.09 and 0.18±0.04 for nitric oxide treatment, respectively (p<0.05). These results were confirmed by TUNEL. FLS proliferation was not affected by Ang II treatment. Ang II protective effect was not observed in the presence of the AT1 receptor antagonist losartan. An 85% reduction of p65 NF-kB expression was obtained by RNA silencing and led to the loss of Ang II anti-apoptotic effect. Similarly, in the presence of the effector caspase inhibitor z-VAD-fmk, Ang II failed to protect FLS from apoptosis. Ang II pre-treatment determined a decrease in caspase 3 cleavage and consequently in its activation.

Conclusions: We have shown that the octapeptide Ang II, whose role in inflammation has been recently highlighted, can protect FLS from apoptosis. We have also observed that this effect requires Ang II binding to AT1 receptor and the ensuing activation of NF-kB. Moreover, we have demonstrated that this activated pathway involves caspase activation. Finally, we have verified that Ang II can prevent caspase 3 cleavage and activation. We propose that Ang II may represent a mediator involved in skewing the balance between survival and death of FLS, thus contributing to their expansion in course of RA.

020 ANTI-RHEUMATIC DRUGS TARGETING TNF-ALPHA AND IL-6 PATHWAYS INDUCE APOPTOSIS OF FIBROBLAST-LIKE SYNOVIOCYTES

L. Pattacini, B. Casali, L. Boiardi1, D. Nicoli, E. Farnetti, C. Salvarani1.Molecular Biology Lab; 1Rheumatology Unit

Aim: To evaluate the effect of biologic (anti-TNF-a and anti-IL-6) drugs on the apoptosis of fibroblast-like synoviocytes (FLS).

Materials and methods: FLS were obtained from synovial fluids of 5 osteoarthritis (OA) and 5 rheumatoid arthritis (RA) patients. Peripheral blood mononuclear cells (PBMC) were collected by Ficoll centrifugation. The adherent fraction was isolated by overnight culture in the absence of serum. For mixed culture experiments, 500,000 FLS were seeded in 6-well plates, exposed to either 10 μg/ml Infliximab, 10 μg/ml Adalimumab, 10 μg/ml Etanercept or 25 μg/ml Tocilizumab. Thereafter, 1,000,000 PBMC or their adherent fraction were added. In some experiments a 0.4 μm pore filter was used to keep the two cell types separated. After 7 days, apoptosis induction was determined by morphological changes detection using light microscopy and by an ELISA allowing the detection of nucleosome release from the nucleus to the cytoplasm. The results were confirmed by TUNEL assay. We evaluated by Western blotting the phosphorylative status of tyrosine 397 residue of focal adhesion kinase (FAK), as well as the levels of MAPK, Bcl-2 and bax and the activation of caspase 3 in FLS that had undergone apoptosis.

Results: We observed that only when the entire PBMC population was co-cultured in direct contact with SF, apoptosis of RA FLS, but not of OA FLS, was induced by the four aforesaid drugs although to a different extent in the three analyzed samples. In fact, one sample resulted resistant to Infliximab, Adalimumab and Tocilizumab but sensitive to Etanercept, with a 1.7 fold increase in apoptosis induction, one responded only to the two anti-TNF-a antibodies (1.3 fold increase) and three of them showed a comparable response for all tested drugs. These results were in agreement with the morphological evaluation of FLS apoptosis, characterized by cell rounding and detachment and were confirmed by TUNEL assay. We then decided to investigate the role of FAK in apoptosis induction. FAK is activated through tyrosine 397 auto-phosphorylation and is involved in the anchorage of the cell to the substrate. We found that in FLS undergoing apoptosis and detached from the substrate, this phosphorylation was decreased, whereas the protein levels did not change. The expression of the anti-apoptotic protein Bcl-2 was reduced in treated cells. We failed to detect any change in bax and MAPK levels. Caspase 3 resulted to be activated by the different treatments by proteolytic cleavage.

Conclusion: In our study, we have shown that the four biologic agents either in use or in clinical trials for RA treatment can exert their effects at least in part through their capacity to induce FLS apoptosis in the presence of PBMC. Moreover, we have shown that these drugs determine the inactivating dephosphorylation of FAK as well as the decrease in Bcl-2 levels and caspase 3 cleavage.

021 RECRUITMENT OF B LYMPHOCYTES INTO THE PERIPHERY IS ASSOCIATED WITH DISEASE ACTIVITY IN PATIENTS WITH REFRACTORY RHEUMATOID ARTHRITIS

Y. K. O. Teng, M. Hashemi, N. L. Levahrt, R. E. M. Toes, T. W. J. Huizinga, J. M. van Laar.Dept. of Rheumatology, Leiden University Medical Center, The Netherlands

Introduction: B-cell depletion is a new and successful treatment for patients with refractory rheumatoid arthritis (RA). However, the exact role of B cells in the pathogenesis of RA is still unclear, although circulating autoantibodies and synovial infiltration of plasma cells are common findings in RA patients. In order to elucidate the possible contribution of B lymphocytes to the pathogenesis of RA, the current study investigated the composition of lymphocyte subsets in three compartments, i.e. bone marrow, peripheral blood and synovial tissue, from refractory RA patients.

Aim: To investigate the relative contribution of lymphocyte subsets to the severity of disease activity in refractory RA patients.

Methods: Twenty-six RA patients failing at least 1 TNF-blocking agent were included in the study. Clinical disease activity was determined by the Disease Activity Score of 44 joints (DAS44). Mononuclear cells were isolated from peripheral blood (PBMC) and bone marrow aspirates (BMMC). Both PBMCs and BMMC were stained for flow cytometric analyses. Synovial tissue specimens were obtained by arthroscopy of clinically involved knee joints, stained for expression of CD3, CD79a, CD20, CD68, Ki-67, CD38, CD138 and analysed semiquantitatively. Autoantibodies (anti-cyclic-citrulinated-peptide-antibodies [ACPA] and rheumatoid factor [RF]) were measured by ELISA. Patients with a DAS44>3,7 (EULAR criterium for persistent disease activity) were compared to patients with a DAS44<3,7 by Mann-Whitney U-test.

Results: All patients had clinically active disease with a median DAS44 [range] of 3.62 [2.17–5.23]. Patients with a DAS44 >3.7 had a significantly higher percentage of circulating antibody-secreting cells (ASCs: CD3-CD38bright), both of the IgD+ subset (0.022% [0.0–0.081] vs 0.005% [0.0–0.045]; p = 0,04) and IgD- subset (0.26% [0.024–0.96] vs 0.095% [0.034–0.378]; p = 0.05). There were no significant differences in other peripheral blood lymphocyte subsets between these patients groups. In the bone marrow two differences were only observed in the B-lymphocyte subsets and not in other lymphocyte subsets. RA patients with a DAS44 >3.7 had significantly lower percentages of naïve (CD3−CD19+IgD+) B cells (1.86% [0.61–7.93] vs 5.77% [1.16–13.1]; p = 0,04) and memory (CD3−CD19+CD27+) B cells (1.43% [0.61–5.57] vs 2.64% [0.80–5.09]; p = 0,05) in bone marrow. In synovial tissue, we observed more cellular infiltration of all stained cell types in patients with DAS44 >3.7. There was a trend (p<0.10) towards higher CD68 and Ki-67 expression, indicative of more synovial inflammation. Patients with a DAS44 >3.7 had a significant higher titre of ACPA (894 AU/ml [162-1280] vs 200 AU/ml [20-729]; p = 0.001) but not of RF (63 IU/ml [14–205] vs 44 IU/ml [1–192]; p = 0.47). Moreover, ACPA titres were significantly correlated with DAS44 in these patients (r = 0.70; p<0.0001).

Conclusion: The current study indicates that RA patients with high disease activity show characteristics of B cell hyperactivity and suggest that B cells are recruited into the inflamed synovium and thereby critically involved in the inflammatory processes of RA.

022 GENETIC DISSECTION OF CONGENITAL HEART BLOCK IN A RAT MODEL

L. Strandberg1, S. Salomonsson1, M. Jagodic2, K. Bechanovic2, S-ESonesson3, T. Olsson2, M. Wahren-Herlenius1.1Rheumatology Unit, Department of Medicine; 2Neuroimmunology Unit, Department of Neuroimmunoloy; 3Department of Women and Child Health, Karolinska Institutet, Stockholm, Sweden

Aim: Congenital heart block occurs in children of Ro52 autoantibody positive mothers. However, mothers of affected children can later give birth to healthy children despite persisting Ro52 autoantibodies, indicating that other factors are involved in the penetrance of the disease. The aim of this study was to investigate the MHC and non-MHC genetic influence in heart block development in an immunization-induced rat model of congenital heart block.

Methods: Four rat strains, three of the four sharing the same MHC region (RT1) haplotype (DA.1AV1, PVG.1AV1, LEW.1AV1), and two of the four with similar background genes but different MHC genes (LEW.L and LEW.1AV1) were studied. Female rats were immunized with Ro52 or control protein and mated. The resulting pups (208 from Ro52 immunized mothers, and 105 from the control immunized mothers) were analysed for congenital heart block with electrocardiography. Serum samples were taken throughout the study from both mothers and pups and epitope mapping of the antibody specificities using recombinant Ro52 and a panel of overlapping and mutated Ro52 peptides was performed.

Results: Analysis of the ECGs of the 313 born pups from the four strains showed that in the 3 strains sharing the same MHC region haplotype (DA.1AV1, PVG.1AV1, LEW.1AV1), AV block developed in 45%, 44% and 47% of the pups, respectively. In the Lew.L strain only 10% of pups were affected by AV block. These results indicate that genes in the MHC region are important for penetrance of the disease (p<0.001).

Conclusion: Our study is the first to explore genetic influence in development of congenital heart block in animal models. Results show that genes found in the rat MHC region are of significant importance for the penetrance of congenital heart block, while non-MHC genes appear to have less influence on the disease.

023 IDENTIFICATION OF SINGLE-CHAIN ANTIBODY FRAGMENTS SPECIFIC FOR HUMAN SYNOVIAL MICROVASCULAR ENDOTHELIUM BY IN VIVO PHAGE DISPLAY

P. S. Kamperidis, L. Lee, M. Blades, T. Garrood, A. Nissim, A. George, C. Pitzalis.King’s College London, UK

Introduction: The microvascular endothelium (MVE) plays a major role in inflammation in Rheumatoid Arthritis (RA) by keeping a steady influx of inflammatory cells to the joint. There is evidence to suggest that MVE expresses molecules in a tissue specific manner, thus attracting specific lymphocyte subsets. Since MVE is known to lose much of its tissue specific molecules when cultured in vitro, it is vital to study it in vivo. Thus, a model has been developed in our department in which human tissues transplanted into SCID mice can be targeted directly, using in vivo antibody phage display. The rationale of the technique is based on the capacity to isolate, by multiple rounds of selection, high affinity antibody fragments expressed on the surface of filamentous bacteriophage in association with its coat protein. A large phage-scFv repertoire (Tomlinson library) has been used for the purposes of this project.

Aims: To identify single-chain antibody fragments (svFv) specific for human synovial MVE. The ultimate goal is to use high affinity antibodies for joint-specific delivery of therapeutic/diagnostic agents.

Methods: In vitro validation: a) Three rounds of selection were performed against collagen type II; b) ELISA and competition assays were used to verify collagen specificity of the final phage-pool, as well as individual phage-scFv or pure-scFv clones; c) Individual clones were identified by sequencing; d) scFvs were purified by affinity chromatography. In vivo selection: a) Three rounds of selection were performed against synovial MVE using the human/SCID-mouse transplantation model; b) Sequencing allowed the identification of individual clones from the final phage-pool; c) Those individual clones were pooled and their synovium homing capacity tested by re-injecting them in double transplanted SCID mice.

Results: In vitro validation: a) A clear enrichment (3000-fold) in the collagen-binding phage was observed in the final round, while the background binding to the negative control was negligible; b) Collagen-binding of the phage-pool and individual clones from the final round was specific; c) A number of different collagen-specific clones were identified, and d) produced independently of the phage particles and confirmed again to bind specifically to type II collagen. In vivo selection: a) An enrichment of synovium-homing phage was observed in the third round of in vivo selection; b) Nine clones from the final pool third round were identified, pooled and tested in recirculation studies; c) Recirculation studies in vivo in double transplanted (synovium and skin-control) SCID mice demonstrate higher numbers (over 100 fold) of phage from synovial transplants compared to skin grafts.

Conclusion: Antibody phage display has the potential to produce high affinity antibodies, fast and against any antigen/self-antigen. We have validated the technology in vitro, while in vivo studies have yielded encouraging results towards the identification of high affinity scFv clones specific for synovial MVE. Future work will involve characterization of the clones by sequencing and their utilization as joint specific delivery systems.

024 MODULATION OF HUMORAL IMMUNE RESPONSES BY TNFALPHA BLOCKADE: LESSONS FROM A CARDIAC ALLOGRAFT MODEL

S. Brouard1, F. Moizant1, B. Vanhove1, P-PTak2, J-PSoulillou1, D. Baeten1.1INSERM U643, Nantes, France; 2Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands

Objectives: Whereas TNFalpha blockade is very effective for the treatment of various types of chronic inflammatory arthritis, the exact mechanisms of action in vivo have not yet been clearly defined. We recently suggested that TNFalpha blockade may interfere with the maturation of the humoral immune response by impairing isotype class switching. The aim of this study was to assess this issue functionally in a rat mismatched cardiac allotransplantation model, in which graft rejection is known to be mediated by alloantibodies as well as CD4+ T lymphocytes.

Materials and methods: LEW.1W hearts were ectopically transplanted in LEW.1A rats, which were treated with anti-rat TNFalpha or control antibody (3G8). Graft rejection was monitored. Transplanted hearts were obtained 5 days after transplantation and were assessed by histology, immunohistochemistry, and quantitative RT-PCR for cytokines (Th1/Th2), TLRs, and regulatory molecules.

Results: Graft survival in the LEW.1W to LEW.1A rat allotransplantation model was prolonged from 6 days to 13 days by a single IP injection with anti-TNFalpha (8 mg/kg) at day 0. Upon multiple injections (day 0, 3, and 6), the mean graft survival was further prolonged to 23 days. Higher dosage of anti-TNFalpha (15 mg/kg) or concomitant treatment with suboptimal doses of cylcosporin or rapamycin did not have a significant additional effect. At day 5, histology of the graft showed a better conserved histological architecture in anti-TNFalpha treated recipients compared to controls (Banff grade 2-3A versus 3A-3B). Accordingly, immunohistochemical analysis showed a lower number of infiltrating leucocytes in the anti-TNFalpha treated grafts (p = 0.015), with a similar trend for T lymphocytes, B lymphocytes, and macrophages. Of major interest, IgG deposition was significantly lower in anti-TNFalpha treated grafts than control grafts (p = 0.001), without significant differences for IgM deposition. At the mRNA level, no Th1/Th2 cytokine deviation was observed in anti-TNFalpha treated grafts, with a tendency towards decreased levels of all investigated cytokines (IL-2, IFNgamma, TNFalpha, IL-4, IL-10, IL-13). There were no differences at the mRNA level for the regulatory molecules TGFbeta, IDO, HO-1, and FoxP3. Among TLR1-10, there was a downregulation of TLR7 (p = 0.041) with a similar trend for TLR8 (p = 0.093) in the anti-TNFalpha treated grafts. In contrast, TLR4 mRNA tended to be upregulated (p = 0.065).

Conclusions: TNFalpha blockade results in a significant prolongation of graft survival in this allotransplantation model. This is associated with a marked decrease of IgG deposition in the grafts, without alteration of the T cell profiles or regulatory responses. Ongoing analyses of alloantigen-specific antibodies will confirm if the efficacy of TNFalpha blockade in this model is related to the modulation of the humoral response.

025 ANTI-TNFALPHA INDUCED APOPTOSIS CONTRIBUTES TO THE INDUCTION OF AUTOANTIBODIES BUT NOT THE DECREASE OF SYNOVIAL INFLAMMATION IN SPONDYLOARTHROPATHY (SPA)

L. De Rycke1, T. Cantaert1, B. Vandooren1,2, E. Kruithof2, C. A. Wijbrandts1, F. De Keyser2, E. M. Veys2, P. P. Tak1, D. Baeten1.1Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, Amsterdam, Netherlands

Objective: Infliximab, but not etanercept, induces apoptosis of activated macrophages and T lymphocytes in vitro, but the in vivo relevance remains controversial. Therefore, we assessed the induction of apoptosis and related this to modulation of synovitis and autoantibody induction in spondyloarthritis (SpA).

Methods: Synovial biopsies and serum were obtained from SpA patients treated with infliximab (n = 20) or etanercept (n = 20). Synovial apoptosis was assessed by TUNEL and active caspase 3 staining. Nucleosomes and anti-nucleosomes were assessed by ELISA.

Results: Infliximab and etanercept downmodulated synovial inflammation over a 12-week period with a decrease of macrophages and T lymphocytes. However, neither TUNEL nor active caspase 3 staining indicated an increase of synovial apoptosis at weeks 1 and 12. Serum levels of nucleosomes increased significantly at 2 (+89%; p = 0.002) and 6 (+26%; p = 0.014) weeks of infliximab but not etanercept treatment. Accordingly, anti-nucleosome antibodies were strongly increased at week 12 (+94%; p<0.001) and 34 (+124%; p<0.001) of infliximab but not etanercept treatment. As for anti-dsDNA antibodies, this increase was largely restricted to antibodies of the IgM isotype. The early increase of serum nucleosomes was correlated with the subsequent rise in IgM anti-nucleosome antibodies at week 12 (r = 0.383; p = 0.008) and at week 34 (r = 0.453; p = 0.014).

Conclusion: The increase of serum nucleosomes upon treatment with infliximab, but not etanercept, suggests the induction of apoptosis in vivo. This is strongly associated with the induction of autoantibodies restricted to the non-pathogenic IgM isotype, but does not contribute to the efficient downmodulation of synovial inflammation by either TNFalpha blocker.

TC and LDR contributed equally.

026 COEXISTENT MEFV AND CIAS1 MUTATIONS MANIFESTING AS FAMILIAL MEDITERRANEAN FEVER PLUS DEAFNESS

D. Singh-Grewal1,3, B. Bennetts2, 3, J. Chaitow1, I. Aksentijevich4, J. Christodoulou2.1Dept of Rheumatology, The Children’s Hospital at Westmead, Sydney; 2Western Sydney Genetics Program, The Children’s Hospital at Westmead, Sydney; 3Discipline of Paediatrics and Child Health, University of Sydney, Australia

Aims: To report the first cases of coexistent mutations in the MEFV and CIAS1 presenting clinically with typical Familial Mediterranean fever (FMF) with progressive sensorineural deafness, a feature more commonly seen in the cold induced autoinflammatory syndromes such as Muckle-Wells Syndrome.

Cases: Patient 1—the son of non-consanguineous Egyptian parents was investigated for splenomegaly at age one year. No cause was identified. He was noted to have speech delay at age three and a half years with a mild conductive deficit on audiometry. Tympanostomy tubes were inserted. At five years of age he had a sensorineural loss of 60 dB bilaterally in the 2000–3000 Hz range. Temporal bone CT scan was normal. Hearing aids were fitted and his speech improved significantly. Neither parent was deaf. At six years of age, he developed recurrent episodes of fever, abdominal pain, arthritis and elevated inflammatory markers. FMF was diagnosed and he responded well to colchicine therapy. Patient 2—the daughter of non-consanguineous Egyptian parents neither of whom had FMF or deafness. She was found to have anaemia and splenomegaly without any cause found at five years of age and bilateral sensorineural hearing loss of 60 dB at frequencies greater than 1000 Hz with speech delay at six years of age despite previously normal audiometry. She was fitted with hearing aids. A CT scan of the temporal bones showed features consistent with a left-sided vestibular aqueduct syndrome but no obvious abnormality of the right inner ear despite bilateral sensorineural hearing loss. No mutations of the connexin 26 or PDS gene were found. At age eight years, FMF was diagnosed after recurrent episodes of abdominal pain, fever, arthritis and raised inflammatory markers. Compliance with colchicine was poor, and she died of renal amyloidosis at 13 years. Patient 3 was the maternal uncle of patient 2 and had deafness and FMF since childhood.

Results: Sequencing of MEFV exon 10 PCR products from cases showed a c.2076_2078delATA mutation which results in the deletion of an isoleucine residue at position 692 (p.I692del). Affected patients also had the c.442G>C (p.E148Q) mutation in cis. Further sequencing revealed that both were compound heterozygotes carrying the c.2177T>C (p.V726A) mutation and the complex p.I692del/p.E148Q allele. Sequencing of exon 3 of the CIAS1 gene revealed that Patient 1 carried the p.Q703K missense nucleotide change and Patient 2 the reduced penetrance p.V198M mutation in this gene. Patient 3 refused CIAS1 sequencing.

Conclusion: In summary, we present the first report of patients with concurrent mutations of the MEFV and CIAS1 genes. These patients display a phenotype of FMF with the associated feature of progressive sensorineural deafness as seen in cold induced autoinflammatory syndromes. We postulate that the finding of deafness is due to synergistic heterozygosity between individually non-disease causing mutations in the CIAS1 gene and disease causing mutations in the MEFV gene.

027 INCREASED VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) LEVELS REFLECT DISEASE ACTIVITY IN SPONDYLOARTHRITIS

B. Vandooren1,2, E. Kruithof2, L. De Rycke1, E. M. Veys2, P. P. Tak1, D. Baeten1.1Division of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands; 2Department of Rheumatology, Ghent University Hospital, Belgium

Objectives: We previously demonstrated that soluble, synovium-derived mediators such as MMP3 and MRP8/14 are valuable and sensitive biomarkers for peripheral arthritis and global inflammation, respectively, in spondyloarthritis (SpA). Since marked hypervascularity is one of the major features of SpA synovitis in comparison with other types of arthritis such as rheumatoid arthritis (RA), the present study explored the value of VEGF and soluble endothelial adhesion molecules as potential biomarkers in SpA.

Materials and methods: Serum was collected from 60 SpA with peripheral synovitis, 14 SpA with exclusive axial disease, 30 RA, and 20 healthy controls. All patients had active disease. In the SpA cohort with peripheral synovitis, paired serum and synovial fluid (SF) was available in 17 patients and serum before and after 12 weeks of TNFalpha blockade in 34 patients (infliximab: n = 14; etanercept: n = 20). Levels of soluble E-selectin, ICAM-1, VCAM-1, and VEGF were determined by ELISA and were correlated with disease activity parameters.

Results: SpA with peripheral arthritis depicted higher serum levels of VEGF (p = 0.037) and E-selectin (p = 0.028) than healthy controls, with even higher levels of E-selectin in RA (p = 0.030). ICAM-1 was only elevated in RA compared to healthy controls (p<0.001) and VCAM-1 levels were not elevated at all in arthritis. Within the SpA cohort with peripheral arthritis, none of the 4 soluble factors were different between ankylosing spondylitis (n = 17), psoriatic arthritis (n = 24), and undifferentiated SpA (n = 19). The soluble factors do not originate exclusively from the peripheral joint as indicated by the similar serum levels in SpA with peripheral arthritis and exclusive axial disease, and the similar (VCAM-1) or lower (E-selectin and ICAM-1; p<0.001 for both) levels in SF than in serum. Only for VEGF, the levels tended to be 2× higher in SF than in serum (p = 0.105) with a good correlation between these levels (r = 0.826; p<0.01). Of the 4 soluble factors, only VEGF correlated significantly and consistently with disease activity in SpA as assessed by CRP (r = 0.509), ESR (r = 0.644), swollen joint count (r = 0.421), and patient’s (r = 0.463) and physician’s (r = 0.454) global assessment of disease activity. Finally, serum VEGF but not the other factors decreased significantly towards normal levels upon treatment with infliximab (p = 0.011) and etanercept (p = 0.001).

Conclusions: The facts that serum VEGF levels are increased, correlate with disease activity, and are normalized by effective treatment in SpA, identify serum VEGF as a potential biomarker in SpA. Ongoing studies are assessing the value of serum VEGF levels for the prediction and monitoring of treatment response. BV and EK contributed equally.

028 EFFECTS OF INTRAVENOUS ILOPROST ON TGF B1, IL2, IL10 IN SYSTEMIC SCLEROSIS AND PERIPHERAL ARTERIAL DISEASE

A. Mazzone, P. Faggioli, L. Giani, R. Pacifici1, S. Pichini1, M. Rondena, P. G. Zuccaro1.AO Ospedale Civile di Legnano, Legnano, Milano, Italy; 1Istituto Superiore di Sanità Roma

Aim: Transforming Growth Factor-Beta1 (TGFb1) has been shown to be a potent stimulator of collagen production by fibroblasts, and could play a role in the pathogenesis of Systemic Sclerosis. TGFB1 is also involved in atherosclerotic plaque formation with fibroblast proliferation and synthesis of collagen. The current study was undertaken to ascertain if iloprost treatment modifies the cytokine network, TGFb1, Interleukin-2 (IL2) as expression of Th1 lymphocytes and Interleukin-10 (IL10) as anti-inflammatory cytokine expression of Th2 lymphocytes.

Materials and methods: Forty patients (40) with Systemic Sclerosis (dSS), and 25 with Peripheral Artery vascular Disease (PAD) IIB/III stage of Fontaine’s classification were enrolled. Iloprost was administered for 5 days in dSS patients and for 21 days in Pad patients. Plasma levels of TGFb1, IL2, IL10 were detected at baseline and after 5 days in both groups. For quantitative measurement of human interleukin-2 (IL-2), interleukin-10 (IL-10), and transforming growth factor-beta 1 (TGF-b1) in plasma, specific solid phase enzyme-linked immunosorbent assay (ELISA) employing the multiple antibody sandwich principle, were used (Biosource International, Camarillo, CA, USA). Mean intra- and inter-assay coefficients of variation were always less than 5%. Statistical analysis was performed with SSPS program. Non-parametric comparison of SSc vs PAD has been performed with Anova. Baseline vs post therapy results have been analysed with Wilcoxon paired test.

Results: All patients were included in the analysis. The basal level of TGF b1, IL2 and IL10 were assayed before to start iloprost therapy. After infusion of iloprost a significant decrease of TGFb1, (TGFb1 841.013±82 pg/ml vs 527.625±61 p<0.001) and a significant variation of IL2 ( 46.708±3.5 pg/ml vs 63.737±5.2 pg/ml p<0.001) and IL10 (2908.67±135.2 vs 2292.72±116 pg/ml) were observed in all patients.

Conclusions: The study provides further evidence that iloprost reduces TGFb1 secretion from Th1 e consequently collagen production from fibroblasts. The modulation by TGFb1 of inflammatory stimuli can promote the decrease of IL10 and down regulation of IL2, stressing a key role of iloprost in stabilizing dSS and PAD.

029 PHOSPHODIESTERASE 4 (PDE4) INHIBITORS ARE POTENT MODULATORS OF SPONTANEOUS PRO-INFLAMMATORY CYTOKINE AND CHEMOKINE RELEASE FROM SYNOVIOCYTE CULTURES AND MACROPHAGES ACTIVATED VIA T CELL CONTACT

A. Crilly, S. Robertson, J. Reilly, J. A. Gracie, P. F. Life1, I. B. McInnes.Division of Immunology, Infection and Inflammation, Biomedical Research Centre, Glasgow University, Glasgow, UK; 1GlaxoSmithKline Medical Research Centre, Stevenage, UK

Background: Phosphodiesterase 4 (PDE4) is a cellular regulator of cAMP which potentiates the inflammatory response of immune cell types, including T cells and macrophages. TNF alpha production is particularly sensitive to PDE4 inhibition, both in vitro and in vivo, and it is principally on this basis that PDE4 inhibitors have been suggested to have potential utility in the treatment of rheumatoid arthritis (RA). Importantly, LPS has almost exclusively been used as the stimulus to study TNF modulation by PDE4 inhibitors and data generated from more disease-relevant systems is lacking. Using an RA synoviocyte culture system and an in vitro live cell contact model of synovitis, we have looked at the ability of two novel PDE4 inhibitors to regulate pro-inflammatory cytokine and chemokine release.

Methods and materials: RA synovial membranes were obtained from patients undergoing joint arthroplasty. Inflamed synovium was removed and digested with liberase 3 to give a single cell suspension of synoviocytes. Cells were washed and the concentration adjusted to one million cells per ml. The synoviocytes were then cultured in 96 well tissue culture plates in the presence or absence of PDE4 inhibitors [concentration range:300–0.03 nM]. Culture supernatants were collected after 24 hours and assayed for pro-inflammatory cytokines and chemokines by ELISA and luminex. For cell contact experiments, CD14+ and CD3+ cells were isolated from buffy coats. CD14+ cells were plated into 96 well tissue culture plates while CD3+ cells were stimulated for 6 days in the presence of TNF alpha (25 ng/ml), IL-2 (25 ng/ml) and IL-6 (100 ng/ml) to generate cytokine activated T cells (TcK). Activated TcK were then added back to the autologous CD14+ cells in the presence or absence of PDE4 inhibitors. Cells were cultured for 24 hours after which time supernatants were harvested and assayed as above.

Results: PDE4 inhibitors significantly reduced TNF alpha release from RA synoviocyte cultures (n = 4). Both drugs were comparable in their ability to reduce TNF alpha secretion with an IC50 of between 3 and 0.03 nM, p<0.05, depending on the tissue studied. Secretion of MIP-1 alpha; MIP-1 beta; MCP1 and RANTES from these cultures was consistently reduced along with IL-12p40-70 and GM-CSF. Using an in vitro live cell contact assay (n = 5) similar regulation of TNF alpha and chemokine release was observed. In this system the T cell derived cytokines IFN gamma and IL-17 were also reduced by PDE4 inhibitors.

Conclusions: These data uniquely demonstrate PDE4 inhibitors to be robust and potent inhibitors of cytokines and chemokines in cellular assay systems of relevance to RA immunopathology. PDE4 inhibition offers a potential therapeutic option for RA.

030 PERSISTING INFLAMMATION IN MUSCLE TISSUE AFTER INFLIXIMAB TREATMENT IN PATIENTS WITH REFRACTORY INFLAMMATORY MYOPATHIES

C. Grundtman, M. Dastmalchi, H. Alexanderson, H. Einarsdottir, S. Barbasso Helmers, K. Elvin, I. Nennesmo, I. E. Lundberg.Dept of Medicine, Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden

Objective: To investigate the efficacy of infliximab combined with methotrexate or azathioprine in patients with treatment resistant inflammatory myopathies. A role for tumour necrosis factor (TNF) in the pathogenesis of myositis has been suggested by presence of TNF both on protein and mRNA level as well as increased expression of TNF-receptors in muscle tissue of myositis patients.

Methods: Thirteen patients with refractory polymyositis, dermatomyositis, or inclusion body myositis were treated with four infliximab infusions (5 mg/kg body weight) during a period of 14 weeks. Outcome measures included disease activity score, according to The International Myositis Assessment and Clinical Studies Group (IMACS), functional index of myositis, magnetic resonance imaging (MRI), and repeated muscles biopsies. The muscle biopsies were investigated by immunohistochemistry and evaluated with both conventional microscope and digital image analyzing system for cellular markers (CD68, CD163, Ki67, CD3, CD4, and CD8), pro-inflammatory cytokines, (TNF, IL-1alpha, IL-1beta, IL-6, HMGB-1, IFN-gamma, MxA) MHC class I and MHC class II antigen in muscle fibres, and endothelial cell markers (MAC, CD31).

Results: Nine patients completed the study whereas four patients discontinued, three due to adverse events and one due to a discovered malignancy. Three of the completers improved by at least 20% in three or more variables of the disease activity core set, four patients were unchanged and two worsened by more than 20%. No patients improved in muscle strength. In three of the completers, increased muscle inflammation was recorded on MRI scans. Signs of inflammation were detected in the muscle biopsy taken before infliximab treatment in all patients and were still evident in all biopsies taken after treatment. There were no changes in the number of macrophages, T lymphocytes, expression of MHC class I or II in muscle fibres, in expression of pro-inflammatory cytokines or MAC expression in capillaries.

Conclusions: The persisting inflammation in muscle tissue after infliximab treatment for 4 months together with absence of improved muscle strength makes it unlikely that TNF has a major role in the disease mechanisms in refractory myositis. Furthermore, as some cases worsened clinically and increased signals were seen on MRI scans we advocate caution with the use of infliximab in refractory inflammatory myopathies.

031 IS CHEMERIN INVOLVED IN THE PREFERENTIAL ACCUMULATION OF PLASMACYTOID DENDRITIC CELLS IN RHEUMATOID ARTHRITIS SYNOVIUM?

M. C. Lebre1, B. A. Zabel2, S. Aarrass1, E. C. Butcher2, P. P. Tak1.1Div. Clinical Immunology/Rheumatology, AMC/University of Amsterdam, Amsterdam, The Netherlands; 2Lab. of Immunology and Vascular Biology, Dept. Pathology, Stanford University School of Medicine, CA, USA

Background and aim: Rheumatoid arthritis (RA) synovium is characterized by a dense infiltrate, consisting of macrophages (Mø), T and B cells, plasma cells, dendritic cells (DC), and other cells. Inflammatory chemokines present in RA synovium may contribute to the accumulation of these immune cells. Chemerin is a potent chemotactic agent that binds to chemokine-like receptor 1 (CMKLR1). CMKLR1 is specifically expressed by tissue Mø, circulating plasmacytoid (p)DC and immature monocyte-derived DC, while chemerin is expressed by endothelial cells. We have recently shown that RA synovial tissue (ST) is enriched with pDC. As pDC do not respond to inflammatory chemokines we investigated whether the chemerin/CMKLR1 system might be involved in the recruitment of pDC to the inflamed joint.

Methods: Chemerin and CMKLR1 expression in ST (RA, psoriatic arthritis [PsA], and osteoarthitis [OA] patients) was detected by immunohistochemistry and quantified by digital image analysis. Moreover, in RA ST double immunohistochemistry analysis was performed. CMKLR1 expression by circulating pDC was assessed by FACS. Chemerin activity in synovial fluids (SFs) was measured by investigating the ability of CMKLR1-transfectants chemotaxis and chemerin expression by quantitative western blot analysis. Chemerin’s C-terminal processing was identified in RA and PsA SFs using heparin chromatography, PAGE separation, MALDI-TOF and tandem MS/MS analysis.

Results: Chemerin and CMKLR1 expression in RA was significantly higher compared with PsA+OA STs. Chemerin expression was confined to blood vessels whereas CMKLR1 expression was observed in dispersed cells throughout the synovial sublining. Double immunostainings revealed that CD31+ and von Willebrand factor+ endothelial cells expressed chemerin. In addition, only CD68+ and a few CD163+ Mø, BDCA4/CD304+ pDC, MRP8+ and MRP14+ cells expressed CMKLR1 while BDCA1/CD1c+ myeloid DC, CD3+ T cells, CD19+ B cells and CD15+ neutrophils were CMKLR1 negative. Circulating blood pDC in RA showed increased expression of CMKLR1 compared with healthy controls and non-RA patients. In all diagnostic groups studied, SF activity and expression of chemerin was observed. Identification of the C-terminal of chemerin in RA demonstrated that its sequence is the same as the form identified in serum (active form).

Conclusion: The in vivo distribution of chemerin in RA ST strongly suggests that chemerin might be involved in the migration/accumulation of pDC and Mø in the inflamed joint. Importantly, since pDC do not respond to inflammatory chemokines, chemerin is likely the key chemoattractant responsible for the migration of pDC into the synovium. Blocking the novel chemerin/CMKLR1 system represents an attractive candidate for future drug development as it could disrupt disease perpetuation.

032 PROTECTION AGAINST COLLAGEN-INDUCED ARTHRITIS WITH DIFFERENTIALLY MODULATED DENDRITIC CELLS

L. van Duivenvoorde, W. Han, A. Bakker, P. Louis-Plence, L-MCharbonnier, F. Apparailly, E. van der Voort, C. Jorgenson, T. Huizinga, R. Toes.Department of Rheumatology, Leiden University Medical Center, The Netherlands; INSERM U475, Montpellier, France

Introduction: Dendritic cells (DC) are crucial for the initiation of T cell immunity and therefore play an important role in the initiation and regulation of immune responses in arthritis. Full mobilization of effector T cells depends on the proper maturation of DC. Current evidence indicates that the type of T cell response induced is crucially dependent on the activation status of the DC. In this study, we explored the immunologic effects of differentially matured DC on the development of collagen-induced arthritis (CIA), a well-defined model for RA.

Methods: CIA is induced after injection of bovine collagen type II (CII) in CFA. It is a typical B cell-mediated autoimmune disease, as CII-specific antibodies are sufficient and required for disease induction. Especially IgG2a antibodies are thought to be important, as IgG2a is able to efficiently recruit effector-mechanisms. Here, we investigated the possibility to protect mice against arthritis in an Ag-specific manner employing differentially modulated dendritic cells (DC).

Results: DC modulated with TNF, IL-10 or dexamethasone, but not LPS activated DC, were able to decrease disease severity and incidence. Even in a therapeutic setting, when measurable autoantibodies (against murine CII) are present in serum, IL-10 modulated DC are capable of suppressing CIA. Moreover, protection against disease correlates with lower anti-mCII IgG2a/IgG1 ratios compared to control mice. However, differentially modulated DC installed different modes of protection, since T cell responses induced by these differentially modulated DC were different. TNF and IL-10 stimulated DC skews T cells towards the Th2 cell subset, whereas dexamethasone modulated DC induce the production of IL-10 production without the concomitant production of IL-5.

Conclusion: These data indicate that targeting DC represent an effective way to modulate arthritis and indicate that different types of DC, although leading to similar clinical effects, mediate protection via different mechanisms.

033 TLR3, TLR7 AND TYPE I INTERFERONS ARE INVOLVED IN CHRONIC INFLAMMATION DURING RHEUMATOID ARTHRITIS

M. F. Roelofs, F. Brentano, L. A. B. Joosten, S. Abdollahi-Roodsaz, M. H. Wenink, B. Oppers-Walgreen, M. C. de WaalMalefijt, P. L. C. M. van Riel, D. Kyburz, W. B. van den Berg.Dept. of Rheumatology Research & Advanced Therapeutics, Radboud University Nijmegen Medical Centre, the Netherlands/Centre of Experimental Rheumatology, University Hospital, Zurich, Switzerland

Toll-like receptors (TLR) are essential in the initial recognition of invading microorganisms. TLR3 and TLR7, which serve as receptors for viral nucleic acids, are able to induce production of type I interferons (IFNa/b), which is essential for a successful antiviral immune response. In terms of autoimmunity, it is interesting that endogenous molecules, such as RNA released from necrotic synovial fluid cells, could also activate TLR3. Moreover, it has been shown that both TLR3 and TLR7 are abundantly expressed in RA synovial tissue, suggesting a role for these receptors in the pathogenesis of RA. Here we investigated whether the synovial expression of TLR3 and TLR7 was associated with the expression of IFNa, TNFa, IL-1b, IL-12, IL-17 and IL-18. In addition we studied in what way these receptors and cytokines were associated to each other in vitro. Using immunohistochemistry on synovial tissue specimens from 34 RA patients with active disease, we found that TLR3 and TLR7 expression in synovial tissue was associated with the presence of IFNa, IL-1b and IL-18, but not with TNFa, IL-12 and IL-17. In order to investigate whether IFNa, IL-1b and IL-18 were able to induce upregulation of TLR3 and TLR7 in vitro, we incubated monocytes, monocyte-derived dendritic cells (DC) and RA synovial fibroblasts (RASF) with these cytokines and subsequently determined TLR3 and TLR7 expression using real-time PCR. Whereas both IL-1b and IL-18 were not able to regulate the TLR expression, incubation with IFNa did result in significant upregulation of TLR3/7 mRNA levels. To check whether this upregulation was functional, we pre-incubated monocytes, DC and RASF with IFNa and subsequently stimulated TLR3 or TLR7 using poly(IC) or loxoribine respectively. This resulted in significantly enhanced levels of IL-6, TNFa and type I IFN, indicating that the IFN-induced TLR upregulation was functional in terms of cytokine production. Although high amounts of IL-1b mRNA were produced upon TLR3/7 stimulation, no biologically active IL-1b was released, probably due to a lack of Caspase-I activity. Addition of the Caspase-I activator ATP only resulted in low amounts of biologically active IL-1b. Interestingly, IFNa- primed cells produced significantly higher levels of active IL-1b, which suggests a role for IFNa in synovial IL-1b production. Altogether, we showed that the expression of TLR3/7 was associated with IL-1b, IL-18 and IFNa in RA synovial tissue. Furthermore, we demonstrated a dual role for IFN type I in vitro, which could explain the association between TLR and IL-1b/IL-18 in synovial tissue. First, involvement in TLR3/7 regulation and second, involvement in the production of biologically active IL-1b by ATP. These results suggest the involvement of antiviral immune responses in RA and type I interferons as a key player in chronic inflammation.

034 RHEUMATOID ARTHRITIS SYNOVIAL T CELLS ARE RESISTANT TO CORTICOSTEROID INDUCED APOPTOSIS

D. Makrygiannakis, C. Grundtman, E. af Klint, O. Snir, L. Klareskog, A. Irinel Catrina.Department of Rheumatology, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden

Background: Rheumatoid arthritis (RA) is characterized by synovial chronic inflammation with local accumulation of both macrophages and lymphocytes. Synovial RA lymphocytes have a mature memory phenotype suggesting T cell activation, but are anergic and resistant to apoptosis. We previously demonstrated that intraarticular corticosteroids decrease synovial T cell infiltrates and we aimed to investigate if this is mediated through modulation of apoptosis.

Methods: Active caspase-3, TUNEL, CD3, CD68 and CD163 were evaluated by immunohistochemistry in serial sections of synovial biopsies obtained from 12 RA patients before and after 2 weeks of an intraarticular knee injection with triamcinolone hexacetonide. Biopsies were evaluated by double blind semi-quantitative analysis and image analysis. We characterized synovial apoptotic cells by dual immunofluorescence detection with TUNEL and surface markers (CD3 and CD163). We then tested the in vitro effect on apoptosis of 3 different corticosteroid compounds (triamcinolone hexacetonide, dexamethasone and methylprednisolone acetate). After 24 h incubation non-isolated and isolated RA synovial fluid (SF) T cells were evaluated for annexin V expression by flow cytometry. We also studied the effect of corticosteroids (dexamethasone) on phytohemaglutinin (PHA) stimulated non-isolated RA SF T cells. Statistical analysis of the biopsy data was performed using the Wilcoxon test followed by Bonferroni correction. In vitro data was analyzed by one-way analysis of variance with Dunett post-hoc analysis.

Results: Intraarticular corticosteroids reduced the T cell but not the macrophage synovial infiltrate. Apoptosis levels, as expressed both by active caspase-3 and TUNEL, were generally low and unchanged following 2 weeks of treatment. Apoptosis was virtually absent from lymphoid aggregates both before and after corticosteroid therapy. This was confirmed by dual immunofluorescence, showing that less than 2% of the CD3 positive cells were also TUNEL positive, both before and after IA corticosteroid therapy. In vitro non-isolated RA SF T cells were resistant to corticosteroid induced apoptosis, independent of the compound used. In contrast all three compounds were able to increase the apoptosis rate of isolated RA SF T cells, to different extents. Interestingly no change in the apoptosis rate of PHA stimulated non-isolated RA SF T cells was observed following 48 hours incubation with dexamethasone.

Conclusion: Our findings demonstrate that RA synovial T cells are resistant to corticosteroid induced apoptosis probably due to the potent anti-apoptotic synovial environment. Specific targeting of the T cells or T cell survival signals might be beneficial for rheumatoid inflammation.

035 SMOKING UPREGULATES PEPTIDYLARGININE 2 ENZYME EXPRESSION AND CITRULLINATION

D. Makrygiannakis1, M. Hermansson1, A-KUlfgren1, A. P. Nicholas2, A. J. Zendman3, A. Eklund4, J. Grunewald4, M. Skold4, L. Klareskog1, A. Irinel Catrina1.1Department of Rheumatology; 4Department of Respiratory Medicine, Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden; 2University of Alabama at Birmingham and Birmingham Veterans Administration Medical Center, Birmingham, Alabama; 3Department of Biochemistry, Radboud University Nijmegen, Nijmegen, The Netherlands

Background: We have previously reported the increased risk of developing anti- citrulline positive RA in smoking individuals genetically characterized by the presence of shared epitope. We suggested that citrullination induction by smoking might be the first step in the pathogenic chain of RA, by identification of citrullinated proteins in bronchoalveolar fluid (BAL) from smokers and their absence in the non-smokers. We wanted to confirm our previous study and to extend it by investigating the expression of peptidylarginine (PAD) enzymes and citrullinated proteins in BAL cells and bronchial mucosal biopsies from healthy individuals either smokers or non-smokers.

Methods: We obtained BAL cells and bronchial mucosal biopsies through bronchoscopy from 14 healthy smokers and 16 healthy non-smokers. We used 2 antibodies recognizing citrullinated proteins, a rabbit polyclonal antibody against modified citrulline (AMC) and a mouse monoclonal antibody (F95). We used 2 rabbit polyclonal antibodies recognizing the PAD2 enzyme (Cosmobio and PAD2SN665) and a rabbit polyclonal antibody recognizing the PAD4 enzyme (PAD4SN823). Immunohistochemistry was used to detect expression of the various proteins. Appropriate controls were used for every staining. BAL samples and biopsy specimens were evaluated by double blind semi-quantitative analysis. We also performed blind quantitative analysis of BAL samples. Statistical analysis was performed using the Mann-Whitney test for histological scores.

Results: The majority of BAL cells were macrophages with a median of 94% macrophages in the smoker samples and 86% macrophages in the non-smoker samples. Analysis of BAL samples showed that 56% (5/9) smokers and 7% (1/15) non-smokers were AMC positive while 17% of the smokers (2/12) and none out of 7 non-smokers were F95 positive. The pattern of expression for citrullinated proteins was predominantly cytoplasmatic.

All samples were positive for PAD2 and PAD4. We observed significant up-regulation of the PAD2 enzyme with both PAD2 antibodies (median positive cells in smokers 86% and 64% and in non-smokers 63% and 43%, for Cosmobio and PAD2SN665, respectively) but not of the PAD4 enzyme (median positive cells in smokers 75% and in non-smokers 64%). Similar to citrullinated proteins PAD2 and PAD4 expression was predominantly cytoplasmatic. Analysis of bronchial mucosal biopsies showed that 100% (10/10) smokers and 85% (12/14) non-smokers were F95 positive. Citrullinated proteins were detected mainly intracellular but also extracellular in the epithelial and the lamina propria areas. All biopsies were positive for PAD2 and PAD4. We observed up-regulation of the expression of PAD2 but the difference reached significance only for one of the two antibodies used. PAD enzyme expression had a similar distribution with the expression of citrullinated proteins with a mainly intracellular (cytoplasmic for PAD2, cytoplasmic and nuclear for PAD4) but also extracellular pattern.

Conclusion: This study confirms our previous results and provides evidence that smoking enhances PAD2 enzyme expression in the lung mucosal and alveolar (BAL cells) compartment with consequent generation of citrullinated proteins in the latter. Taken together, these findings suggest smoking as one of the environmental factors leading to autoimmunity in genetically susceptible individuals, resulting in RA development.

036 IMATINIB MESYLATE (GLEEVEC) PREVENTS EFFICIENTLY THE DEVELOPMENT OF SKIN FIBROSIS

J. H. W. Distler1,2, A. Jüngel2, L. C. Huber2, B. A. Michel2, G. Schett1, S. Gay2, O. Distler2.1Department of Internal Medicine III, University of Erlangen-Nuremberg, Germany; 2Center of Experimental Rheumatology and Human Integrative Physiology, University Hospital Zurich, Switzerland

Aim: Imatinib mesylate is a selective small molecule tyrosine kinase inhibitor that binds to the ATP-binding pocket and blocks selectively the tyrosine kinase activities of Abl, c-kit, and PDGFR. Imatinib is a well tolerated, orally administered drug that is widely used for treatment of bcr-abl positive chronic myelogenous leukemia and gastrointestinal stromal tumors.

Methods: SSc and normal dermal fibroblasts were incubated with imatinib at concentrations from 0.01 to 1 μg/ml for time periods from 12 hours to 7 days. The highest concentration used in our experiments was within the mean plasma level of 0.72 ug/ml achieved after standard doses of imatinib. The expression of col 1a1, col 1a2 and fibronectin 1 was analyzed by Real-time PCR. Results were confirmed on the protein level by Western blot and with the SirCol collagen assay. Proliferation capacity was assessed with the MTT assay. Cell viability was analyzed by determination of the mitochondrial membrane potential using JC-1 and by annexin V/ propidium iodide staining. Imatinib mesylate was also tested in concentrations of 50 μg/mg/d and 150 μg/mg/d i.p. in the model of bleomycin induced skin fibrosis. After 4 weeks, mice were sacrificed, the dermal thickness was determined, the mRNA levels of col 1a1, col 1a2 and fibronectin 1 were analyzed and immunohistochemistry for a-smooth muscle actin was performed.

Results: In SSc dermal fibroblasts stimulated with TGFb, imatinib reduced strongly the synthesis of mRNA for col 1a1, col 1a2 and fibronectin-1. This effect was dose-dependent with a reduction as high as by 88±4% for col 1a1, 92±1% for col 1a2 and 92±2% for fibronectin 1 at 1 μg/ml imatinib. Similar results of imatinib were observed in normal dermal fibroblasts stimulated with TGFb and in dermal fibroblasts stimulated with PDGF. The basal synthesis of col 1a1, col 1a2 and fibronectin 1 in unstimulated cells was inhibited to a lesser extent (col 1a1: 66±15%; col 1a2: 67±14%; fibronectin 1: 66±10%). The effects of imatinib under basal conditions were more pronounced in SSc fibroblasts than in healthy controls. Similar results were obtained on the protein level. Imatinib did not affect the proliferation capacity of dermal fibroblasts. In addition, the number of apoptotic or necrotic cells was not increased by imatinib even after long-term incubation for one week indicating that in the concentrations used imatinib had no toxic effects on dermal fibroblasts. Treatment with imatinib prevented also the development of dermal fibrosis in bleomycin treated mice in a dose-dependent manner. The dermal thickness decreased from 168±14 % in mice treated with bleomycin only to 119±14% and 105 %±6% in animals treated additionally with imatinib at doses of 50 μg/mg/d and 150 μg/mg/d, respectively. Furthermore, the induction of mRNA for col1a1, col1a2 and fibronectin was significantly reduced in mice treated with imatinib. Consistent with these findings, the number of myofibroblasts decreased from 28±5 in bleomycin treated animals to 15±2 myofibroblasts were detected and 9±2 myofibroblasts in mice treated with 50 μg/mg/d and 150 μg/mg/d imatinib.

Conclusion: These data indicate that imatinib mesylate can reduce efficiently the synthesis of extracellular matrix proteins without toxic effects on fibroblasts. Treatment with imatinib prevented completely the development of dermal fibrosis in physiological doses. Because of its potent anti-fibrotic effects, its favourable pharmacokinetics and its good tolerance, imatinib might be a promising new drug for the treatment of SSc.

037 QUIPP: A NOVEL TYROSINE PHOSPHOPROTEIN IMPLICATED IN TOLL LIKE RECEPTOR SIGNALLING

M. J. Peirce, J. Testar, M. Brook, S. Begum, R. Wait, T. Hussell, A. P. Cope.The Kennedy Institute of Rheumatology, Imperial College London, UK

Background: Engagement on macrophages of members of the Toll-like receptor (TLR) family by pathogen associated molecular patterns (PAMPs, eg LPS), or endogenous associated proteins (eg Hsp70), provide pivotal cues which define the character of a developing immune response to infection or trauma. While many TLR signalling components are now known (eg, MyD88, TRAF6, p38 MAP kinase, NFkB), how TLR specific responses may be generated using a shared panel of signalling intermediates is poorly understood. We hypothesised the existence of hitherto uncharacterised signalling proteins which might contribute to the diversity of responses following engagement of different TLRs. Thus, using a phosphotyrosine-targeted proteomic approach we identified a novel protein, currently denoted QUIPP, from a plasma membrane-enriched fraction of RAW 264.7 murine macrophages. We set out to characterise the role of QUIPP, a novel protein implicated in TLR signalling.

Materials and methods: Plasmids encoding FLAG-tagged QUIPP or a control protein (bacterial alkaline phosphatase, BAP) were nucleofected in to cells of the murine macrophage cell line RAW 264.7 and stable transfectants selected in G418-containing medium. Tagged proteins were immunoprecipitated with anti-FLAG sepharose beads and associated proteins detected by Western blot. Cytokine production induced by TLR ligands was measured by ELISA and COX2 expression by western blot. QUIPP mRNA expression in a variety of murine tissues was measured by QT-PCR. QUIPP expression in primary splenocytes from Balb/c mice injected (IP) with zymosan or infected with influenza were measured by western blot.

Results: QUIPP mRNA was selectively expressed in cells of the haematopoietic lineage, predominantly monocyte/macrophages and B but not T lymphocytes. QUIPP was inducibly tyrosine phosphorylated in RAW cells following LPS stimulation and this was associated with transient interaction with the Src family tyrosine kinase, Lyn. RAW cells stably over expressing QUIPP produced enhanced levels (2-4-fold increase) of TNF alpha in response to the TLR4 ligand LPS but not to PAM3-cys or poly I:C, ligands for TLR2 and TLR3, respectively. In vivo zymosan injection led to a profound and long lasting (at least 12 weeks) down regulation of QUIPP expression in the adherent (monocyte/macrophage) splenocyte population while expression in B cells, purified from the same spleens, was unaffected. In whole splenocytes from influenza infected mice QUIPP expression was profoundly but transiently down regulated at time points (2–4 days post infection) that correlated with both peak weight loss and cellular infiltration of the flu-infected lungs.

Conclusion: QUIPP is a novel tyrosine phosphoprotein that may be an important regulator of macrophage responses to some but not all TLR ligands in vitro. Expression of QUIPP is subject to regulation in vivo in response to inflammatory and infectious insults.

038 TCR-ZETA DIM T CELLS DEFINE A POPULATION OF CIRCULATING EFFECTOR CELLS THAT MIGRATE TO INFLAMED TISSUES

C. L. Gorman, Z. Zhang, A-CVermi, C. Monaco, F. Marelli-Berg, F. Dazzi, A. P. Cope.The Kennedy Institute of Rheumatology, Imperial College London, UK

Background: The T cell receptor zeta chain is a master sensor and regulator of lymphocyte responses. Loss of TCR-zeta expression has been documented in infectious, inflammatory and malignant diseases, suggesting that it may serve to limit T cell reactivity and effector responses at sites of tissue damage. Previous experiments from our laboratory have suggested that TCR-zeta dim T cells define a population of effector T cells. These observations prompted us to explore in more detail the relationship between TCR-zeta dim expression and key effector functions in T cells.

Materials and methods: We analysed by flow cytometry the expression of TCR-zeta in peripheral blood (PB), synovial fluid (SF) and tissue (ST) from patients with active rheumatoid arthritis. Similar analysis was undertaken using PBMC from patients before and fourteen weeks after anti-TNF (infliximab) therapy. Clinical responses were defined according to EULAR response criteria. Unfractionated PBMC were subjected to transendothelial migration assays using TNF stimulated human umbilical vein endothelial cell monolayers overlaid onto gelatin in Transwell inserts. Migration of specific cell subsets in the presence or absence of exogenous chemokines was determined after 24 hr by flow cytometric analysis of cells in the lower chamber, relative to those non-migrating cells remaining in the upper chamber.

Results: T cells expressing low levels of the TCR-zeta chain are enriched at sites of inflammation such as the rheumatoid synovial joint, and during acute flares of disease are greatly increased when compared to PB from the same patient. Treatment with anti-TNF leads to accumulation of TCR-zeta dim T cells in PB at 14 weeks to an extent that predicts the clinical response at 30 weeks. In vitro, TCR-zeta dim T cells have a greatly increased capacity to migrate when compared to their TCR-zeta bright counterparts, an effect more striking for the CD4+TCR-zeta dim subset. Unlike whole PBMC, TCR-zeta dim T cells are relatively refractory to exogenous stimulation with CXCL-10, CCL5 and CXCL-12. Crucially, we found that those TCR-zeta dim T cells accumulating in PB of RA patients after anti-TNF therapy in vivo retain the capacity for robust migration across activated endothelium in vitro.

Conclusion: Exclusion of TCR-zeta dim effector T cells from inflamed tissues appears to be associated with good clinical responses to anti-TNF. Persistence of these effector cells, in spite of therapy, may go some way to explain disease flares following withdrawal of anti-TNF therapy. The data suggest targeting TCR-zeta dim effector cells by means of “second-hit” therapeutic regimens may be required to achieve sustained periods of biologic free remission. At the molecular level, the results suggest that the TCR-zeta chain may play a crucial role in transducing motility regulating signals.

039 MYELOID RELATED PROTEINS MRP-8/MRP-14 REGULATE CHRONICITY OF JOINT INFLAMMATION AND CARTILAGE DESTRUCTION DURING ANTIGEN-INDUCED ARTHRITIS

P. L. E. M. van Lent1, A. B. Blom1, A. Sloetjes1, T. Vogl2, W. Nacken2, J. Roth2, W. B. van den Berg1.1Dep of Rheumatology, Radboud University Medical Centre; 2Dep of Dermatology, UKM, Muenster, Germany

Purpose: In earlier studies we showed that activated macrophages are crucial in regulating joint inflammation and cartilage destruction during experimental arthritis (AIA). The most prominent proteins released by activated macrophages are the “calgranulins”, myeloid related proteins MRP-8[S100A8]) and MRP-14[S100A9]. Both proteins which form a heterodimer accumulate in inflammatory fluids during RA and are postulated to be involved in the pathogenesis of RA. This prompted us to investigate the role of MRP-8/-14 in joint inflammation and cartilage destruction during antigen-induced arthritis.

Methods: MRP-14 deficient mice (myeloid cells also deficient for MRP-8) were used. AIA was induced by immunizing mice with mBSA followed by injection of the antigen into the right knee joint. Joint inflammation and cartilage destruction was measured by 99mTc uptake and histology. MMP-mediated cartilage destruction was measured using immunolocalisation of the neoepitope VDIPEN. mRNA levels of several matrix metalloproteinases (MMP) were measured in synovial specimen using Q-PCR. In vitro, peritoneal macrophages were stimulated by recombinant MRP-8 or MRP-8/-14 heterodimers.

Results: Three weeks after immunization with the antigen mBSA, cellular (T cell responses as measured by lymphocyte proliferation) and humoral (total IgG, IgG1,2a and 2b anti-mBSA levels measured by ELISA) immunity were comparable in MRP-14-/- and their controls excluding deficient immunity as a disturbing factor in the knockout mice. Joint swelling, measured by 99mTc uptake, was significantly lower in MRP-14 knockout mice (70% when compared to WT controls) at day 7 (but not at day 3) after AIA induction. Cellular infiltrate and exudate were 63% and 80% lower. Cartilage destruction was additionally measured in various cartilage layers of the knee joint. Proteoglycan depletion was significantly lower (between 50–95%). MMP-mediated cartilage destruction was clearly present in arthritic controls (VDIPEN between 15–25%) but absent in cartilage layers of day 7 arthritic MRP-14-/-. In line with this, mRNA levels of various MMPs (MMP3, 9 and 13) were significantly lowered in arthritic synovia of MRP-14-/-. Interestingly, intra-articular injection of recombinant MRP-8 (5 μg) in normal mice strongly induced arthritis. mRNA levels of various MMP-3,-9 and -13 in the inflamed synovium became significantly elevated and PG depletion of cartilage layers was observed already at day 1 after injection. In vitro stimulation of macrophages by rMRP-8 or the heterdimer MRP-8/-14 elevated mRNA levels of MMP-3,-9 and particularly MMP-13 (between 10–17 times), suggesting that MRP-8 and MRP-8/-14 are involved in upregulation of MMPs.

Conclusions: MRP-8/-14 are important in chronicity of joint inflammation and cartilage destruction during antigen-induced arthritis, the latter probably by upregulation of MMPs.

040 SELECTIVE ELIMINATION OF SYNOVIAL MACROPHAGES BY BORON NEUTRON CAPTURE THERAPY PREVENTS ONSET OF MURINE EXPERIMENTAL ARTHRITIS

P. van Lent1, G. Krijger2, A. Sloetjes1, G. Koning2, S. Nievaart3, R. Moss3, W. van den Berg1.1Department of Rheumatology, St Radboud University Medical Centre Nijmegen, The Netherlands

Purpose: During RA large amounts of activated macrophages accumulate in the joint, driving inflammation and cartilage destruction. Selective removal of activated macrophages in the inflamed joint may be an effective therapeutic approach. In the present study, we investigated whether synovial macrophages can be selectively eliminated by 10boron (10B) encapsulated into liposomes combined with neutron irradiation (boron neutron capture therapy: BNCT) and to investigate its therapeutic effects in experimental arthritis.

Methods: Disodium dodecahydrododecaborate (Na2B12H12), having a 10B enrichment of 99.7%, was encapsulated into small (110–200 nm) unilamellar liposomes. B encapsulated liposomes were injected into the mouse knee joint. Boron concentrations were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES). Immune complex arthritis was induced in mice by injecting the antigen lysozyme into knee joints which previously were given antibodies directed against lysozyme. Swelling of the knee joint was measured using the 99mTc uptake method. Total knee joints were taken at various days after induction of arthritis and joint inflammation and cartilage destruction were determined using histology.

Results: When boron-containing liposomes are injected directly into the mouse knee joint, high concentrations of boron were measured in the synovial layer up to 24 hours after injection. No boron was detected in the ipsilateral paw indicating that boron does not leak out of the knee joint. Neutron irradiation, 24 hours after injection of 10 ul boron-liposomes into the knee joint, showed selective depletion of synovial lining macrophages which was maximal at day 5 after injection. This selectivity was in line with in vitro studies showing that macrophages but not fibroblasts died by apoptosis (85–95%) when boron-liposomes were combined with neutron irradiation. When experimental arthritis was induced in the macrophage depleted joint, swelling of the knee joint was significantly lower when compared to controls (53% and 79% lower at days 1 and 3 after arthritis induction, respectively). Histology of total knee joints showed that influx of inflammatory cells was highly decreased and severe cartilage destruction measured as chondrocyte death was almost completely prevented.

Conclusion: Boron neuron capture therapy using 10B containing liposomes causes selective depletion of synovial lining macrophages and largely prevents onset of joint inflammation and severe cartilage destruction of experimental arthritis.

041 A NOVEL APPROACH TO EVALUATE SYNOVIAL INFLAMMATION: VOLUMETRIC COLOUR CODED SHAPE MAPPING

M. G. H. Van de Sande1, C. Lavini2, C. A. Wijbrandts1, M. C. de Jonge2, M. Maas2, P. P. Tak1.1Department of Clinical Immunology and Rheumatology, 2Department of Radiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

Objective: To develop a novel dynamic contrast enhanced (DCE) MRI method for the evaluation of synovitis, to correlate MR parameters with synovial tissue vascularity, and to evaluate differences between RA and non-RA patients.

Materials and methods: Early arthritis patients with inflammation of at least 1 knee joint (<1 year’s duration) underwent MRI and arthroscopy of the knee joint. Clinical data were collected at start and after 1 year of follow up. The DCE-MRI protocol for the knee joint was performed with a 1.5 Tesla scanner (GE Signa) and consisted of 20 subsequent 3D-Spoiled GRE sequences. The 5 characteristic curve shapes (I-II-III-IV-V) (van Rijswijk et al Radiology 2004; 493) were categorized and displayed in maps. Arthroscopic synovial tissue biopsy samples were collected by standard procedures within one week after MRI. To evaluate vascularity in synovial tissue biopsies, immunohistochemical staining was performed to detect von Willebrand Factor (VWF); stained sections were evaluated by digital image analysis.

Results: The study group consisted of 14 patients, of which 7 RA and 7 non-RA at t = 0 and 9 RA and 5 non-RA at t = 1 year. In each patient all different curve types were seen, although there were differences between patients. We found a correlation between shape curve type II and VWF (R 0.75, p = 0.002) and between total enhancement and VWF (R 0.73, p = 0.003),. In addition, there was a difference in curve type 2 and total enhancement between RA and non-RA patients at t = 1 (p = 0.02).

Conclusion: DCE-MRI shape curves are associated with synovial tissue vascularity. DCE-MRI colour coded shape curves might be a novel tool that could differentiate RA patients from non-RA patients in an early phase of the disease. Future research will focus on the clinical relevance and possible predictive value of results obtained by DCE-MRI.

042 MAPPING OF CITRULLINATED ALPHA-ENOLASE ANTIBODIES TO AN IMMUNODOMINANT EPITOPE WITH HIGH SEQUENCE SIMILARITY TO BACTERIAL ENOLASE

K. Lundberg1, A. Kinloch1, H. Allison2, S. Sriskandan3, D. Moyes1, P. Venables1.1The Kennedy Institute of Rheumatology, Imperial College; 2School of Biological Sciences, Liverpool University; 3Imperial College, Faculty of Medicine, London, UK

Background: Anti-citrullinated protein antibodies (ACPAs), targeting proteins containing the post-translational conversion of arginine to citrulline, are specific and sensitive for the diagnosis of rheumatoid arthritis (RA), making citrullinated proteins strong candidates for driving the autoimmune response. Little is known about the antigens reactive with ACPAs or the origin of this antibody response, although we have recently identified citrullinated alpha-enolase as a candidate antigen and in the present study explored the possible role of bacterial infection in its generation.

Material and methods: Serum from patients with RA (n = 102), disease controls (n = 92) and healthy individuals (n = 92), were tested by ELISA for reactivity with 11 citrullinated alpha-enolase peptides. Antibodies specific for the immunodominant peptide were affinity purified from rabbit antiserum and from RA-patient sera. Porphyromonas gingivalis and Streptococcus pyogenes lysates were in vitro treated with peptidyl arginine deiminase (PAD). Antibody cross-reactivity between citrullinated human alpha-enolase and bacterial enolase was investigated by western blots.

Results: Serum samples from patients with RA identified an immunodominant epitope within citrullinated alpha-enolase, with a diagnostic sensitivity of 40% and a specificity of 97%. The importance of citrulline in this antibody response was demonstrated by the low degree of reactivity to the arginine-containing control peptide (<5%). The epitope was 100% identical to the corresponding sequence in citrullinated enolase encoded by P gingivalis and 87% with that of S pyogenes. Affinity purified antibodies, specific for the immunodominant epitope on citrullinated human alpha-enolase, cross-reacted with enolase from both bacteria treated with PAD. Untreated lysates of P gingivalis also reacted with the affinity purified anti-peptide antibody and with a commercially available anti-citrulline antibody, indicating endogenous citrullination.

Discussion: Our data suggest that autoimmunity in the subset of RA-patients who have antibodies to citrullinated alpha-enolase could be primed by P gingivalis infection, a common cause of periodontitis and the only known bacterium to synthesise its own PAD. Since chronic periodontitis has also been associated with the MHC shared epitope, we propose that in genetically susceptible individuals P gingivalis infection could break tolerance and, by cross-reacting with citrullinated antigens in the joints, lead to the chronic inflammation that characterises RA.

043 MODULATING OSTEOCLAST FUNCTION BY TARGETING THE ALPHAVBETA3 INTEGRIN

F. Brunton, L. Lee, A. Nissim, A. Grigoriadis, C. Pitzalis.Kings College London, UK

Introduction: The identification of a peptide which targets inflamed synovial vasculature has opened up the possibility of targeted drug delivery in rheumatoid arthritis. Rheumatoid arthritis is an inflammatory arthropathy that culminates in joint destruction. Critical to this process is the role of osteoclasts that lead to the formation of marginal erosions and joint dysfunction. The importance of osteoclasts has been emphasized experimentally in animal models by crosses between c-Fos knockout (which lack osteoclasts) and human TNF transgenic mice (which develop arthritis). The absence of osteoclasts rescues joint destruction normally observed in the TNF transgenic model. Central to osteoclast attachment to bone and resorption is the integrin molecule alpha v beta3. Mice lacking the alphavbeta3 integrin have dysfunctional osteoclasts and accumulate bone mass with age. Thus, reagents targeted against alpha v beta3 are potential therapeutics in targeted drug delivery in rheumatoid arthritis.

Methods: A C7C peptide phage display library and a human scFV antibody library were used to target the alphavbeta3 integrin in vitro. Novel reagents identified were screened for their affinity to the integrin and their antiresorptive effect assessed in vitro on osteoclasts derived from murine bone marrow (BM) or human PBMCs in their capacity to inhibit resorption pit formation on dentine slices. Peptides were assessed against a positive control, the RGD inhibitor Echistatin from the Saw scaled viper Echis Carinatus.

Results: Peptide phage display identified two phage clones with high affinity for the alphavbeta3 integrin. Murine BM cells cultured with CSPRGDHPC showed a decreased number of multinucleated TRAP positive cells at 10 μM. Resorption was also inhibited significantly. Murine BM cultures with the control peptide CDPRPHSGC were comparable to control cultures in terms of osteoclast number and area of resorption. Echistatin inhibited both murine and human differentiation and activity, although a 5–10 fold higher dose was required to see an inhibition in the number of human osteoclasts as identified by actin and VNR staining and an inhibition of resorption. It is yet to be determined if a similar increase in concentration will be required to see an inhibitory effect of CSPRGDHPC on human osteoclasts.

Conclusions: Phage display technology can identify novel reagents against the alphavbeta3 integrin with the capacity to inhibit osteoclast resorption in vitro, thus providing novel reagents for targeted drug delivery to the rheumatoid joint. Work is in progress to select novel reagents using scFV antibody phage display in order to obtain high affinity human antibody fragments that may be more suitable for therapeutic applications in vivo.

044 BINDING OF HIGH AVIDITY ANTI- BETA2-GLYCOPROTEIN I ANTIBODIES TO THE ANTIGEN IN FLUID PHASE

S. Cucnik, T. Kveder, B. Rozman, B. Bozic.University Medical Centre, Department of Rheumatology, Ljubljana, Slovenia

Introduction: The avidity of antiphospholipid antibodies and its clinical significance have been evaluated in several studies. The binding of anti-beta2GPI antibodies to physiologic concentrations of beta2GPI in fluid phase is supposed to be weak due to their low avidity. It is assumed that the density of beta2GPI is important for low affinity antibodies because their avidity is increased by bivalent binding in vivo and in vitro. Previously, we demonstrated that anti-beta2GPI antibodies are also of high avidity. We would expect that high avidity anti-beta2GPI antibodies are able to bind beta2GPI in fluid phase also.

Aim: To evaluate the importance of beta2GPI concentration for binding high avidity anti-beta2GPI.

Material: Plasma, IgG fraction and affinity purified high or low avidity anti-beta2GPI antibodies from an APS patient; - Fab fragment of high avidity anti-beta2GPI.

Methods: Standard, chaotropic and fluid-phase inhibition anti-beta2GPI ELISA on the Costar high binding microtitre plates.

Results: High avidity anti-beta2GPI antibodies showed dose dependent binding to the antigen at the concentration range 1.25–20 mg/L on microtitre plates. Similar results were obtained when using isolated Fab fragment of high avidity anti-beta2GPI. Isolated low avidity IgG anti-beta2GPI antibodies showed negligible binding to the antigen below 5 mg/L. High avidity anti-beta2GPI antibodies in the concentrations found in APS patients are able to bound beta2GPI in fluid phase at physiologic concentrations.

Conclusions: High antigen density resulting in bivalent binding is crucial for the detection of low avidity anti-beta2GPI antibodies in ELISA. High avidity anti-beta2GPI antibodies are able to bind the antigen at low density on microtitre plates as well as in fluid phase at physiologic concentrations.

045 COMBINED TUMOUR NECROSIS FACTOR-ALPHA (TNF-ALPHA) AND TUMOUR NECROSIS FACTOR RECEPTOR GENOTYPES COULD PREDICT RHEUMATOID ARTHRITIS PATIENTS’ RESPONSE TO ANTI-TNF-ALPHA THERAPY AND EXPLAIN CONTROVERSIES

A. Chatzikyriakidou, I. Georgiou, P. V. Voulgari1, A. I. Venetsanopoulou1, A. A. Drosos1.Genetics Unit, Department of Obstetrics and Gynaecology; 1Rheumatology Clinic, Department of Internal Medicine, Medical School, University of Ioannina, 45110, Greece

Aim: Anti-tumour necrosis factor-alpha (anti-TNF-alpha) therapy has been a breakthrough in the management of chronic inflammatory diseases. However, patients show high variability in their response to TNF-alpha blockers and the prognostic value of certain polymorphisms remains controversial. The aim of the present study was to investigate the complex genotypic interactions among TNF-alpha and TNF receptor (TNFR1 and TNFR2) genes in patient response to anti-TNF-alpha therapy.

Materials and methods: Genomic DNA from 58 patients with rheumatoid arthritis was genotyped for 6 polymorphisms: 36A>G (TNFR1, exon 1), 676T>G (TNFR2, exon 6), −857C>T (TNF-alpha, promoter), −308G>A (TNF-alpha, promoter), −238G>A (TNF-alpha, promoter), and +489G>A (TNF-alpha, intron 1) by polymerase chain reaction-restriction fragment length polymorphism analysis. All patients were receiving anti-TNF-alpha therapy (infliximab). The disease activity score for 28 joint indices was evaluated in order to determine patient response to TNF-alpha blocker.

Results: Independent polymorphisms cannot predict good or poor response to anti-TNF-alpha treatment. However, the complex genotypic analysis revealed statistical significant difference (p = 0.008) in the distribution of the genotypic association of the wild type 676T allele of TNFR2 gene with the wild type −857C/+489G allele (in strict linkage disequilibrium; p = 0.000) of TNF-alpha gene between the good and poor responders. Moreover, taking into account the role of −857C>T polymorphism in TNF-alpha gene transcription, this polymorphic variant has a higher predictive value than the +489G>A in the combined genotypic analysis with the 676T>G polymorphism.

Conclusions: The compound genotype of polymorphisms −857C>T (TNF-alpha) and 676T>G (TNFR2) appears to be a powerful tool in the prognosis of patient response to anti-TNF-alpha therapy.

046 PRODUCTION AND SECRETION OF THE LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2 (LPPLA2) FROM BLOOD MONOCYTES OF PATIENTS WITH EARLY RHEUMATOID ARTHRITIS: EFFECT OF DISEASE MODIFYING ANTIRHEUMATIC DRUGS ADMINISTRATION

E. C. Papavasiliou, A. N. Georgiadis1, A. A. Drosos1, A. D. Tselepis.Laboratory of Biochemistry, Department of Chemistry; 1Rheumatology Clinic, Department of Internal Medicine, Medical School, University of Ioannina, 45110 Ioannina, Greece

Aim: Lipoprotein-associated phospholipase A2 (LpPLA2) is a Ca2+-independent phospholipase A2 that circulates in plasma bound on lipoprotein particles. LpPLA2 hydrolyzes platelet activating factor, as well as oxidized phospholipids, ie, lipids that play important roles in inflammation and atherogenesis. Thus this enzyme could be implicated in inflammatory disorders as well as in atherogenesis. The main cellular source of the plasma LpPLA2 is monocytes/macrophages and this enzyme represents a marker of inflammatory stimulation of these cells. Early immunointervention with disease-modifying antirheumatic drugs (DMARDs) may control the inflammatory process of rheumatoid arthritis (RA) and may reduce the articular damage.

Materials and methods: We investigated the spontaneous production and secretion of LpPLA2 from peripheral blood monocytes (PBMs) in culture, during their differentiation into macrophages. PBMs were isolated from 9 healthy volunteers as well as from 9 patients with early RA before and after 4 months of treatment with methotrexate (15 mg/week) and prednisone (7.5 mg/day). Adherent PBMs were cultured in RPMI medium supplemented with 10% human serum at 37°C for 72 h. The total LpPLA2 activity and that secreted into the medium were determined.

Results: The results of the present study show that PBMs from healthy volunteers during their differentiation into macrophages produce and secrete LpPLA2 spontaneously. The maximum of LpPLA2 production and secretion occurs at 48 h of culture, attaining the level of 209.6±52.0 and 192.5±40.0 nmol/mg protein/h, respectively. Importantly, the LpPLA2 activity produced and secreted into the medium by PBMs from patients with active early RA was significantly lower (88.6±11.5 and 82.6±12.5 nmol/mg protein/h, respectively) compared to that produced by PBMs from healthy volunteers, p<0.01 for both comparisons. Treatment with DMARDs restored the ability of patient’s PBMs to spontaneously produce and secrete LpPLA2 (195.0±33.5 and 177.6±26.6 nmol/mg protein/h of enzyme activity at 48 h of culture, respectively).

Discussion: We suggest that PBMs from patients with active early RA are defective in their ability to spontaneously produce and secrete LpPLA2 during their differentiation into macrophages. Treatment with methotrexate and prednisone restores the enzyme production and secretion by PBMs, a phenomenon that may represent a new antiinflammatory effect of these drugs.

047 THE SYNOVIAL CITRULLINOME OF RHEUMATOID ARTHRITIS PATIENTS

J. J. B. C. van Beers, R. Raijmakers, A. van der Heijden, G. J. M. Pruijn, A. J. W. Zendman.Department of Biomolecular Chemistry, Radboud University Nijmegen, Nijmegen, The Netherlands

Background: Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by chronic inflammation of the synovial joint. A specific feature in RA patients is the presence of autoantibodies against citrullinated proteins. Citrulline residues are generated post-translationally from arginine by peptidylarginine deiminase (PAD, EC 3.5.3.15). Several candidate citrullinated antigens, such as fibrinogen and vimentin, have already been detected in synovial material from RA patients. However, their role in the pathogenesis of RA remains unknown.

Objective: To identify new citrullinated candidate autoantigens in RA patients and to elucidate their function in the pathogenesis of RA.

Methods: Synovial fluid samples and synovial biopsies from RA patients were centrifuged to separate insoluble and soluble components into pellet and supernatant fractions, respectively. The majority of the proteins in the pellet fraction were solubilized in a buffer containing 2% SDS. Western blotting with anti-modified citrulline (AMC) antibodies was used to monitor the presence of citrullinated proteins in both pellet and supernatant fractions. Polypeptides in these synovial samples were fractionated by SDS-PAGE. The resulting gels were cut into slices, digested with trypsin and subsequently analyzed with MALDI-TOF mass spectrometry. Identity of the protein fragments was determined by database screening (Mascot search). The presence of possible protein modifications, eg, citrullination, was subsequently predicted by analyzing the mass spectra by the FindMod tool.

Results: The analysis of RA synovial samples by AMC antibodies revealed several patterns of citrullinated proteins, which were generally found in both the insoluble and soluble fractions. In a subset of RA patients no citrullinated proteins were detected in synovial samples. By mass spectrometry many proteins were identified in all synovial samples analysed. The polypeptide composition of the samples from individual patients showed a partial overlap and no significant differences between citrullinated protein containing samples and samples devoid of citrullinated proteins. As expected, for many proteins various proteolytic fragments were identified. For a number of proteins, the MALDI-TOF data predicted multiple putative citrullinated sites. Our data confirmed the presence of known citrullinated candidate autoantigens in RA (eg, vimentin and fibrin). Additional experiments will have to verify the citrullination status of the putatively citrullinated synovial proteins.

Conclusions: : Potential novel citrullinated antigens have been detected in synovial (fluid and tissue) samples of RA patients. Further research is needed to determine the role of these proteins in the pathogenesis of RA.

Funded by -Dutch Arthritis Association; grant number 05-1-302 - Netherlands Organization for Health Research and Development; grant number 916.56.071

048 COMPARATIVE ANALYSIS OF MESENCHYMAL STEM CELLS FROM NORMAL BONE MARROW AND SYNOVIAL FLUID: IMPLICATIONS FOR CARTILAGE REGENERATION STRATEGIES

E. Jones1, A. English1, A. Crawford2, J. Mundy2, K. Henshaw1, D. Corscadden1, P. Emery1, P. Hutton2, D. McGonagle1.1The Academic Unit of Musculoskeletal Disease, Leeds Institute for Molecular Medicine, University of Leeds, UK; 2Centre for Biomaterials & Tissue Engineering, University of Sheffield, UK

Aims: Abnormal tissue repair processes are a common feature of osteoarthritis, but the identity, biology and topography of stem cells involved in these responses remains poorly understood. In healthy humans, the study of joint stem cells is even more difficult, and large animals have often been used as models.1 We have previously demonstrated the presence of mesenchymal stem cells (MSCs) in synovial fluid (SF) of patients with arthritis.2 Here we hypothesised the existence of a normal SF MSC population that may be inherently chondrogenic and hence particularly suitable for cartilage repair strategies.

Methods: Bone marrow (BM) and SF was harvested from 16 cows. Immediately following initial tissue processing, single cell-derived cultures were established using limiting dilution analysis. These were grown for at least 24 population doublings prior to functional assays of tri-potentiality, which were performed in conventional in vitro assays or following long-term culture on polyglycolic acid (PGA) scaffolds. The MSC cell phenotype was investigated following pre-enrichment with Anti-CD271 MicroBeads.34 Standard polyclonal cultures were also established and tested for tri-potentiality.

Results: In common with human BM MSCs, bovine BM MSCs expressed low-affinity nerve growth factor receptor (CD271); however bovine SF MSCs did not. Growth rates and expandability of single cell-derived clones from the SF was significantly higher compared to the BM (27/27 clones expandable a million-fold compared to 11/22, respectively). When seeded onto PGA scaffolds only some BM-derived clones formed cartilage constructs with hyaline characteristics which were similar in size to control constructs of PGA seeded with bovine chondrocytes. In contrast, constructs engineered from all the SF-derived clones tested, had hyaline-like characteristics and were similar in size to the control constructs formed from bovine chondrocytes. Compared to donor-matched BM, standard polyclonal SF-derived cultures were always superior in their chondrogenic capacity, both in a pellet system and on PGA scaffolds. Conversely, both clonal and polyclonal cultures from the SF were significantly (over 20-fold) less adipogenic than donor-matched BM-derived cultures.

Conclusions: These data suggest distinct tissue-specific roles of MSCs in different locations. Higher adipogenesis from BM MSCs is not unexpected giving their homeostatic role in BM fat production. The exceptional proliferative capacity of SF MSCs is however surprising. This, coupled with apparent high chondrogenicity of SF MSCs, is consistent with a potential physiological role for these stem cells in repair/maintenance of the cartilage surface in health. Altogether, SF represents a potentially attractive source of chondrogenic cells for cartilage repair therapies in trauma and arthritis, without the inherent risks associated with more traditional procedures such as cartilage or bone marrow biopsy.

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049 THE ROLE OF THE LEVEL OF BAFF IN CHRONICALLY INFLAMED JOINTS IN RHEUMATOID ARTHRITIS PATIENTS

E. Zanova, U. Musalowicz, W. Rudnicka, E. Kontny, A. Radzikowska, W. Maslinski.Department of Pathophysiology and Immunology, Institute of Rheumatology, Warsaw, Poland

Aims: The B cell-activation factor (BAFF), a member of the tumour necrosis family, is expressed by several types of cells including monocytes/macrophages, dendritic cells and T lymphocytes. BAFF regulates B cell homeostasis and immunoglobulin production through its receptors BCMA, TACI and BAFF-R. BAFF-triggered signalling might counterbalance the pro-apoptotic signals induced by the B cell receptor (BCR). Thus, BAFF is known as a positive regulator of B cell function. The aim of this work was to compare the levels of BAFF in serum, bone marrow and synovial fluid from the patients with rheumatoid arthritis, osteoarthritis. As an additional control, serum levels of BAFF in healthy donors were measured.

Materials and methods: Peripheral blood serum was obtained from RA, OA and healthy donors. Serum from bone marrow was obtained from RA and OA patients. Synovial fluid was withdrawn from RA and OA. Quantikine Human BAFF/BLyS/TNFSF13B Immunoassay ELISA kit was used for the quantitative determination of human BAFF belonging to the TNF family

Results: We report that the levels of BAFF were higher in synovial fluids from patients with RA in comparison to OA. BAFF levels were higher in blood in comparison to bone marrow from the same patients. However, no differences between BAFF levels in blood and bone marrow from RA and OA patients were observed.

Conclusions: High levels of BAFF in synovial fluid suggest its local production in chronically inflamed joints in rheumatoid arthritis patients. Thus, locally overproduced BAFF can contribute to the activation, maturation and enhancement of immunoglobulin production by joints infiltrating B-cells in rheumatoid arthritis.

The work was supported by EURO-RA project No. MRTN-CT-2004-005693

050 DIFFERENT EFFECT OF TNF BLOCKADE BY INFLIXIMAB AND ETANERCEPT ON NALP3 AND NALP3 INFLAMMASOME REGULATORS IN RHEUMATOID ARTHRITIS PATIENTS

R. Mathews, S. Churchman, L. Church, L. Coulthard, L. Dickie, S. Nizam, D. Bryer, B. Saleem, P. Emery, M. McDermott.Leeds Institute of Molecular Medicine, Leeds, UK

Background: The proinflammatory cytokines, IL-1 and TNF, are key to the pathogenesis and persistence of rheumatoid arthritis (RA), with associated joint damage. Blockade of TNF in these patients, using current biological response modifiers (infliximab, etanercept, and adalimumab), has had profound therapeutic effects with marked benefit to patients. RA has traditionally been described as being due to adaptive immune responses, but there has been a gradual appreciation of the role of innate immunity, acting as precursors to subsequent adaptive responses. The NALP3 inflammasome is a highly conserved and specific response system, composed of a proinflammatory protein complex involved in regulation of innate immunity and, in particular, the production of IL-1. This study investigates the expression and activation of the various components of the NALP3 complex in RA patients and the effects of TNF blockade on this pathway.

Methods: The patients studied had failed at least two traditional disease modifying anti-rheumatic drugs, before being initiated onto either infliximab or etanercept. Protein and mRNA expression analysis was carried out on peripheral blood mononuclear cells (PBMC), pre- and 2 hours post-infusion, at weeks 0, 2, and 14 for 10 patients receiving infliximab, and at 0, and 12 weeks for 10 patients receiving etanercept. ASC (caspase-1-activating adaptor for IL-1 generation) and pyrin (a negative regulator of the NALP3 inflammasome) protein expression in cytosolic fractions derived from PBMC was analysed by western blotting (WB). Quantitative PCR was used to detect ASC and pyrin messenger RNA from PBMC of RA patients.

Results: Infliximab had marked effects on the expression of ASC, with significant increases in both gene and protein expression levels; there was a rapid increase in ASC mRNA at 2 hours post infusion (p<0.05), which was maintained at pre-infusion weeks 2 and 14. The protein expression, determined by WB, reflected this immediate change in ASC. In contrast to this, pyrin expression was unaffected by infliximab treatment. However, patients receiving etanercept showed variability in pyrin expression, with both increased and decreased expression levels observed in different patients after 12 weeks. However, ASC expression was unperturbed in etanercept treated RA patients, in contrast to patients receiving infliximab.

Conclusions: Both infliximab and etanercept share the proinflammatory cytokine TNF as a common therapeutic target, but our findings demonstrate that TNF blockade, using these two biologic response modifiers, have different impacts on the NALP3 inflammasome. The data show increased ASC mRNA and protein expression immediately post infusion in infliximab treated patients, which is maintained, or even further increased at week 14. The range of changes in pyrin protein expression after etanercept suggests that this therapy affects regulation of pyrin rather than ASC. These data provide insights into different mechanisms of action of infliximab and etanercept.

051 BOSENTAN MODULATES THE EXPRESSION OF ADHESION MOLECULES BY T CELLS AND ENDOTHELIAL CELLS IN SYSTEMIC SCLEROSIS ASSOCIATED PULMONARY ARTERIAL HYPERTENSION

F. Iannone1, S. Guiducci2, M. T. Riccardi1, M. Cinelli2, M. Covelli1, M. Matucci-Cerinic2, G. Lapadula1.1DiMIMP Rheumatology Unit, Bari, Italy; 2SOD Internal Medicine 1 and Rheumatology, Florence, Italy

Aim: To assess changes of serum levels of endothelial activation markers and expression of adhesion molecules on peripheral blood (PB) T cells in patients with PAH-SSc during bosentan therapy.

Methods: Thirty-five SSc patients and 25 healthy donors (HD) were enrolled in the study after giving informed consent. Ten out of 35 patients had isolated PAH assessed by Doppler echocardiography and 6-minute walking distance (6-MWD) between 60 and 450 m, and treated with bosentan at the dosage of 62.5 mg twice daily for 4 weeks followed by 125 mg twice daily. In all SSc patients and HD, PB T cells were isolated by density gradient centrifugation and expression of LFA-1, VLA-4, L-selectin on CD3 T cells was assessed by specific monoclonal antibodies and following flow cytometry analysis. In PAH-SSc patients T cell subsets were assessed at baseline and then after 6 and 12 months of bosentan therapy. As endothelial activation markers, soluble vWF (von Willebrand Factor), P-selectin, PECAM, VCAM-1, and ICAM-1 were assessed by ELISA in all subjects, and in PAH-SSC also after 12 months. Results are shown as mean ±1 SD (standard deviation). For statistical comparison analysis of variance (ANOVA) with the LSD (least square differences) range test was used.

Results: Bosentan improved exercise ability and cardiac haemodynamics in PAH-SSc patients. 6-MWD raised from 267±33 m at baseline to 359±27 m at 6 months and to 376±40 m at 12 months (p<0.05 vs baseline) of therapy. Bosentan induced also a decrease of PAP levels (baseline 45.7±7 mmHg, 6 months 41.4±4 mmHg, 12 months 39.0±4 mmHg), but the difference was not statistically significant. SSc without PAH patients had significantly increased levels of soluble ICAM-1 (p<0.01), VCAM-1 (p<0.01), P-selectin (p<0.01), vWF (p<0.05) in comparison with HD and normal percentages of T cells expressing adhesion molecules. PHA-SSc patients had significantly higher levels of ICAM-1 (p<0.01), VCAM-1 (p<0.05), P-selectin (p<0.01), vWF (p<0.05) than HD at baseline and decreased to normal values after 12 months of therapy with bosentan. CD3-LFA1 T cells were significantly higher in PHA-SSc at baseline than in HD and SSc (p<0.05) and decreased at 12 months of therapy. On the other hand, CD3-Lselectin T cells were significantly lower in PHA-SSc at baseline than HD and SSc (p<0.01) and raised at normal levels after 12 months of therapy.

Conclusions: This study confirms that endothelial activation occurs in SSc and suggests that changes of T-cell/endothelium interplay takes place in PAH-SSc. Bosentan, beside improving PHA-SSc, seems to be capable to restore endothelium function in these patients.

052 SYSTEMIC ADENOVIRAL MEDIATED OVEREXPRESSION OF SUPPRESSOR OF CYTOKINE SIGNALLING (SOCS)-3 GENE MARKEDLY AMELIORATES COLLAGEN-INDUCED ARTHRITIS (CIA) IN MICE

S. Veenbergen, M. B. Bennink, A. S. K. de Hooge, O. J. Arntz, L. A. B. Joosten, W. B. van den Berg, F. A. J. van de Loo.Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, The Netherlands

Introduction: SOCS proteins are negative intracellular regulators of cytokine- and growth-factor signalling and evidence is emerging that SOCS may exert anti-arthritic effects. For example, SOCS1 deficiency aggravates antigen-induced arthritis in the IFN-gamma knockout mice. Similarly, defective SOCS induction due to mutations in the signal transducer gp130 receptor chain augments arthritis as a result of hyperstimulation of synovial cell activation and T-cell proliferation.

Objective: In this study, we explored the anti-arthritic effect of systemic SOCS3 gene transfer in mice developing collagen-induced arthritis, and elucidated the mode of action.

Methods: The SOCS3 gene was obtained from cDNA of the B9 cells and cloned into the pShuttle transfer vector (AdEasy) for production of recombinant adenoviruses. DBA1/J mice were immunized at day 0 and 21 with bovine collagen type II, and at day 22 mice received an intravenous injection of 3×10E8 ffu adenoviruses. The liver was isolated to determine class I and class II acute phase proteins by RT-QPCR. CD4+CD25bright expression and Foxp3 in magnetic cell sorted splenic CD3+ T cells was determined by flow cytometry and QPCR. Cytokines were measured using protein-multiplex assays (BioPlex).

Results: Treatment of mice with Ad5.SOCS3 markedly suppressed arthritis development as observed macroscopically in the paws. Histology showed reduced joint inflammation and cartilage proteoglycan loss and this confirmed the anti-arthritic effect of SOCS3. Interestingly, SOCS3-gene transfer significantly reduced the circulating levels of TNFa (−72%), a proinflammatory cytokine that plays a pivotal role in arthritis. The liver is the main target organ of circulating adenoviruses and forced SOCS3 expression clearly inhibited STAT3 phosphorylation and significantly reduced the acute phase reaction during CIA. Furthermore, we observed that the spleen was also efficiently transduced by adenoviruses leading to SOCS3 expression in antigen-presenting cells. This resulted in enhanced mRNA expression of T-bet (Th1), GATA-3 (Th2) and FoxP3 (Treg) in spleens, however, the antigen-specific proliferation of splenic lymphocytes was reduced. A possible explanation for the impaired antigen-response could be an increase of regulatory T-cell activity by SOCS3. Indeed, we found an increased TGF-beta/IFN-gamma mRNA ratio (4×) in the liver and enhanced levels of CD4+CD25+FoxP3+ T-cells in the circulation. All data is in favour of enhanced regulatory T-cell activity by SOCS3-treatment. Conversely, the numbers of Th17 cells were reduced by SOCS3 treatment. Protein multiplex immunoassay analysis showed decreased levels of IL-4 (−41%), IL-17 (−70%), and IFN-gamma (−43%) in conditioned media of hyperstimulated (PMA+ionomycin) splenic lymphocytes from Ad5.SOCS3 animals. Furthermore, SOCS3 gene transfer also suppressed a mediator upstream of IL-17. IL-23/p19 mRNA expression in splenocytes and IL-23 protein levels (−80%) in the circulation were reduced.

Conclusion: Adenoviral-mediated SOCS3 gene transfer clearly ameliorated experimental arthritis and we identified an upregulation of T-cells with a regulatory phenotype (CD25+FoxP3+) and downregulation of the proinflammatory Th17 cells in these mice.

053 SOLUBLE IL-18 RECEPTOR-BETA BLOCKS REGULATORY T CELL INDUCTION BY IL-18 LEADING TO EXACERBATION OF EXPERIMENTAL ARTHRITIS

S. Veenbergen, M. B. Bennink, R. L. Smeets, O. J. Arntz, L. A. B. Joosten, W. B. van den Berg, F. A. J. van de Loo.Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, The Netherlands

Interleukin-18 (IL-18) is known to stimulate T helper 1 maturation in synergy with IL-12 and thereby promoting collagen-induced arthritis (CIA) when given during immunization. Our previous experiments showed that neutralization through systemic treatment with either neutralizing antibodies or IL-18 binding protein isoform c (IL-18BPc) results in amelioration of CIA. The objective of this study was to determine the immunoregulatory role of a novel identified soluble form of the IL-18Receptor accessory protein (sIL-18R-beta) in arthritis. Tissue distribution analysis revealed high expression of sIL-18R-beta in lymphoid tissues, and little expression in immune privileged organs suggesting a role for sIL-18R-beta in immunity. To investigate the effect of sIL-18R-beta overexpression on the development of experimental arthritis, we injected 3×10e8 ffu of adenovirus encoding sIL-18R-beta into DBA1/J mice just before clinical manifestation of murine CIA. Surprisingly, systemic overexpression of sIL-18R-beta resulted in marked exacerbation of CIA that coincided with significant reduced circulating levels of CD4+CD25+Foxp3+ regulatory T (Treg) cells and reduced circulating IFN-gamma and the regulatory IL-10 cytokine as measured by flow cytometry and Luminex multi-analyte technology, respectively. These results suggest that IL-18 might also have anti-inflammatory properties besides the well-known pro-inflammatory properties. Therefore, we examined whether IL-18 is able to induce regulatory T cells by determining expression of the regulatory T cell markers CD25bright and Foxp3 via flow cytometry and RT-QPCR. Stimulation of magnetic-activated cell sorting separation (MACS) purified murine CD3+ spleen T cells with IL-18 for 4 days induced CD25bright and Foxp3 expression at both mRNA and protein level. Next, we tested whether the induction of Treg by IL-18 was mediated by IFN-gamma and whether IL-12 has a synergistic effect. Interestingly, blocking of IFN-gamma by neutralizing antibodies had no effect on the IL-18 induced Foxp3 expression. The combination of IL-12 and IL-18 did not lead to enhanced Foxp3 protein expression. In addition, we confirmed that IL-12 can enhance the IL-18 induced T helper 1 (Th1) formation by increased mRNA expression of T-bet (Th1 marker) when compared to IL-18 stimulation alone. These results show that IL-18 can induce CD4+CD25bright Foxp3+ T cells independent of IL-12 and IFN-gamma. Next, we tested whether the immunoregulatory properties of IL-18 could be affected by soluble IL-18R-beta. Therefore, IL-18 stimulated CD3+ T cells were incubated with sIL-18R-beta overexpressed murine antigen presenting cells. Co-culturing of CD3+ T cells with these sIL-18R-beta producing APCs completely blocked Foxp3 induction by IL-18. This study shows that IL-18 has a much broader range of immunoregulatory properties by promoting the differentiation of Treg cells besides the induction of Th1 cells. We showed that sIL-18R-beta inhibits the Treg formation and thereby promoting CIA, which identifies sIL-18R-beta as a new immunomodulator of IL-18 signalling.

054 PRESENCE AND DISTRIBUTION OF ANTIBODIES SPECIFIC TO DIFFERENT CITRULLINATED ANTIGENS IN SYNOVIAL FLUID OF CCP+ RA

O. Snir1, M. Widhe1, K. Lundberg3, R. Holmdahl2, P. Venables3, L. Klareskog1, V. Malmström1.1Dept of Medicine Solna, Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden; 2Medical Inflammation Research, Lund University, Lund, Sweden; 3The Kennedy Institute, Imperial College, London, UK

Background: Autoantibodies against CCP are present in sera of a majority of rheumatoid arthritis (RA) patients. Lately, specific citrullinated proteins have been suggested as possible RA autoantigens. However, their relevance in disease pathogenesis and their internal relationship have not been investigated.

Aim: To examine the association between antibodies towards four different citrullinated antigens known to be present in the sera of RA patients—the C1 epitope of type-II collagen (CII), alpha-enolase, fibrinogen and vimentin.

Methods: 98-paired samples of serum and synovial fluid from seropositive and seronegative RA patients were examined for their anti-CCP IgG antibody levels. In addition, the levels of IgA and IgG antibodies towards native and citrullinated C1-CII and peptide 1 of alpha-enolase were measured by ELISA. We will further extend the cohort of RA patients, and also include healthy control subjects. Furthermore, we will also investigate antibody levels towards citrullinated fibrinogen, fibrinogen peptide 1 and mutated citrullinated vimentin (MCV, Orgentec).

Results: So far samples from 98 patients have been analyzed, and 69% of them were anti-CCP positive. Among the CCP-positive patients half were IgG positive for the Cit-CII peptide both in serum and synovial fluid. In comparison we also found IgG towards non-citrullinated C1-CII in 20% of the serum and 45% of the synovial fluids, but these levels were consistently lower than the antibodies to the citrullinated form. Within the CCP-negative patients 20% have serum IgG Ab against CII and 14% in the fluid. Interestingly, a few patients also had Cit-CII IgG antibodies although they were CCP-negative. Regarding the alpha-enolase responses, antibodies could only be found towards the citrullinated form of the peptide. Among the CCP-positive patients almost half were cit-enolase IgG positive in both the serum and fluid. The IgA responses to cit-enolase were less frequent than IgG. Only 20% of the CCP-positive patients showed positive IgA responses against cit-enolase in the serum and 12% in the fluid. For all the antigens tested, IgA responses could only be detected in samples with positive IgG responses, but were consistently lower than IgG.

Summary: This study shows that the specificity of the autoantibodies in CCP+ RA patients varies between individuals, e.g. they are directed against different citrullinated autoantigens. Also, the results indicate that many RA patients display antibodies towards more than one autoantigen, which need to be confirmed with inhibition studies. We also show that there is a strong association between CCP antibodies and antibodies to citrullinated antigens like the C1-CII and alpha-enolase. Finally, the association between the IgA and IgG responses against citrullinated peptides raises the question whether the breakage of tolerance could happen at a mucosal site (e.g. lungs or intestines).

055 UPREGULATION OF ANTIPHOSPHOLIPID (APL) ANTIBODY RESPONSES BY CYCLOPHOSPHAMIDE

P. G. Vlachoyiannopoulos1, E. Zintzaras2, G. E. Katsifis1, S. Toya1, A. Tzioufas1, H. M. Moutsopoulos1.1Department of Pathophysiology, School of Medicine, National University of Athens, Greece; 2Department of Biomathematics, University of Thessaly School of Medicine, Papakyriazi, Greece

Background: We have observed some cases of lupus nephritis patients, successfully treated with cyclophosphamide (CYP), which subsequently developed antiphospholipid antibodies (aPL) or full blown antiphospholipid syndrome (APS). To evaluate the significance of this phenomenon we undertook a retrospective evaluation of our patient population.

Methods: Patients with systemic lupus erythematosus (SLE) (n = 320), either treated (n = 117) or non-treated with CYP (n = 203), were evaluated for initial and cumulative clinical and serological characteristics with emphasis on ECLAM score, anti-dsDNA responses, C3, C4, aPL, anti-B2-glycoprotein I(anti-B2GPI) antibody levels which were measured every four months to evaluate disease progression. Seroconversion regarding aPL reactivity was considered the development of aPL antibodies detected by ELISA in patients which were negative for aPL before CYP or any other therapy. Positive values were absorbance values higher by 5SD from the mean of 100 normals. At least two measurements of aPL 6 weeks to 4 months apart were demanded before seroconversion could be considered. Kaplan Meier survival analysis and log-rank test were used to compare the two groups in terms of seroconversion.

Results: The group treated with CYP was characterized by higher prevalence of nephritis, central nervous system involvement and vasculitis. CYP therapy was associated with significant decrease of anti-dsDNA responses (mean ±SD); from 86.94±178.87 units before to 19.6±27.6 units after therapy (p = 0.000074) as well as ECLAM scores (mean ±SD); from 5.8±1.6 before to 2.3±1.1 after (p = 0.0001). On the contrary the C3 and C4 levels (mean ±SD) were significantly increased: from 61.8±28.3 before to 87.3±24.8 after therapy (p = 0.0000) and from 15.7±9.5 before to 23.5±12.5 after (p = 0.0000), respectively. However, 22 out of 117 CYP treated vs only 2 out of 203 non CYP treated patients became positive for aPL after instituting the CYP therapy (p<0.05); odds ratio = 23.274 with 95% CI (5.36–101.01). This association remained significant after adjustment by ECLAM at least measurement, prednisolone dose and disease duration.

Conclusions: Although CYP therapy downregulates anti-dsDNA and suppresses SLE activity, it upregulates aPL antibody responses, probably by suppressing regulatory T-cell populations. The latter remains to be further investigated.

056 EVIDENCE FOR INTERACTION OF B2-GLYCOPROTEIN I (B2GPI) WITH CD40 LIGAND (CD40L): THEIR ROLE IN THE PATHOGENESIS OF ANTIPHOSPHOLIPID SYNDROME (APS)

M. P. Sikara, P. G. Vlachoyiannopoulos.Department of Pathophysiology, School of Medicine, National University of Athens, Greece

Background: The antiphospholipid syndrome (APS) is an autoimmune disease characterized by vascular thrombosis and/or recurrent pregnancy loss. It has been suggested that anti-B2GPI antibodies, occurring in the context of APS, activate endothelial cells and platelets by stimulating them to express adhesion molecules and tissue factor (TF) and to secret proinflammatory cytokines. Evidence that anti-B2GPI recognize CD40 was shown but the molecular events operating in this recognition are unknown.

Aim: To study the interaction between anti-B2GPI/B2GPI complex and the CD40-CD40L pathway, at molecular level, in platelet protein extracts.

Materials and methods: Immunoprecipitation: Platelets from healthy donors preincubated with monomeric B2GPI and activated with ionophore, were immunoprecipitated (IP) by anti-CD40 and anti-B2GPI antibodies. The precipitated proteins were electrophoresed in a 10% polyacrylamide gel under non-reducing conditions. Western Blot (WB) analysis with anti-B2GPI and anti-CD40 respectively, was subsequently performed. In addition, IP with anti-CD40L followed by WB with anti-B2GPI and IP with anti-B2GPI followed by WB with anti-CD40L were also performed. In vitro binding assays: Binding of biotinylated-B2GPI to immobilized CD40L and b-lactoglobulin as irrelative protein (negative control) was performed using microtitre plates. The wells were coated with 50 μl of CD40L and b-lactoglobulin (5 μg/ml) and incubated overnight at 4 C. The plates were washed and blocked with 4% BSA-PBS. 50 μl of various concentrations (0-10 μg/ml) of biotinylated B2GPI was added and incubated overnight at 4°C. After washing, streptavidine-peroxidase was added. Finally, OPD substrate solution was added and the optical density was measured at 450 nm.

Results: The IP experiments with anti-CD40 and anti-B2GPI did not reveal any specific interaction between CD40 and B2GPI, suggesting probably that CD40 is not associated with B2GPI. IP with anti-CD40L precipitates a protein band of 47 kDa which is recognized in WB by anti-B2GPI and IP with anti-B2GPI precipitates another protein band of 50KDa which is recognized in WB by anti-CD40L. These protein bands were absent in immunoprecipitates obtained by normal IgG (negative control experiment). In addition, electrophoresis of the antibodies used in IP (anti-CD40L and anti-B2GPI) confirmed that the above two bands did not represent immunoglobulin heavy chains. The in vitro binding assay demonstrates that B2GPI can bind directly to CD40L in comparison with the significantly reduced ability to bind to another irrelative protein that is coated on the plate.

Conclusion: Anti-B2GPI precipitates CD40L while anti-CD40L precipitates B2GPI from whole platelet protein extract preincubated with monomeric B2GPI. This result, in combination with the in vitro ability of B2GPI to bind to CD40L, indicates a possible interaction between B2GPI and CD40L in platelets, which may explain platelet activation of APS.

057 POOR SEROLOGICAL RESPONSE UPON INFLUENZA VACCINATION IN PATIENTS WITH RHEUMATOID ARTHRITIS TREATED WITH RITUXIMAB

L. B. S. Gelinck1, Y. K. O. Teng2, G. F. Rimmelzwaan1, B. J. F. van den Bemt1, F. P. Kroon1, J. M. van Laar2.1Dept. of Infectious Diseases, Leiden University Medical Center, The Netherlands; 2Dept. of Rheumatology, Leiden University Medical Center, The Netherlands

Objectives: Annual influenza vaccination is recommended for immunocompromised patients at the beginning of each winter season, under the assumption that it will provide protection against the circulating viral strains during the influenza season12 We examined the impact of rituximab on influenza vaccination outcomes in rheumatoid arthritis (RA) patients.

Methods: Four RA patients treated with Rituximab, 25 RA patients treated with tumour necrosis factor alpha (TNF)-blocking agents, and 20 healthy controls, were vaccinated with a trivalent influenza vaccine (fig 1). For B-cell counts blood samples were obtained at baseline and at days 28, 84 and 168 after starting rituximab treatment. Absolute B-lymphocytes were detected by flowcytometry using TruCOUNT tubes with a mixture of monoclonal antibodies against CD45, CD3 and CD19. Haemagglutination inhibition (HI) titres were measured (in duplo) just before vaccination and 28 days later in order to determine the response upon vaccination.

Results: The influenza vaccine was administered between 87–140 days after the first rituximab infusion. B-cells were completely depleted (<1×106 cells/L) in all four patients at day 84 after the first rituximab infusion. Significantly lower postvaccination titres were found in the RA patients treated with rituximab, compared to both control groups (fig 1). Postvaccination protection rates were also significantly lower in the rituximab group (25–50%) compared to both control groups (84% for all three antigens in the RA group and 85–100% in healthy controls, p<0.05). No major side effects were observed after vaccination nor any effect on disease activity.

Conclusions: Rituximab impairs the ability to respond adequately to influenza vaccination. This is the first study to demonstrate significantly lower postvaccination titres and protection rates, for all three influenza vaccine strains, in RA patients treated with rituximab, compared to a group of RA patients treated with anti-TNF and a group of healthy controls. In conclusion, our findings suggest that influenza vaccination, although not completely ineffective, will most likely not sufficiently protect rituximab treated RA patients from influenza infection during the whole winter season.

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058 BASIS FOR A NOVEL PHOTODYNAMIC THERAPY OF RHEUMATOID ARTHRITIS: POLYMERIC PHOTOSENSITIZER PRODRUGS ACTIVATED BY THROMBIN AND UROKINASE IN ARTHRITIC SYNOVIAL TISSUES

N. Busso1, D. Gabriel2, M. A. Campo2, V. Chobaz-Péclat1, R. Gurny2, A. So1, N. Lange2.1CHUV-Laboratoire de Rhumatologie, Lausanne, Switzerland; 2Section of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland

Background: Photodynamic therapy (PDT) of arthritis requires a selective accumulation of photosensitizers (PS) in the diseased synovium following administration. Subsequent activation with light then leads to selective destruction of the tissue making it an attractive alternative to radioactive synovectomy. We have previously shown that PS prodrugs, such as 5-aminolevulic acid hexylester, selectively induce porphyrins in disease-associated tissues due to increased enzymatic activities in the inflamed synovium.1 Due to their increased levels in arthritic joints, thrombin and urokinase might represent another potential target to locally activate PS.23 Therefore, we have conceived two novel polymeric photosensitizer prodrugs (PS-T and PS-U) that only become activated when digested by these target enzymes.

Purpose: The present study was designed to determine the optimal conditions for the specific activation of these photosensitizer prodrugs in diseased synovia, thus leading to selective photodestruction in arthritic tissue.

Material and methods: Specific activation of PS-T and PS-U by the target enzyme was shown by fluorimetric HPLC analysis. Then, the phototoxicity of activated or non-activated PS-T (from 0.5 µM to 10 µM) was tested on synovial cells from rheumatoid arthritis patients. Furthermore, the optimized PS prodrugs were injected systemically into arthritic mice and the biodistribution was studied by fluorescence imaging, HPLC analysis and confocal fluorescence microscopy.

Results: HPLC analysis revealed that both PS prodrugs become specifically activated when incubated with thrombin or urokinase, respectively, showing the expected fragments of the released PS. No activation was seen without the presence of the enzyme. PDT of synoviocytes using activated PS prodrugs led to growth inhibition in a dose-dependent manner. Finally, fluorescence imaging and confocal fluorescence microscopy showed the selective accumulation of the activated PS in the target tissue.

Conclusion: PS-T and PS-U become selectively activated in arthritic synovial cells and tissues and lead to cell death in vitro.

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059 PAR2 UP-REGULATES CCL19 EXPRESSION AND INDUCES CELL ACTIVATION

M. Karababa1, M. Frasnelli1, J-YChatton2, A. Glück3, B. Cenni3, J. Hamilton4, A. So1, N. Busso1.1Laboratoire de Rhumatologie, CHUV; 2Cellular Imaging Facility, Lausanne, Switzerland; 3Novartis, Basel, Switzerland; 4University of California at San Francisco, USA

Proteinase-Activated Receptors (PARs) constitute a recently described family of seven transmembrane G protein-coupled receptors that are activated by proteolysis. The mechanism of PARs activation is common for all members (PAR1-4). Serine proteases cleave at specific sites within the extracellular N-terminus to unmask a tethered ligand domain that interacts with its receptor in the extracellular loop II to initiate signalling. All PARs, with the exception of PAR3, can be activated by specific synthetic peptides that mimick the specific tethered ligand sequence allowing investigation of each PAR. However little is known about the function of the different PARs, which have been shown to be involved in pro- and anti-inflammatory responses. Therefore mice deficient for PAR1, PAR2, PAR3 and PAR4 were used in a Antigen-Induced Arthritis (AIA) model using methylated-BSA (mBSA) as antigen to evaluate the role of the different PARs in the severity of this autoimmune inflammatory disease. Only PAR2 deficient mice showed a significant reduced severity of arthritis: 1/ the clinical scoring of the knee, 2/ the concentration of anti-mBSA IgG, 3/ the proliferation of T cells in response to mBSA are reduced in a PAR2−/− mice compared to the PAR2+/+ mice. These data suggest that PAR2 is important in the immune response. To investigate the mechanism of PAR2 activation, a genome wide approach was used to determine genes regulated by PAR2 activation in murine synoviocytes. CCL19; encoding a chemokine involved in the migration of immune cells to lymphoid organ and Dendritic Cells (DC) activation, appeared as the most up-regulated gene during PAR2 activation, which could therefore explain the modulation of immune system through PAR2 activation. Then, the function of PAR2 was investigated in human DC and showed that PAR2 activation induced functional DC maturation: 1/ PAR2 activation increased specific cell surface markers expression (molecules of co-stimulation CD80 and CD86), 2/ T cell proliferation via PAR2-induced DC was comparable to LPS-induced DC in a mixed lymphocyte reaction. Moreover activation of PAR2 induces CCL19 up-regulation in human DC. Taken together these data suggest that PAR2 activation could be involved in the initiation of adaptive immune response.

060 TGF-BETA ENHANCES EXPRESSION OF MATRIX METALLOPROTEINASES IN MESENCHYMAL STROMAL CELLS INDEPENDENT OF THEIR ATTACHMENT TO ECM PROTEINS

K. Warstat, G. Klein, W. K. Aicher.University of Tübingen, Germany

Recent work showed that mature fibroblasts attached to laminin (LM) show significant MMP-3 expression upon induction with TGF-β. As mesenchymal stromal cells (MSC) attach to different proteins in situ, we investigated MMP expression patterns in MSC after attachment to proteins and TGF-β stimulation.

Materials and methods: Fibroblasts were isolated from the synovial membranes of RA and OA patients. MSC were isolated from bone marrow of patients undergoing endoprosthetic surgery. Fibroblasts and MSC were seeded into polystyrol-flasks coated with different proteins and peptides (BSA, LM-111, LM-211/221, peptide from laminin alpha-4 chain, LM-511) and treated with 10 ng/mL rhTGF-β. Untreated cells served as controls. After 24 h of induction, cells were harvested, RNA was isolated and analysed by quantitative RT-PCR. Matrix metalloproteinases were detected in supernatants of cells by Luminex® cytokine multiplex assay (CMA). Expression of integrins was examined by immunofluorescence using monoclonal antibodies 4B4 and G0H3 for β1 integrin and α6 integrin, respectively.

Results: In fibroblasts grown on LM-111 coated flasks, mRNA levels for MMP-1,-3,-9 and -13 were weakly enhanced. Stimulation of fibroblasts with TGF-β in uncoated flasks resulted in a significant increase of TIMP-3 (4.98 fold ±2.62, p<0.02, n = 6), MMP-3 (4.84 fold ±2.75, p⩽0.0006, n = 10) and MMP-10 mRNA (3.25 fold ±3.09, p<0.04, n = 14). In CMA, after stimulation with TGF-β, enhanced protein levels of MMP-3 (untreated cells: 9.83±10.65 ng/mL, n = 7, stimulation with TGF-β: 39.87±35.47 ng/mL, p⩽0.05, n = 7) and MMP-10 (untreated cells: 2.6±1.74 ng/mL, n = 7, treatment with TGF-β: 31.36±27.4 ng/mL, p<0.02, n = 7) were detected. Attachment to LM-111 and costimulation with TGF-β further enhanced expression of MMP-3 (12.35 fold ±8.04, p<0.00002, n = 16) and MMP-10 mRNA (11.69 fold ±20.0, p<0.05, n = 14) significantly. Similar results were obtained by CMA (MMP-3: 62.76±42.5 ng/mL, p<0.01, n = 7 and MMP-10: 54.27±44.36 ng/mL, p<0.02, n = 7). In MSC, upon stimulation with TGF-β, a significant upregulation of MMP-10 mRNA (10.25 fold ±12.6, p<0.05, n = 9) and MMP-13 (14.33 fold ±11.93, p<0,01, n = 8) was detected. In contrast to our findings on fibroblasts, adhesion of MSC to LM-111 or any coating investigated had no effect on expression of MMPs. In fibroblasts, immunofluorescence data show that the LM-111 receptor α6 integrin and β1 integrin is highly expressed and slightly upregulated by TGF-β. In contrast, MSC seem to show a lower staining using monoclonal antibody G0H3 for the detection of α6 integrin.

Conclusion: Fibroblasts attach to LM-111 via α6 β1 integrin. Costimulation by LM-111 plus TGF-β leads to a significant increase in MMP-3 and MMP-10 mRNA. MSC seem to express low amounts of α6 integrin. Attachment of these cells to LM-111 does not affect their expression of MMPs or TIMPs, but they upregulate MMP-10 and MMP-13 when exposed to TGF-β. We conclude that MSC differ significantly from synovial fibroblasts with respect to the regulation of MMPs upon attachment to extracellular matrix proteins.

The project was supported by DFG grants Ai16/10-3 and Ai16/14-1.

061 IN HLA-B27+ RATS THE DEVELOPMENT OF INFLAMMATORY BOWEL DISEASE (IBD) IS PREVENTED BY TNF-ALPHA BLOCKADE

A. F. Milia1, M. Cinelli1, S. Generini1, M. Manetti2, L. Polidori2, L. Ibba-Manneschi2, M. Matucci-Cerinic1.1Dept. Internal Medicine, Division of Rheumatology; 2Dept. Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy

Aim: Inbred rats transgenic for HLA-B27 and human b2microglobulin (HLA-B27/hb2m, TG) spontaneously develop a multisystemic disease resembling human IBD and spondyloarthropathy (SpA). TNFa has a crucial role in sustaining chronic inflammation and SpA. The aim of this study was to assess the effect of TNFa blockade on spontaneous IBD in HLAB27/hb2m TG rats.

Materials and methods: Twelve TG rats, 9 weeks old, received i.p. injection of mAb anti-TNF-alpha (n = 6) or an isotype negative control IgG2a,k (n = 6) (kindly supplied by Centocor, 15 mg/kg once a week), up to the age of 18 weeks. A second group was monitored up to the age of 18 weeks and then randomly assigned, for 9 weeks, to the treatment with mAb anti-TNF-alpha (n = 6) or with IgG2a,k (n = 6). Each rat was weekly weighted and monitored for clinical manifestation of gastrointestinal inflammation (score from 1-normal to 3-water stool). Three non-TG F344 rats were used as control in each set. After sacrifice, the colon was examined for pathological changes and for TNF-R1 (p55) and -R2 (p75) expression, by immunohistochemistry and RT-PCR.

Results: At 11 weeks of age, both IgG2a,k-treated and untreated TG rats presented clinical signs of IBD (diarrhoea, score 3), without differences between the groups. On the contrary, in anti-TNF-alpha-treated TG rats no signs of IBD were detected and the stool score remained 1. In the late-treated group, clinical signs of IBD improved after the first week of treatment and stool score normalised in the following weeks. Histopathological analysis of IgG2a,k-treated TG colon showed relevant signs of inflammation with a loss of epithelial cells and enlarged tubular glands. Prominent inflammatory infiltration was observed in the lamina propria, among the glands and around the glandular crypts reaching the muscularis mucosae and the submucosa. Early treatment with anti-TNF-alpha prevented colon inflammation observed in the IgG-treated TG rats. The late treatment, while improving clinical signs, did not significantly reduce the pathological signs of inflammation. Immunohistochemical analysis showed a weak staining for p55 in the epithelial cells of the colon of IgG-treated TG rats, while was comparable to F344 controls in anti-TNF-alpha treated rats. On the contrary, p75 immunostaining was strongly evident in the IgG-treated TG rats, whereas was absent in the anti-TNF-alpha treated animals, as the F344. These results were confirmed by RT-PCR, that showed a downregulation for p55 and an upregulation for p75 in the inflamed colon of the TG rats. No differences, in the mRNA levels of the two receptors, were observed between early and late treatment with anti-TNF-alpha antibody.

Conclusion: TNF-alpha blockade is effective in preventing the development of inflammation in the early phase of IBD. TNF-R2 seems to substain the inflammatory action of TNF-alpha in the colon of the HLA-B27 TG rats.

062 A QUANTITATIVE AGGREGATIONAL SCORE FOR RHEUMATOID SYNOVIUM—A POTENTIAL BIOMARKER?

F. Humby1, A. Manzo1, S. Oakley1, M. Blades1, I. Portek2, J. Edmonds2, M. Lassere2, B. Kirkham1, C. Pitzalis1.1Department of Rheumatology, GKT School of Medicine, London, UK; 2Department of Rheumatology, St George’s Hospital, Sydney, Australia

Introduction: There is a pressing need for better prognostic markers in RA to identify those at greatest risk of rapid disease progression. As in other inflammatory and/or proliferative conditions, the histopathology of the affected tissue, in the specific case of RA the synovial membrane (SM), may represent the source of good biomarker(s). Histomorphologically RA SM has been classified into an aggregate or diffuse pattern, with an associated specific clinical phenotype.1 Here we report a novel histological scoring system that quantifies both the aggregational tendency and degree of diffuse infiltrate within RA SM, using the technique of digital image analysis.

Methods: We investigated 52 RA patients (disease duration 6 months to 12 years) from an earlier study (the DAMAGE study).2 RA SM obtained arthroscopically from 3 sites within the knee joint: suprapatellar pouch (SPP), cartilage pannus junction (CPJ) and medial peri-meniscal area (MPM). 5 µm thick sections were stained with H&E. Sections were digitized using an image analysis package slaved to a fully motorized microscope. Using a digitizing tablet the boundary of the section was outlined (A) as were areas within the tissue occupied by mononuclear cells (MNCs) in aggregate form (AG). Nuclei were discriminated using colour thresholding. Scores were calculated as follows: 1. aggregate score (AS)  =  the area fraction occupied by MNCs in aggregate form; 2. diffuse score (DS)  =  the area occupied by MNCs in the remainder of the tissue. Initially 150 sections were analysed by a blinded observer who then repeated the measurements following a 2 week interval. 10 random sections were analysed by a second independent observer. Intra-class correlations (ICCs) for each were determined. For each patient sections from 2 (CPJ SPP) or 3 (CPJ, SPP MPM) biopsy sites were examined, depending on tissue available, and mean scores calculated and correlated with CRP.

Results: The ICC for AS and DS were 0.92 and 0.97 respectively for repeated measurements by the same observer and 0.92 and 0.93 for the second observer, demonstrating reliability in measurement. CRP correlated significantly with DS and AS (r2 0.57 and 0.77). The correlation was higher if 3 sites compared to 2 sites were used.

Conclusions: The histological score described is a simple reliable method to assess RA synovium. Its correlation with CRP suggests a good relationship with disease activity. The capacity of SM histomorphology to predict disease outcome is under investigation.

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063 FAILED UPAR-INTEGRIN-ACTIN CYTOSKELETON ASSOCIATION IN SYSTEMIC SCLEROSIS ENDOTHELIAL CELLS RESULTS IN IMPAIRED ANGIOGENESIS

M. Manetti1, F. Margheri2, S. Serratì2, A. F. Milia3, A. Del Rosso3, G. Fibbi2, B. Kahaleh4, M. Matucci-Cerinic3, M. Del Rosso2, L. Ibba-Manneschi1.1Department of Anatomy, Histology and Forensic Medicine; 2Department of Experimental Pathology and Oncology; 3Department of Internal Medicine, Division of Rheumatology, University of Florence, Florence, Italy; 4Medical College of Ohio, Toledo, USA

Aim: The GPI-linked urokinase-type plasminogen activator receptor (uPAR) has an important role in the regulation of the actin cytoskeleton and cell motility in many cell types. uPAR is present in a three-domains full-size (D1D2D3) and a two-domains cleaved form (D2D3). It participates in the angiogenesis process by modulating the adhesive properties of endothelial cells to extracellular matrix (ECM) and ECM degradation. In Systemic Sclerosis microvascular endothelial cells (SSc-MVECs), cell-surface uPAR is truncated by the overexpression of matrix metalloproteinase 12 at the D1-D2 connection, negatively regulating the angiogenesis process. Since integrins are associated with the invasive activity of uPAR in angiogenesis, this study was undertaken to show whether full-size and cleaved uPAR are differentially associated with integrins and actin cytoskeleton. Moreover, we analysed the signalling pathway involving Rac and Cdc42, since it mediates uPAR-dependent cytoskeletal rearrangements and cell motility through integrin engagement-delivered signals to actin cytoskeleton.

Materials and methods: SSc-MVECs and normal MVECs (N-MVECs) were isolated from human skin biopsy samples and studied by confocal laser scanning microscopy and immunoprecipitation to assess the mechanisms of association of cleaved and full-size uPAR with integrins and the actin cytoskeleton. The integrin composition of the MVECs was studied by RT-PCR. Cell migration and capillary morphogenesis were studied on fibrinogen substrates. Involvement of Rac and Cdc42 was evaluated by Western blotting.

Results: Only full-size uPAR showed a connection with actin cytoskeleton in N-MVECs. Such connection was mediated by uPAR-associated αMβ2 and αXβ2 integrins and was absent in SSc-MVECs. The cleaved receptor was not associated with β2 integrins or with actin. β3 integrins were associated with both full-size and cleaved uPAR at focal contacts. The uncoupling of uPAR from β2 integrins in SSc-MVECs impaired the activation of Rac and Cdc42, thus inhibiting their mediation of uPAR-dependent cytoskeletal rearrangements and cell motility. Invasion and capillary morphogenesis on fibrinogen-coated substrates indicated that uPAR ligation by uPA empowers the β23 integrin-dependent invasion of fibrinogen, and that this system is impaired in SSc-MVECs.

Conclusion: uPAR cleavage and the following loss of β2 integrin-mediated uPAR connection with actin cytoskeleton account for reduced angiogenic properties of SSc endothelial cells.

064 ASSOCIATION BETWEEN BIOACTIVE CIRCULATING TUMOR NECROSIS FACTOR LEVEL AND TUMOR NECROSIS FACTOR-308 POLYMORPHISM GENE IN RHEUMATOID ARTHRITIS

H. Marotte, B. Arnaud, J. Diasparra, A. Pachot, B. Mougin, P. Miossec.Unité Mixte Hospices Civils de Lyon-bioMérieux, Hôpital Edouard Herriot, Lyon, France

Objective: TNFalpha-308 polymorphism has been associated with high production of TNFalpha, but this remains controversial. Since bioactive TNFalpha has to be taken into account, we explore the association between this polymorphism and the circulating TNFalpha bioactivity in RA patients.

Patients and methods: 53 RA patients with active RA were enrolled. Genotyping for TNFalpha at position -308 was performed by ELOSA. A functional assay for TNFalpha activity was designed using the ability of RA synoviocytes to produce IL-6 in response to exogenous TNFalpha (5 ng/ml) combined with plasma at 10% in order to increase the sensitivity of synoviocyte response. We have previously demonstrated that IL-6 production by synoviocytes increased in a dose dependent manner with TNFalpha concentrations and correlates with the circulating TNFalpha bioactivity. IL-6 production in 48 hr supernatants and plasma concentrations of TNFalpha were measured by ELISA.

Results: The 53 patients have the main characteristics of RA population—77% of women, mean ± SD age: 47.4±10.9 years, disease duration: 10.0±8.2 years, 72% were RF positive, and 70% were Shared Epitope positive. All patients had active disease despite methotrexate treatment, as indicated by a mean DAS28 score of 5.1. The distribution of TNFalpha -308 genotypes was as follows: G/G n = 36 (67.9%); A/G, n = 15 (28.3%); A/A n = 2 (3.8%). According to the TNFalpha -308 genotype, levels of circulating TNFalpha bioactivity were higher for combined A/A or A/G genotypes (44.4±23.3 ng/ml vs. 27.4±20.9 ng/ml; p = 0.05) than the common G/G genotype. Level TNFalpha by ELISA was 6.47±9.75 pg/ml for the G/G genotype and 5.00±6.40 pg/ml (NS) for A/A or A/G genotypes. No association was observed between levels of circulating TNFalpha bioactivity and the SE status. No correlation was observed between ESR, CRP, protein TNFalpha level, and circulating TNFalpha bioactivity.

Conclusion: These data suggest an association between the functional TNFalpha level and the TNFalpha -308 polymorphism. They are in line with a genetic contribution to circulating active TNFalpha.

065 IL-17A AND IL-17F INDUCES SYNOVIOLIN EXPRESSION IN RA FIBROBLAST-LIKE SYNOVIOCYTES: DIFFERENTIAL EFFECTS ON MAPK, AP-1 AND NFKAPPAB ACTIVATION

M-LToh, Y. Zhou, H. Marotte, P. Miossec.Department of Immunology and Rheumatology, Mixed Unit Civil Hospital of Lyon-Biomerieux, Edouard Herriot Hospital, Lyon, France

Objective: Synoviolin, a novel E3 ubiquitin ligase, is implicated in synovial hyperplasia and murine arthritis. Although IL-17A has an established role in RA pathogenesis, the role of IL-17A in comparison with IL-17F and their regulation of synoviolin and intracellular signalling pathways is not known.

Methods: Synoviolin expression was analysed by real-time RT-PCR. p65, p50 NFkappaB and c-fos, c-jun AP-1 activation were measured by Trans AM Transcription Factor Assay Kits and real-time RT-PCR. TRAF6, MEKK3, Myd88 and MAPK were analysed by real-time RT-PCR, Western blot. RA FLS were treated with IL-17A or IL-17F.

Results: In RA FLS, IL-17A or IL-17F induced sustained synoviolin expression over 24 h. IL-17A induced stronger synoviolin expression compared to IL-17F. IL-17A or IL-1 were more potent inducers of synoviolin followed by TNF and IL-17F. Neutralising anti-IL-17R and anti-IL-17RC Abs reduced IL-17-induced synoviolin expression. In RA FLS, IL-17A and IL-17F activated common upstream signalling molecules TRAF6, MEKK3 but not Myd88. IL-17A and IL-17F equipotently induced JNK and ERK MAPK activation. In comparison with IL-17A, IL-17F induced more potent p38 MAPK and c-fos AP-1 activation. In contrast to IL-17F, IL-17A induced more robust p65 and p50 NFkappaB activation.

Conclusion: IL-17 may play a role in RA FLS proliferation and dysregulated cell growth through synoviolin regulation. IL-17A has more potent effects on synoviolin expression and NFkappaB activation, compared to IL-17F on p38 MAPK and c-fos AP-1. These results support underlying molecular mechanisms for both shared and potentially differential biological effects of IL-17A and IL-17F in RA FLS.

066 TRAIL INDUCES RA SYNOVIAL FIBROBLAST CELL DEATH MEDIATED BY CASPASES AND REGULATED BY THE AKT PATHWAY

R. Audo1, M. Hahne1, B. Combe2, J. Morel2.1Institute de Génétique Moléculaire de Montpellier, CNRS UMR5535, 34293 Montpellier; 2Department of Immuno-rheumatology, CHU Lapeyronie, 34295 Montpellier, France

Rational: A hallmark of rheumatoid arthritis (RA) is the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLSs), and the RAFLS have been proposed as a therapeutic target. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has been described as a pro-apoptotic factor on RAFLSs and, therefore, suggested as a potential drug. We have previously showed that exposure to TRAIL induces apoptosis only in a portion of RAFLSs. In the surviving cells, TRAIL induced RAFLS proliferation and complicating then the proposed strategy to use TRAIL in the treatment of RA. To evaluate possibilities to overcome TRAIL resistance in RAFLS, we study the intracellular pathways implicated in TRAIL-induced signalling.

Material and methods: We examined the implication of caspases in TRAIL induced apoptosis. RA FLS were stimulated with TRAIL and pro-caspases cleavage was assessed by western blot. We evaluated the participation of caspases activation but also ERK1/, p38 MAP Kinase and Akt in the regulation of TRAIL-induced apoptosis and proliferation. RAFLS were pre-treated for an hour with specific chemical inhibitors and cultured with TRAIL for 24 hours. Apoptosis was then analyzed by flow cytometry on basis of the annexin V-FITC binding and TOPRO-3 up-take. Proliferation was measured using incorporation of tritiated [3H] thymidine.

Results: TRAIL-induced apoptosis in RAFLS is mediated by caspase 8 and 3, as their pro-forms were cleaved after TRAIL treatment and their inhibitions reduced TRAIL induced cell death, while proliferation was increased. A pan-caspases inhibitor (z-VAD-FMK) almost completely blocked TRAIL-induced apoptosis, but also blocked TRAIL-induced proliferation. Inhibition of MAP or PI3 kinases decreased significantly TRAIL-induced proliferation of RAFLS. More importantly, Akt inhibitors not only abrogated TRAIL-mediated RAFLS proliferation, but also increased cell death induced by TRAIL. Because MAPK and Akt pathways are involved in TRAIL induced proliferation, we also tested the participation of the caspases on MAPK and Akt pathways activation after TRAIL treatment. This hypothesis was not confirmed since inhibition with pan caspase inhibitor did not have any effect on TRAIL-induced MAP and PI3 kinases activation. Finally, TRAIL-induced appears not to be mediated by IL-1β since no IL-1β production was detected by ELISA following TRAIL stimulation.

Conclusion: Inhibition of PI3 kinase/Akt signalling pathway abrogates TRAIL-mediated RA FLS proliferation and increases RAFLS susceptibility to TRAIL-induced apoptotis. Taken together, the use of Akt inhibitors in combination with TRAIL might be a useful strategy, in order to induce RAFLS cell death and to control synovial hyperplasia.

067 DIFFERENTIAL ROLE OF TLR2 AND TLR4 IN EXPRESSION OF ARTHRITIS IN IL-1 RECEPTOR ANTAGONIST DEFICIENT MICE MEDIATED BY SKEWED IL-23-IL-17 AXIS

S. Abdollahi-Roodsaz1, L. A. B. Joosten1, M. I. Koenders1, I. DevesaGiner1, M. F. Roelofs1, T. R. D. J. Radstake1, M. J. H. Nicklin2, F. Ribeiro Dias3, W. B. van den Berg1.1Rheumatology Research & Advanced Therapeutics, Radboud University, Medical Centre, Nijmegen, The Netherlands; 2University of Sheffield, UK; 3Universidade Federal de Goiás, Goiânia, Brazil

Toll-like receptors (TLRs) activate innate and adaptive immune systems through recognition of pathogen-associated molecular patterns and several endogenous ligands produced upon tissue damage and stress during ongoing disease. Since both microbial and host-derived TLR ligands have repeatedly been found in arthritic joints, these receptors may contribute to the initiation as well as the chronic progression of rheumatoid arthritis.

We studied the involvement of TLR2 and TLR4 activation in initiation and progression of arthritis using interleukin-1 receptor antagonist deficient (IL-1Ra−/−) mice, which spontaneously develop an autoimmune T-cell mediated arthritis from the age of 5 weeks.

Spontaneous onset of arthritis in IL-1Ra−/− mice was dependent on TLR activation by microbial flora, as germ-free mice showed absolutely no signs of arthritis during 20 weeks of follow-up. To investigate the contribution of distinct TLRs in the progression of arthritis, we generated IL-1Ra−/− mice also lacking TLR2 or TLR4 genes. These TLRs differentially contributed to disease expression, since IL-1Ra−/− TLR2−/− mice developed much more progressive and destructive arthritis compared to IL-Ra−/− TLR2+/+ littermates. Interestingly, TLR4−/− IL-1Ra−/− mice showed a clear suppression of clinical and histological characteristics of arthritis.

IL-17 has been described to play a crucial role in IL-1Ra−/− arthritis and TLRs are known as main inducers of IL-23, a prominent cytokine involved in Th17 cell survival.

Therefore, we studied the effects of TLR gene deficiency on development of Th17 cells and production of IL-23 and IL-17 to clarify the mechanism by which TLR2 and TLR4 differentially regulated the disease expression. Stimulation of bone-marrow-derived dendritic cells (BMDCs) from wild type mice with 100 ng/ml PamCys or LPS (TLR2 and TLR4 ligands, respectively) for 24 hours resulted in similar IL-23 production. On the other hand, IL-1Ra−/− BMDCs produced less IL-23 than wild type DCs upon TLR2 stimulation and more IL-23 than wild type DCs upon TLR4 stimulation.

To study the effect of different IL-23 levels on IL-17 production by IL-1Ra−/− T cells, we stimulated total splenocytes from wild type and IL-1Ra−/− mice (non-diseased) with anti-CD3 antibodies in combination with TLR2 or TLR4 ligands for 72 hours. In line with IL-23 concentrations, IL-1Ra−/− T cells produced lower amounts of IL-17 when cultured with TLR2-activated APCs and higher amounts of IL-17 when cultured with TLR4-stimulated APCs. FACS analysis of Th17 (CD4+/IL-17+) cells in both spleen and draining lymph nodes revealed a reduction of approximately 70% in IL-1Ra−/− TLR4−/− mice compared to IL-1Ra−/−TLR4+/+ littermates. Furthermore, specific CD3/CD28 stimulation of non-adherent splenocytes for 96 hours confirmed lower IL-17 production in IL-1Ra−/− TLR4−/− compared to IL-1Ra−/− TLR4+/+ mice. These findings suggest important roles for TLR2 and TLR4 in regulating Th17 development and thereby explain more severe arthritis in TLR2−/− and less severe disease in TLR4−/− mice.

068 INHIBITION OF RHEUMATOID SYNOVITIS BY TREATMENT WITH LIVER X RECEPTOR AGONIST IN COLLAGEN-INDUCED ARTHRITIS MODEL

M-CPark, J-HKim.Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea

Objective: In addition to the regulation of cholesterol homeostasis, liver X receptors (LXRs) have recently been characterized as regulators of macrophage inflammatory pathways, and synthetic LXR agonists such as GW3965 inhibit the macrophage response to bacterial pathogens and antagonize the induction of a number of proinflammatory genes. The aim of this study was to investigate the effects of GW3965 on the inflammatory response in mice with collagen-induced arthritis (CIA).

Methods: CIA was induced in DBA/1J mice by an intradermal injection of 100 microliter of an emulsion of bovine type II collagen (CII; 100 microgram) in Freund’s complete adjuvant (CFA) at the base of the tail. On day 21, a second injection of CII in CFA was administered. Beginning on day 25 (arthritis onset) and continuing until day 35, mice (n = 10 per group) are treated with GW3965 (at 30 mg/kg) or vehicle (10% DMSO at a dosage of 10 mg/kg/day), and GW3965 is orally administered daily to the mice for 7 days. Clinical, radiographic, histopathologic, and laboratory assessments were performed.

Results: Mice immunized with CII in CFA developed erosive arthritis of the hind paws. Macroscopic evidence of CIA first appeared as periarticular erythema and edema of the hind paws. The incidence of CIA was 100% by day 27 in the CII-challenged mice, and the severity progressed over the 35-day study period. Histopathologic features of CIA included erosion of cartilage at the joint margins. GW3965 treatment ameliorated the clinical signs on days 26–35 and improved the histologic findings in the joint and paw. The degree of oxidative and nitrosative damage was significantly reduced in GW3965-treated mice, as indicated by formation of nitrotyrosine, and activation of poly(ADP-ribose) polymerase. Plasma levels of the proinflammatory cytokines tumor necrosis factor-alpha, and interleukin-1beta were also significantly reduced by GW3965 treatment.

Conclusion: These data demonstrate that LXR agonist exerts an anti-inflammatory effect on chronic inflammation and is able to ameliorate the tissue damage associated with CIA.

069 ANALYSES OF IMMUNOPATHOGENIC MECHANISM INVOLVED IN THE INITIATION AND SUSTAINING OF COLLAGEN INDUCED ARTHRITIS

A. Rapp, K. Dalwigk, A. Savitskaya, M. Bonelli, J. S. Smolen, C. Scheinecker.Department of Rheumatology, Internal Medicine III, Medical University of Vienna (MUW), Vienna, Austria

Aim: Collagen induced arthritis (CIA) represents a well established model for human rheumatoid arthritis (RA) and shares a number of pathological, genetic and immunologic features with RA. We therefore set up experiments to further study the immunological background of CIA in order to elucidate pathogenic mechanisms relevant to RA and to determine optimal time-points as well as target cell populations for therapeutic intervention.

Material and methods: For the induction of CIA DBA/1 mice were injected with type II collagen (CII) and boosted after 10–14 days. Upon the onset of arthritis the animals were scored for clinical signs of arthritis including paw swelling and grip strength. Anti-CII antibody levels were determined by ELISA. At early time points (first clinical signs of arthritis) and late time points (> day 70) respectively animals were sacrificed and paraffin sections of the joints of affected animals and control animals were analyzed for histomorphologic signs of cartilage and bone destruction. In addition, joint-draining as well as non-draining lymph nodes (LN) and spleens were harvested, single cell suspension was prepared and analyzed by flow cytometry (FACS).

Results: At the late stage of the disease >90% of animals developed clinical signs of arthritis and serum anti-CII antibodies. Histological analyses revealed extensive synovitis with massive cellular infiltrations as well as articular cartilage and bone destruction. Phenotypic analyses demonstrated no significant differences in the distribution of proportions of T cells, B cells and DC at early time points. Increased proportions of MHC class II+ cells were observed at late time points in draining LN of animals with CIA as compared to non-draining LN or controls. This was mainly due to an increase in CD19+B220+CD40+ B cells and to a lesser extent in CD11c+ dendritic cells (DC). In contrast, proportions of CD3+ T cells were decreased in draining LN of CIA animals as compared to non-draining LN or controls whereas ratios of CD4+ and CD8+ T cells were not affected. No differences were observed concerning the expression of costimulatory molecules on B cells or DC at both, early and late time points.

Conclusion: Our studies suggest substantial changes in the distribution of cells involved in the initiation and/or sustaining of the inflammatory response in CIA. Further experiments will concentrate on functional analysis as well as on the migratory behavior of distinct cell populations in CIA.

070 FRZB, A MOLECULAR BASIS FOR AN INVERSE RELATIONSHIP BETWEEN OSTEOARTHRITIS AND OSTEOPOROSIS

R. J. U. Lories1, J. Peeters1, A. Bakker1, P. Tylzanowski1, I. Derese1, J. Schrooten2, J. T. Thomas3, F. P. Luyten1.1Laboratory for Skeletal Development & Joint Disorders; 2Department of Metallurgy & Materials Engineering, KU Leuven, Belgium; 3Center for Biologics Evaluation and Research, FDA, Rockville USA

Introduction: Different Wnt family members and receptors play a role in skeletal development and homeostasis. FRZB is a soluble Wnt antagonist, originally identified from a chondro- and osteogenic extract of articular cartilage. Polymorphisms in the FRZB gene have been associated with hip osteoarthritis. An inverse relationship between hip osteoporosis and osteoarthritis has been suggested. Frzb knockout mice show accelerated cartilage loss in induced models of osteoarthritis. Here, we studied mechanisms of cartilage damage, including bone and biomechanical properties in Frzb knockout mice.

Methods: Frzb knockout mice were generated by targeted inactivation of exon1 using a Cre/LoxP strategy. Femoral and tibial bone density was studied by in vivo and ex vivo dual-energy X-ray absorptiometry (DEXA), peripheral quantitative computerized tomography (pQCT), microCT and histology at different ages. Cortical bone stiffness was estimated by compression of the ulnae. In addition, mechanical loading-induced bone adaptation was studied by compression of the ulnae of 17 week old mice, followed by microCT analysis at a resolution of 5 μm. Subchondral bone properties were studied with histomorphometry, pQCT and microCT. Active Wnt signalling in the articular cartilage was studied in normal and mBSA, collagenase and papain induced arthritis using beta-catenin immunohistochemistry. Gene expression of tissue destructive enzymes in the cartilage was studied by real-time PCR. Matrix metalloproteinase-3 activity was tested in vitro.

Results: Gene-targeted deletion of Frzb in mice increases articular cartilage loss in models of arthritis triggered by instability, enzymatic injury or inflammation. Cartilage damage in Frzb knockout mice is associated with increased Wnt signalling, and Matrix Metalloproteinase-3 expression and activity. In addition, the Frzb knockout mice have an increased cortical bone thickness and density, resulting in stiffer bones as demonstrated by a different stress-strain relationship in Frzb knockout mice. Moreover, the periosteal anabolic response to mechanical loading is significantly greater in Frzb knockout mice than in wild-type mice.

Conclusion: The genetic association between osteoarthritis and FRZB polymorphisms is corroborated by increased cartilage proteoglycan loss in Frzb null mice in three different models of arthritis. Loss of Frzb may contribute to cartilage damage by increased expression and activity of MMPs, in both a Wnt dependent and independent manner. FRZB deficiency also results in thicker cortical bone with increased stiffness and higher cortical appositional bone formation after loading. This may contribute to the development of osteoarthritis by producing increased strain on the articular cartilage during normal locomotion. In addition, increased cortical density of long bones in Frzb knockout mice supports our earlier observation that polymorphisms in the human FRZB gene are differentially associated with hip osteoarthritis and osteoporotic hip fractures. Therefore, the role of FRZB in cartilage and bone biology may provide a mechanistic basis for the longstanding clinical observation that osteoarthritis and osteoporosis show an inverse relationship.

071 TYPE I INTERFERONS ARE INVOLVED IN THE DEVELOPMENT OF UVB-INDUCED INFLAMMATORY SKIN LESIONS IN SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) PATIENTS

E. Reefman1, H. Kuiper2, P. C. Limburg1,2, C. G. M. Kallenberg1, M. Bijl1.1Departments of Rheumatology & Clinical Immunology; 2Pathology & Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

Objective: To investigate the involvement of type I interferons and endothelial cell adhesion molecules in the development of UVB-induced systemic lupus erythematosus skin lesions.

Methods: Nineteen SLE patients and 13 controls were irradiated with 2 Minimal Erythemal Doses (MED) of UVB. Subsequently, skin biopsies were analyzed (immuno)histologically, over 10 days, for expression of IFNalpha-induced MxA, numbers of plasmacytoid dendritic cells (pDC), and expression of endothelial cell adhesion molecules, namely, E-selectin, ICAM-1, and L-selectin ligand. Additionally, MxA expression was compared to its expression in 9 established cutaneous LE (CLE) lesions of SLE patients.

Results: Before irradiation IFNalpha-induced MxA was expressed at significantly higher levels in non-lesional skin of SLE patients compared to healthy controls. In patients developing infiltrates upon UVB irradiation, MxA expression increased further, reaching expression levels similar to or exceeding levels in CLE-skin lesions. In these patients, MxA expression was sustained up to day 10, in contrast to patients not developing infiltrates in whom expression decreased. No noteworthy numbers of plasmacytoid dendritic cells (pDC) were detected in non-irradiated skin or at any time after UVB exposure in SLE patients or controls. MxA expression correlated with influx of T-cells and monocytes/macrophages, and with expression of E-selectin and ICAM-1.

Conclusion: Development of UVB-induced SLE skin lesions involves a skewing towards production of Th1-associated cytokines, like IFNalpha. In turn, this may lead to upregulation.

072 HLA GENES ANALYSIS IN A FRENCH SCLERODERMA COHORT: A COMMON AMINO ACID SEQUENCE

J. M. Rak1, P. Pagni1, Y. Allanore2, A. Kahan2, K. Tiev3, J. Cabane3, D. Farge-Bancel4, S. Parlier4, D. Launay5, E. Hachulla5, J. R. Harlé6, R. Didelot7, N. Pero1, D. Reviron8, M. Martin1, J. Roudier1,9, N. C. Lambert1.1INSERM U639, Marseille; 2Service de Rhumatologie, Hôpital Cochin, Paris; 3Service de Médecine Interne, Hôpital St Antoine, Paris; 4Service de Médecine Interne et Pathologie Vasculaire, Hôpital St Louis, Paris; 5Hôpital Claude Huriez, Lille; 6Service de Médecine Interne, Hôpital La Conception, Marseille; 7Centre d’Examen de Santé Assurance Maladie; 8Etablissement Français du Sang; 9Service de Rhumatologie, Hôpital La Conception, Marseille, France

Background: Scleroderma (SSc) is a rare autoimmune disease characterized by excessive collagen deposition in the skin and internal organs and microvascular injury. As many autoimmune diseases, SSc is associated with particular HLA class II alleles. The most consistently described HLA-DR associations with diffuse SSc have been DR11 with Caucasian patients and DR15 with Asian patients.

Objectives: We propose to analyse HLA-DR allele frequencies on a French SSc cohort compared to a control population. We also test the hypothesis that a common motif is present on HLA-DR susceptibility alleles to SSc.

Methods: In collaboration with five French hospitals, we recruited 192 patients with SSc, including 38 men and 154 women. Clinical data comprise history of pregnancies, date of diagnosis, type of SSc (limited or diffuse) and type of autoantibodies. DNA extracted from whole blood samples was HLA typed for HLA-DRB alleles. The current analysis is made on 63 women with limited SSc and 78 women with diffuse SSc and compared with 137 healthy matched controls recruited at the Centre d’Examen de Santé de l’Assurance Maladie, Marseille.

Results: Our results confirm an increase of DRB1*15, HLA DRB1*11 and DRB1*08 alleles in patients with diffuse disease compared to controls. Among patients with limited disease, only DRB1*08 is increased.

Conclusion: HLA-DRB1*11 and some DRB1*08 alleles, which are associated with diffuse SSc encode a common “FLEDR” sequence (amino acid position 67–71) which is present on their DRβ1 chains. Moreover, DRB5 alleles which are coexpressed with HLA-DRB1*15 alleles, associated to SSc, also encode this sequence. As rheumatoid arthritis patients have a particular shared amino acid sequence associated with the disease, SSc patients have a common amino acid sequence on the third hypervariable region of DRβ chains. Results are still under investigations to distinguish other susceptibility motifs in SSc.

073 PROSTAGLANDIN E2 IS AN ESSENTIAL MODULATOR FOR TNF-α-INDUCED EXPRESSION OF PRO-INFLAMMATORY/PRO-DESTRUCTIVE MEDIATORS IN RHEUMATOID SYNOVIAL FIBROBLASTS: REGULATION VIA THE PROSTAGLANDIN E2-RECEPTOR EP2 THROUGH A CAMP-INDEPENDENT PATHWAY

A. Jansen1,2, F. Kojima1, M. Kapoor1, L. Yang1, R. W. Kinne2, L. J. Crofford1, E. Kunisch2.1University of Kentucky, Lexington, USA; 2Experimental Rheumatology Unit, Friedrich Schiller University Jena, Germany

Aim: Tumor necrosis factor (TNF)-α-stimulated synovial fibroblasts (SFB) produce cytokines (e.g. Interleukin [IL]-6) and matrix-degrading proteases (e.g. matrix metalloproteinase [MMP]-1) and strongly contribute to joint inflammation/destruction in rheumatoid arthritis (RA). Prostaglandin E2 (PGE2) is one of the major TNF-α-induced products and acts via the receptor subtypes EP1-4. In RA SFB, predominantly EP2 and EP4 are expressed. Both receptors couple to Gs and increase intracellular cAMP formation. The present study analyzes the regulation of PGE2-signaling via the EP2-receptor and the involvement of the key downstream signaling molecule cAMP.

Materials and methods: Synovial tissues were obtained from 3 patients fulfilling the ACR criteria for RA. Anti-CD14 negatively purified SFB were cultured in DMEM containing 10% FCS. For the experiments, fourth to sixth passage cells were stimulated for 24 hrs with TNF-α (10 ng/ml) in serum-free medium. Levels of mRNA expression of IL-6, IL-8, MMP-1, and macrophage chemoattractant protein (MCP)-1 were determined by reverse-transcription polymerase chain reaction. Intracellular cAMP levels were assessed using an enzyme immunoassay kit.

Results: Expression of IL-6, IL-8, MMP-1, and MCP-1 was upregulated in RA SFB after stimulation with TNF-α. On one hand, TNF-α-induced IL-6 expression was decreased by NS-398 (a selective COX-2 inhibitor) and this effect was reversed by PGE2. On the other hand, TNF-α-induced expression of IL-8, MMP-1, and MCP-1 expression was further enhanced by NS-398, and PGE2 reversed this induction. The regulatory effects of PGE2 were largely mimicked by butaprost (an EP2 receptor agonist), suggesting that the effects of PGE2 may be mediated via EP2 receptor signaling. Levels of cAMP (one of the major downstream signaling molecules of EP2) were elevated in a time-dependent manner after stimulation with TNF-α, exogenous PGE2, or butaprost. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine (DDA) partially inhibited the elevation of cAMP levels following stimulation with either TNF-α or PGE2. However, DDA did not show any effect on the expression levels of IL-6 or MMP-1 in the presence of TNF-α, alone or together with PGE2 or butaprost. The phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) further enhanced elevated levels of cAMP by PGE2. However, IBMX did not enhance the effect of PGE2 on the expression of IL-6 and MMP-1.

Conclusion: TNF-α-induced expression of pro-inflammatory IL-6 and pro-destructive MMP-1 in RA SFB may be modulated by PGE2 via the EP2-receptor through a cAMP-independent pathway. The regulations of pro-inflammatory/pro-destructive mediators by PGE2 may play a pivotal role in pathophysiological events associated with RA.

074 RADIOLOGICAL DAMAGE IN RHEUMATOID ARTHRITIS IS ASSOCIATED WITH IL-6 AND IL-10 GENOTYPES INDEPENDENT ON AUTOANTIBODY PRODUCTION

I. Marinou1, J. Healy1, D. Mewar1, D. J. Moore1, M. C. Dickson2, M. H. Binks2, D. S. Montgomery2, K. Walters1, A. G. Wilson1.1Section of Musculoskeletal Sciences, School of Medicine & Biomedical Sciences, The University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK; 2GlaxoSmithKline R&D, Stevenage, UK

Objectives: The role of genetics in RA susceptibility is clearly established, but less is known of the genetic influences on disease severity. We hypothesised that functional genetic variants associated with cytokine production may influence RA severity. The aim of this study was to seek associations between functional single nucleotide polymorphisms (SNPs) in 3 candidate genes and severity of radiographic damage in RA.

Methods: Radiographic damage was assessed using the modified Larsen score (LS) in a cross-sectional cohort of 965 white Caucasian RA patients all fulfilling the ACR criteria for RA and minimum disease duration of 3 years. The presence of rheumatoid factor (RF) was determined using nephelometry and anti-cyclic citrullinated peptide antibody (anti-CCP) levels were measured using a commercial ELISA (DIASTAT™ Anti-CCP, Axis-Shield, UK). Taqman SNP genotyping assays were designed for functional genetic variants in IL-1, IL-6 and IL-10. The Kruskal-Wallis test was used to compare median LS across different genotypes followed by Cuzick’s test for trend when a gene-dose effect was observed. Pearson’s X2 was used to test for association between genotypes and autoantibody status.

Results:1. Associations of functional SNPs in IL-10 and IL-6 genes with radiographic severity of RA (table 1). Carriers of the IL-10 −592C allele previously associated with lower production of this anti-inflammatory cytokine had more severe radiographic joint damage compared with those carrying only the A allele (p = 0.006). A similar pattern was observed for IL-10 −1082 with patients carrying at least one copy of G allele had increased x-ray damage (p = 0.01). Likewise in carriers of the IL-6 −174G allele previously associated with increased transcriptional activation of this gene, more severe joint destruction was observed (p = 0.005). 2. Functional SNPs in IL-10 and IL-6 are not associated with autoantibody status. We determined if the presence of autoantibody-producing B cells confound the association between genotype and disease severity. No association between genotype and antibody status was observed suggesting that the association with x-ray damage is not confounded by autoantibody status (data not shown). 3. Associations of functional SNPs with RA severity are influenced by RF and CCP status (table 2). Stratification by RF and anti-CCP status showed that the IL-10–592C associations with LS was restricted to either RF-ve (p = 0.002) or anti-CCP-ve patients (p = 0.002). However, the IL-6–174G variant remained significantly associated with radiological damage only in RF+ve (p = 0.01) or anti-CCP+ve (p = 0.004) patients.

Conclusion: The genetic associations of IL-10 and IL-6 with radiographic damage suggest that they may be useful novel biomarkers of RA severity. The interaction between genotype and antibody status in the association with disease severity suggests that genetic variation affecting IL-10 and IL-6 production may be secondary severity factors that act downstream from the effect of autoantibody-producing B-cells.

075 NOVEL FOXP3 STAINING AND MIGRATORY PHENOTYPE OF SYNOVIAL REGULATORY T CELLS IN JUVENILE IDIOPATHIC ARTHRITIS (JIA)

H. Moncrieffe, K. Nistala, L. R. Wedderburn.Rheumatology Unit, UCL Institute of Child Health, University College London, UK

Aim: To characterise regulatory T cells and their migratory phenotype from the synovial fluid of children with JIA.

Introduction: Human regulatory T cells (T reg) have been classically defined as CD4+CD25hi and are present within the joints of children with JIA. We have previously demonstrated that T reg are found at a higher frequency in those patients with a milder, self-remitting form of the disease termed persistent oligoarticular JIA. This is consistent with T regs having a role in control of this disease. The factors which influence the migration of these cells into the joint are not defined. We have previously shown that synovial T cells as a whole express high levels of CCR5 and CXCR3.

Materials and methods: Mononuclear cells from peripheral blood (PBMC) and synovial fluid (SFMC) of patients with JIA were analysed as well as PBMC from control subjects. Cells were analysed for coexpression of FOXP3 and CD25. CD4+CD25hi cells were analysed for expression of a set of chemokine receptors, including CCR5. A standard transwell system was used to assay migration of CD4+ enriched T cells to chemokines including CCL5 (RANTES).

Results: We present a novel staining profile of FOXP3 versus CD25 in SFMC compared to peripheral blood of JIA patients and healthy controls. There is uncoupling of CD25hi staining from FOXP3 in CD4+ SFMC, resulting in a population of FOXP3+CD25low T cells. We show inflammatory chemokine receptors such as CCR5 are present on a greater proportion of CD4+CD25hi cells from the joint than from PMBC of JIA or control subjects. We also demonstrate that FOXP3+ cells from synovial fluid have a significantly higher migration rate towards inflammatory chemokines such as CCL5 than FOXP3+ cells from JIA or control PBMC.

Conclusions: Our data demonstrate for the first time that CD25 expression on synovial T cells, unlike on T cells from peripheral blood, does not wholly correlate with FOXP3. This finding has important implications for the estimate of T reg numbers within synovial fluid using CD25 staining alone. We show that FOXP3+ T regs from the joints of children with arthritis have a highly inflammatory migratory phenotype. We are investigating the effects of this phenotype on the functional regulatory activity of these cells.

HM and LRW are funded by SPARKS UK. KN is a Barbara Ansell ARC research fellow.

076 ASSOCIATION OF VON WILLEBRAND FACTOR SERUM LEVELS AND SUBCLINICAL ATHEROSCLEROSIS IN PATIENTS WITH RHEUMATOID ARTHRITIS

G. G. Ristic1, V. Subota2, T. Lepic3, D. Stanisavljevic4, B. Glisic1, M. Petronijevic1, M. Cirkovic1, D. Z. Stefanovic1.1Department of Rheumatology and Clinical Immunology; 2Institute for Medical Biochemistry; 3Department of Neurology, Military Medical Academy, Belgrade, Serbia; 4Institute for Medical Statistics, Faculty of Medicine, Belgrade, Serbia

Background: Increased plasma level of von Willebrand factor (vWF) is a marker of endothelial damage, an initial step in accelerated atherosclerosis of rheumatoid arthritis (RA).

Objectives: To evaluate the prevalence of subclinical atherosclerosis in low-risk RA patients and to determine its association with vWF serum levels and other laboratory and clinical parameters.

Methods: The subclinical atherosclerosis was assessed by ultrasonography measurements of intima-media thickness (IMT) in the carotid arteries. Study population included 42 non-diabetic, normotensive female patients with RA, but low cardiovascular risk (mean age 45.3±10.0 years) and 32 healthy female control subjects (mean age 45.2±9.7 years) matched regarding age, body mass index, lipid status, and smoking habits. The IMT was evaluated on both sides in the common carotid (CC), bifurcation (BI), and internal carotid (IC) arteries. Mean and maximal (max) IMT were calculated from three measurements at each site. Subclinical atherosclerosis was defined as mean IMT+2SD of the controls at any measured point: >0.791 mm at CC, >1.042 mm at BI, and >0.684 mm at IC. The atherosclerotic plaque was defined as IMT>1.5 mm. Clinical work-up included determination of the disease activity score (DAS 28), physical disability score (mHAQ), and patient’s global assessment. Laboratory analyses included erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), lipid status, and vWF.

Results: RA patients had significantly higher maximal and mean IMT (mm) than healthy controls for all measurements (Max-CC: 0.764±0.148 vs. 0.703±0.100, Mean-CC: 0.671±0.119 vs. 0.621±0.085, Max-BI: 1.055±0.184 vs. 0.941±0.161, Mean-BI: 0.889±0.168 vs. 0.804±0.124; Max IC 0.683±0.108 vs. 0.613±0.093, Mean IC 0.577±0.101 vs. 0.535±0.076. The subclinical atherosclerosis was demonstrated in significantly higher proportion of RA patients than controls (15/42 (35.7%) vs. 1/32 (3.1%), p<0.01). Relation of clinical and laboratory findings and subclinical atherosclerosis is shown in table 1.

Univariant analysis demonstrated significant association of subclinical atherosclerosis in RA patients and the age (P<0.01), smoking (cigarettes/day) (P<0.05), and vWF (P<0.05). Atherosclerotic plaques (IMT>1.5 mm) were found in 12/42 RA patients (28.6%) in contrast to 3/32 controls (9.4%), P<0.05. In subjects with atherosclerotic plaques vWF activity was significantly higher than in those without plaques (123±57 vs. 99±45, P<0.05).

Conclusion: Despite low incidence of traditional risk factors for atherosclerosis RA patients had significantly higher IMT than healthy controls. Association of increased serum levels of vWF and subclinical atherosclerosis in RA patients indicate its potential predictive value for cardiovascular risk assessment.

077 FAS−670 GENE PROMOTER POLYMORPHISM CONFERS SUSCEPTIBILITY TO SYSTEMIC SCLEROSIS

L. Ibba-Manneschi1, V. Liakouli2, A. Pacini1, M. Manetti1, B. Tolusso3, P. Cipriani2, A. Toscano1, S. Guiducci4, M. Cinelli4, C. Fatini5, R. Abbate5, S. Bombardieri6, G. Ferraccioli3, C. M. Montecucco7, G. Valentini8, M. Matucci-Cerinic4, R. Giacomelli2.1Dept. Anatomy, Histology and Forensic Medicine, University of Florence, Florence; 2Dept. Internal Medicine and Public Health, University of L’Aquila, L’Aquila; 3Rheumatology unit, University of Rome “La Cattolica”, Rome; 4Dept. Internal Medicine, Division of Rheumatology; 5Dept. of Medical and Surgical Critical Care, Thrombosis Centre, University of Florence, Florence; 6Rheumatology Unit, University of Pisa, Pisa; 7Rheumatology Unit, University of Pavia, Pavia; 8Rheumatology Unit, Second University of Naples, Naples, Italy

Aim: Systemic sclerosis (SSc) is an autoimmune disease affecting the skin and various internal organs, in which T cell activation and resistance to apoptosis play a pivotal role. Apoptosis is a physiologic process that regulates normal homeostasis and likely contributes to the pathogenesis of autoimmunity by impairing the deletion of autoreactive immune cells. FAS, also known as Apo-1/CD95, is a cell-surface receptor belonging to the TNF receptor superfamily, involved in cell-death signalling. The FAS gene is highly polymorphic, particularly in its promoter region. One of these polymorphisms is caused by a single nucleotide change at position −670 in the enhancer region, and it was recently found to be associated with Rheumatoid Arthritis and Systemic Lupus Erythematosus. This mutation abolishes the binding site of the nuclear transcription element GAS (interferon-Gamma-Activated Sequence) and is likely to alter the expression of the FAS gene. In this study we present preliminary results concerning a multicentric Italian Caucasian population of SSc patients in which both the presence of the FAS−670 polymorphism and its relationship with several clinical items of the disease were assessed.

Materials and methods: We studied 159 SSc patients, 50 with the diffuse (dcSSc) and 109 with the limited (lcSSc) cutaneous form of the disease, and 115 ethnically, age and sex-matched healthy controls. Genomic DNA was extracted from the peripheral blood of each subject and the FAS−670 polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Allelic and genotypic frequencies were compared between patients and controls using a χ2 test. Associations were expressed as odds ratios (OR) with 95% confidence interval (95% CI). A p value < 0.05 was considered statistically significant.

Results: The distribution of genotypes in patients and controls was consistent with Hardy-Weinberg’s equilibrium. A significant difference in genotype distribution for the FAS−670 polymorphism was observed between SSc patients and controls (p = 0.02). With regard to the FAS allelic frequency, we found a significantly higher prevalence of the FAS−670A allele in patients than in controls (p = 0.02). In univariate analysis, by using a recessive model of inheritance (AA vs GA+GG), FAS−670A allele influenced the predisposition to SSc (OR = 2.09, 95% CI: 1.23−3.56, p = 0.006). No significant differences in genotype distribution and allelic frequency were observed between dcSSc and lcSSc patients, although we observed a higher prevalence of the FAS−670A allele in dcSSc patients.

Conclusion: Our findings demonstrate a close association between the FAS−670 polymorphism and the susceptibility to SSc disease in Italian Caucasian patients.

078 INCREASED BAFF SERUM LEVELS IN PATIENTS WITH MYOSITIS

O. Krystufkova1,2, T. Wallerskog2, S. B. Helmers2, V. Malmstrom2, C. Trollmo2, I. E. Lundberg2.1Institute of Rheumatology, Prague, Czech Republic; 2Rheumatology Unit, Department of Medicine, at Karolinska University Hospital, Solna, Karolinska Institutet, Stockholm, Sweden

Background: Autoantibodies are frequently present in patients with myositis. The most frequent myositis specific autoantibody is anti-Jo-1, which is associated with a distinct clinical phenotype including interstitial lung disease (ILD), Raynaud’s phenomenon, arthritis and skin changes. The significance of B cells and mechanisms for autoantibody production in different subsets of myositis are not known. The cytokine BAFF (B-cell activation factor of the TNF family) is crucial for B–cell maturation and survival and also plays a role in T-cell activation and differentiation. High serum levels have been reported in patients with various autoimmune diseases such as Sjögren’s syndrome, SLE and RA. There are today no reports focused on BAFF levels in myositis patients.

Objectives: To investigate if serum levels of BAFF are elevated in patients with myositis and whether they correlate to clinical phenotype and autoantibody profile.

Patients and methods: Sera from 14 patients with polymyositis (PM), 8 with dermatomyositis (DM), 6 with inclusion body myositis (IBM), and 16 matched controls were evaluated for BAFF levels by ELISA. 13 patients had autoantibodies (Jo-1 and/or Ro), of which 9 were anti Jo-1 positive (detected by Innolia Update line blot). Nine patients had ILD. Serum levels of CRP and creatine phosphokinase (CPK) were used as laboratory markers of disease activity.

Results: BAFF levels in serum were elevated in the patients (median: 1.24 ng/ml) when compared to healthy controls (0.8 ng/ml, p = 0.0012). Patients with autoantibodies had significantly higher median levels of BAFF, (2.4 ng/ml) compared to healthy controls (p<0.01) and not significantly higher compared to autoantibody negative patients (1.16 ng/ml). This was also true for patients with ILD (2.7 ng/ml, p<0.01) compared to those without ILD (1.1 ng/ml). There was no difference between PM, DM and IBM patients in BAFF levels (medians: 1.4, 1.5 and 1.1 ng/ml). Patients with high serum levels of BAFF had significantly higher CPK and CRP levels and BAFF correlated weakly with CRP (r = 0.41, p<0.02).

Conclusions: Elevated BAFF levels in sera of myositis patients indicate involvement of BAFF in disease pathogenesis in myositis, at least in a subset of myositis patients with autoantibodies and/or ILD. Thus BAFF could play a role in perpetuating autoimmune mechanisms in these patients.

Supported by EU FP6 project, AutoCure LSHB CT-2006-018661. This research was supported by the European Community’s FP6 funding. This publication reflects only the author’s views. The European Community is not liable for any use that may be made of the information herein.

079 TOLL-LIKE RECEPTOR 9 (TLR-9) AND IL-21 SYNERGISTICALLY PROMOTE B-CELL DIFFERENTIATION TO PLASMA CELLS: IMPLICATIONS FOR PLASMA CELL DIFFERENTIATION IN SLE

E. D. Papadimitraki, N. Goulidaki, C. Choulaki, E. Koutala, I. Kyrmizi, M. Ioannou, I. Tassiulas, D. T. Boumpas.Laboratory of Rheumatology, Clinical Immunology and Allergy, Medical School, University of Crete, Greece

Aim: Excessive production of Interleukin 21 (IL-21), a pleiotropic cytokine with an important role in the differentiation of B-cells into plasma cells, results in lupus-like disease in mice. We have recently reported an increased percentage of TLR-9 expressing memory and plasma cells correlating with the presence of anti-dsDNA antibodies in patients with active SLE. Since the B-cell directed actions of IL-21 are highly dependant upon co-activating stimuli, we sought to explore the combined effects of IL-21 and TLR-9 on B-cell function and to measure serum IL-21 levels in lupus patients.

Materials and methods: Isolated B-cells from healthy donors were stimulated using various combinations of IL-21, IL-6, IL-10 or sCD40L and TLR ligands (ODN 2006, poly I:C, LPS, zymosan) in the presence or absence of hydroxychloroquine and the differentiation of B-cells into memory cells (CD19+CD27+) or plasma cells (IgD-CD38high) was evaluated using flow cytometry. The effects of TLR and IL-21 co-stimulation on B-cell proliferation and apoptosis were studied by measuring CFSE and 7-AAD incorporation respectively; signal transduction pathways involving the prosphorylation of STATs, p38MAPK and ERK will be analyzed by Western Blotting. ELISA will be used to determine levels of IL-21 in the sera of both active and inactive SLE patients.

Results: IL-21 acted synergistically with TLR-9 agonists to promote B-cell differentiation to memory cells and plasma cells (10% with either agent alone vs 35% with the combination for memory B-cells, and 4.3% and 13% for ODN 2006 and IL-21 alone respectively vs 25% with the combination for plasma cells), an effect that was attenuated by hydroxychloroquine (5% inhibition for memory cells and 9% inhibition in plasma cells). The effect was specific for TLR-9, since TLR-2, -3 and -4 agonists failed to induce B-cell differentiation. The effect of IL-21 to induce B-cell differentiation in combination with TLR-9 was more potent than that of IL-6, IL-10 or sCD40L and was partially mediated by the up-regulation of BAFF-R. The combination of TLR-9 and IL-21 also inhibited apoptosis of B-cells (20% apoptotic B-cells after IL-21 or ODN 2006 treatment alone vs 12% with the combination). Experiments addressing signal transduction pathways and detection of serum IL-21 levels in lupus patients are in progress.

Conclusions: The combination of TLR-9 and IL-21 is a potent inducer of B-cell proliferation and differentiation to antibody secreting cells, and might confer survival signals to these cells in the context of SLE.

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080 NORMAL EXPRESSION AND FUNCTION OF THE IMMUNOREGULATORY RECEPTOR PROGRAMMED DEATH-1 (PD-1) IN CD4+ T LYMPHOCYTES OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS: IMPLICATIONS FOR IMMUNOMODULATORY THERAPY

G. Bertsias, A. Raptopoulou, C. Choulaki, E. Koutala, E. Papadimitraki, C. Tsatsanis, H. Kritikos, P. Sidiropoulos, D. T. Boumpas.Laboratory of Rheumatology, University of Crete School of Medicine, Heraklion, Greece

Aim: PD-1 has emerged as a negative regulator of T-cell effector function with important implications in homeostasis of peripheral immune tolerance. SLE is the prototype of human autoimmune disease where breakdown of peripheral tolerance occurs with expansion of autoreactive T lymphocytes. We sought to determine the role of PD-1 in autoreactivity against self-antigens and determine its expression and function in CD4+ T lymphocytes of patients with SLE.

Materials and methods: The expression and function of PD-1 and PD-L1 was determined in an ex vivo model of autoreactivity (autologous mixed lymphocyte reaction, AMRL). PD-1 expression in subsets of isolated CD4+ T cells from SLE patients and healthy controls was examined by flow cytometry and real-time PCR. PD-L1.Ig was used to study PD-1 function in anti-CD3/CD28-stimulated cells in terms of proliferation and cytokine production.

Results: In the AMRL, PD-1 was highly increased on CD4+CD25+ cells (20–40%) and PD-L1 on CD14+ monocytes (>90%). Blockade of the PD-1/PD-L1 pathway—but not the CTLA-4/B7 pathway—resulted in 2-fold increase in AMRL-induced T cell proliferation. Patients with SLE demonstrated increased proportion of PD-1+ CD4+HLA-DR+ DR+ (26.7±2.0% vs. 17.9±1.3%, p = 0.001) and CD4+CD69+ (19.5±2.8% vs. 10.6±1.9%, p<0.05) lymphocytes compared to healthy controls. Kinetics of PD-1 on anti-CD3/CD28-stimulated CD4+ T cells did not differ between patients and controls. In functional assays, PD-1 crosslinking resulted in significant reductions in lymphocyte proliferation (54±6% vs. 47±7%), IFN-g (51±9% vs. 34±11%), and IL-10 (68±6% vs. 47±7%) production in SLE patients and controls, respectively. Experiments assessing the function of other immunoregulatory receptors (CTLA-4, BTLA) in SLE CD4+ T lymphocytes are in progress.

Conclusions: PD-1 has an important role in regulation of human autoreactivity against self-antigens, and demonstrates normal expression and function in CD4+ T lymphocytes in patients with SLE. These findings provide implications for the potential modulation of the PD-1/PD-L1 pathway as a therapeutic option in patients with SLE.

081 IDENTIFICATION OF FOXO1A AS A NEW CHONDROGENIC TRANSCRIPTION FACTOR IN MESENCHYMAL STEM CELLS

D. Noël1,2, F. Djouad1,2, D. Mrugala1,2, C. Bony1,2, C. Jorgensen1,2.1Inserm U475, Montpellier, F-34197 France; 2Université MONTPELLIER1, UFR de Médecine, Montpellier, F-34000 France; 3CHU Montpellier, Hôpital Lapeyronie, Unité Clinique d’Immuno-Rhumatologie, Montpellier, France

Mesenchymal stem cells (MSC) are suitable sources for cell-based therapies in cartilage engineering. However, their potential to regenerate a fully functional tissue relies on the presence of a differentiation factor. The identification of such a specific factor represents the major issue of this study. Bone marrow-derived human MSC were induced to differentiate towards chondrocytes using the micropellet culture technique in presence of chondrogenic medium containing hBMP-2 for 21 days. Total RNA were hybridized on DNA microarrays (Affymetrix U133A). Quantitative RT-PCR was performed on RNA extracted at various time points during chondrogenesis. The C3H10T1/2 murine MSC line was stably transfected with a plasmid expressing the wild type (wt) or a constitutively active form of Foxo1A (Δ) and cultured in chondro-, osteo-, adipogenic conditions or injected intra-articularly. SiRNA interference was performed on the CL1 bipotent cell line. Expression of specific markers was assessed by RT-PCR. Among the 1354 differentially regulated genes during chondrogenesis, 705 genes were upregulated in MSC-derived chondrocytes. We first focused our attention on transcription factors and in particular, on Foxo1A which was shown to be increased by a 6-fold factor using real time PCR, as soon as day 2 of chondrogenesis. We thus derived stable clones of C3H10T1/2 cells over-expressing either the wild type or constitutively active form of Foxo1A, respectively wtFoxo1A or ΔFoxo1A. After 21 days of culture in micropellet without any differentiation factor, we could show the up-regulation of aggrecan, collagen IIB and the down-regulation of collagen I, suggesting that Foxo1A is sufficient to induce chondrogenesis. In parallel, the engineered cells did not show a higher osteogenic potential than naïve C3H10T1/2 cells and even more interesting, a lower adipogenic potential when cultured in specific inducing conditions. We also showed that Foxo1a is necessary for chondrogenesis since the knocking-down of Foxo1A in the CL1 bipotent cell line resulted in the decrease of differentiation as shown by lower levels of collagen IIB and aggrecan mRNA. In vivo, after injection of the engineered cells in the intra-articular space of knee joints, we could detect the formation of cartilage staining positive for aggrecan and collagen II in the areas of engineered cell injection confirming their potential to differentiate into chondrocytes. Our results suggest that Foxo1A is one essential transcription factor involved in the early steps of chondrogenesis. Further studies will aim at determining the mechanism underlining the activation of chondrocyte-specific markers.

082 PROSTAGLANDIN D2 PATHWAY IN RHEUMATOID ARTHRITIS SYNOVIAL CELLS

K. R. Gheorghe, M. Korotkova, C. Trollmo, L. Klareskog, P-JJakobsson.Department of Bioscience, Karolinska Institutet, Novum, Huddinge, Sweden and Department of Medicine, Rheumatology Unit, Karolinska Institutet/Karolinska University Hospital, Solna, Stockholm, Sweden

Objective: Prostaglandin D2 (PGD2) has been shown to have several anti-inflammatory properties both by itself and through its degradation product, 15-deoxy-delta-12-14-PGJ2. In this study we determined if synovial fluid mononuclear cells (SFMC) from patients with rheumatoid arthritis (RA) produce PGD2 and which enzymes are involved in its synthesis. In addition, we investigated the effects of glucocorticoids and tumor necrosis (TNF) blockers on PGD2 production in SFMC and peripheral blood mononuclear cells (PBMC).

Methods: SFMC were isolated from patients with RA and PBMC from healthy blood donors. Lipocalin-type prostaglandin D synthase (L-PGDS) expression was assessed by flow cytometry and PGD2 and PGE2 production in culture supernatants was determined by enzyme immunoassay.

Results: Lipopolysaccharide (LPS) induced a 2-fold increase in PGD2 formation to 49.5±8.9 pg/million cells accompanied by an increase in L-PGDS expression in macrophages, both of which were suppressed to baseline by dexamethasone. Also, TNF blockers reduced the formation of PGD2 but to a lesser extent than dexamethasone (33.9±11 pg/million cells) and did not significantly alter the L-PGDS enzyme expression. In line with our previous data, PGE2 was highly up-regulated by LPS stimulation and its levels were suppressed by both dexamethasone and TNF blockers.

Conclusions: RA SFMC and normal PBMC produced PGD2 in culture after stimulation with LPS through the lipocalin-type PGDS. Dexamethasone decreases both the prostaglandin formation and the enzyme expression, whereas TNF blockers seem to have different effects: while decreasing the PGD2 formation, they do not influence the L-PGDS expression. The biological significance of PGD2 presence in the RA joint has not been defined yet, but it may be beneficial through the anti-inflammatory effects of its degradation product, 15-deoxy-delta-12-14-PGJ2.

083 THREE DIMENSIONAL (3-D) MODEL OF THE PRYSPRY DOMAIN OF PYRIN: IMPLICATIONS FOR DISEASE SEVERITY IN FAMILIAL MEDITERRANEAN FEVER (FMF)

E. Fragouli1, E. Eliopoulos2, P. Sidiropoulos3, I. Aksentijevich4, D. T. Boumpas1,3, G. N. Goulielmos1.1Department of Internal Medicine, University of Crete; 2Laboratory of Genetics, Agricultural University of Athens; 3Department of Rheumatology, Clinical Immunology and Allergy, University Hospital of Heraklion; 4Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskelatal and Skin Diseases, Bethesda, USA

Background: The gene responsible for the FMF is MEFV, encodes for the pyrin protein with the most prevalent mutations localized in exons 2 and 10. Greeks are considered to be at “intermediate risk” and in a recent study of 70 FMF patients, focused on the molecular analysis of the most frequent mutations identified so far, it was found a higher incidence of FMF in the island of Crete, with the carriers’ frequency approaching a rate of 1:14.

Aim: a) to construct a three-dimensional model (3-D model) of the PRYSPRY domain of pyrin and localize the MEFV mutations upon classification according to the clinical severity of FMF, b) to construct 3-D model presenting the direct interaction of the PRYSPRY domain of pyrin with caspase-1, and c) to approach the issue of the necessity of alternative pathogenic pathways leading to FMF, taking into consideration the localization of certain mutations on a constructed 3-D model of pyrin, their impact on the different phenotypes and the ability of pyrin’s interacting molecules to have access to the proper interacting sites.

Materials and methods: The 3-D model of the pyrin PRYSPRY-domain was created with MODELLER and the Swiss-model automated protein structure homology-modelling server. The derived model was checked for folding and packing errors using the QUANTA-CHARM program.

Results: We constructed a three-dimensional model (3-D model) of the PRYSPRY domain of pyrin. The majority of the known MEFV mutations located on this domain have been classified, according to the disease severity caused, and localized on this 3-D model. One novel putative missense mutation, S702C, was identified in exon 10 of the MEFV gene and located on the constructed 3-D model. Flexible loops of caspase-1 are able to interact with mutations that are associated with severe FMF disease (i.e. M694V and M680I), spread on the rim of the hydrophobic cavity of the PRYSPRY domain of pyrin, but seem unable to interact with mutations associated with mild disease, distal to the first ones.

Conclusions: The PRYSPRY pyrin globular domain is formed by a beta sandwich and contains an elongated hydrophobic cavity. Mutations causing severe effects are spread on the rim of the cavity while mutations with modest effects are localized on loops away from the major recognition site. The direct interaction of B30.2 (or PRYSPRY) domain of pyrin with caspase-1 shown to modulate the IL-1b production in FMF, appears to be inefficient in some cases. The finding of a putative interaction between the pyrin-interacting, pro-apoptotic protein SIVA protein and the PRYSPRY domain has prompted us to further explore its contribution to the pathogenesis of FMF.

084 EVALUATION OF NOVEL ASSAY SYSTEMS FOR THE DETERMINATION OF ANTI-DSDNA ANTIBODIES IN PATIENTS WITH SLE

C. Zimmermann, E. Hoefler, J. S. Smolen, G. Steiner.Second Dept. of Medicine, Hietzing Hospital, Vienna, Dept. of Rheumatology, Medical Univ. of Vienna, Austria

Background: Antibodies (Ab) to dsDNA are highly specific serological markers for the diagnosis of SLE. Among the various assay systems available indirect immunofluorescence on Crithidiae luciliae (Crith) is the most specific but also the least sensitive one, while the more sensitive Farr assay is hampered by the use of radioactivity. It was the aim of this study to evaluate two novel assay systems for determination of anti-dsDNA Ab, the EliATM dsDNA assay (Phadia) and the microsphere based FidisTM immunoassay (Biomedical Diagnostics).

Methods: Sera of 380 consecutive patients were included in the study. A distinct rheumatic disease was diagnosed in 248 patients including 88 patients with SLE, 67 with other connective tissue diseases, 40 with rheumatoid arthritis and 53 with other joint diseases.

Results: Among the sera tested 21% were positive by EliA, 20% by Fidis, while only 7% tested positive in the Crith assay. Sensitivities for SLE were 24% (Crith), 56% (EliA), 58% (Fidis), and 60% (Farr), respectively. The Crith assay was 99% specific for SLE, while both novel assays showed 92% specificity which was comparable to the Farr assay. Combined sensitivity of EliA, Fidis and Farr assay was 73%. The strongest inter-assay correlation was seen between EliA and Farr assay (r = 0.77, p<0.001) and both assays showed significant correlation with disease activity as measured by the ECLAM score (r = 0.45, p<0.01).

Conclusion: The two novel assay systems showed sensitivities and specificities comparable to the Farr assay and may therefore be considered reasonable alternatives for routine determination of anti-dsDNA Ab.

085 INTRACELLULAR MG CONCENTRATION IS LOW IN PATIENTS AFFECTED BY FIBROMYALGIA ASSOCIATED WITH SPASMOPHILIA

L. Bazzichi1, C. Giacomelli2, T. Giuliano1, A. Rossi2, A. Consensi1, C. Tani1, C. Neri1, S. Bombardieri1.1Department of Internal Medicine, University of Pisa; 2Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Italy

Aim: Mg++ is involved in the reaction catalyzed by adenylyl cyclase producing cAMP from ATP. In the cells this enzyme is regulated by G protein: G protein inhibitory type (Gi) reduces its activity, while G protein stimulatory type (Gs) increases it. Recent study showed a hypofunctionality of Gi proteins in FM patients, probably responsible of higher cAMP basal levels, so these alterations may reflect on PKA regulation as well as on SERT phosphorylation. The aim of the present study was the evaluation of intracellular Mg concentration in patients affected by primitive fibromyalgia (FM) and patients with spasmophilia associated with fibromyalgia after integration with magnesium salt.

Materials and methods: 45 patients (8 male and 37 female; median age: 48 yrs) affected by FM and spasmophilia and 45 patients (44 female 1 male; median age: 56.5 yrs) affected by primitive FM were enrolled at the division of Rheumatology of the University of Pisa. FM was diagnosed according to ACR Criteria (1990); electromyography (EMGs) after ischemic stimuli is a test utilized to evaluate spasmophilia. Fasting blood samples were collected in the morning after one month of therapy with magnesium pidolate. For each patient, tenderness at tender points was evaluated by means of the Fischer dolorimeter. The total fibromyalgic tender point score (right + left) was used in the statistical analysis. Each positive tender point (TP) had a pain score between 0 and 3. To estimate the impact of fibromyalgia on the quality of life, all subjects received a “Fibromyalgia Impact Questionnaire”. The total score was the sum of 10 FIQ item values and reflected the impact of fibromyalgia in the quality of life and ranged from 0 (no impact) to 100 (maximum impact). Tiredness was evaluated as measured by the FIQ tiredness item which consisted of a visual analogue scale numbered between 0 and 10. Intracellular Mg level was determined by atomic absorption spectrophotometry.

Results: We observed a statistically significant difference between intracellular Mg concentrations in FM patients and FM/spasmophilic ones. The patients with primitive FM showed intracellular Mg level higher than FM patients affected also by spasmophilia (4.610±0.1888 mEq/l vs 3.945±0.0837 mEq/l). We did not find a correlation between Mg values and other clinical parameters.

Conclusion: The different intracellular Mg concentration after short therapy with magnesium salt in FM patients and FM/spasmophilic ones suggests a possible malabsorption of Mg in FM/spasmophilic subjects. The intracellular Mg deficit should be one of the causes of spasmophilia.

086 THE PROBABLE RHEUMATOID ARTHRITIS: METHOTREXATE VERSUS PLACEBO TREATMENT (THE PROMPT) STUDY: A DOUBLE-BLIND RANDOMIZED CONTROLLED TRIAL

H. van Dongen*1, J. van Aken*1, L. R. Lard1, K. Visser1, H. K. Ronday2, H. M. J. Hulsmans2, I. Speyer3, M. L. Westedt3, A. J. Peeters4, C. F. Allaart1, R. E. M. Toes1, F. C. Breedveld1, T. W. J. Huizinga1.1Leiden University Medical Center, Leiden, The Netherlands; 2HAGA Hospital, The Hague, The Netherlands; 3Bronovo Hospital, The Hague, The Netherlands; 4Reinier de Graaf Hospital, Delft, The Netherlands; *Van Dongen and Van Aken contributed equally

Objective: A substantial proportion of patients with rheumatoid arthritis (RA) initially presented with undifferentiated arthritis (inflammatory, non-traumatic arthritis that cannot be diagnosed by current classification criteria, UA). The aim of the PROMPT study was to determine whether patients with UA benefit from treatment with methotrexate (MTX). The primary outcomes were progression to the diagnosis of RA fulfilling American College of Rheumatology (ACR) 1987 criteria and radiographic joint damage.

Methods: The PROMPT study was a double-blind placebo-controlled randomized multicenter trial in 110 UA patients, who fulfilled the ACR 1958 criteria for probable RA. Treatment started with MTX 15 mg/wk or placebo tablets, and dose was increased by 3-monthly calculations of the disease activity score (DAS), aiming at a DAS <2.4. After 12 months, the study medication was tapered to nil. Patients were followed up for 30 months. When a patient fulfilled the ACR criteria for RA (primary endpoint), the study medication was changed to MTX. Joint damage was scored on radiographs of hands and feet.

Results: In the MTX-group 22/55 patients progressed to RA versus 29/55 patients in the placebo-group. However, in the MTX-group patients fulfilled the ACR criteria for RA at a later time point than in the placebo-group (p = 0.04), and less patients showed radiographic progression over 18 months (p = 0.046). Subgroup analysis on auto-antibody status revealed that only patients who were anti-cyclic citrullinated peptide (anti-CCP) positive reached the diagnosis RA at a significantly later time point (p<0.001) and had less radiographic progression (p = 0.03), whereas MTX showed no effect in anti-CCP negative patients. The same observations were made for rheumatoid factor (RF) positive patients.

Conclusion: This study provided evidence for the efficacy of MTX treatment in postponing the diagnosis of RA as defined by the ACR 1987 criteria and retarding radiographic joint damage in UA patients. A post-hoc subgroup analysis showed that MTX was only effective in anti-CCP or RF positive patients. This work was supported by the Dutch Arthritis Foundation (NR-02-01-301) and by the Netherlands Organisation for Scientific Research (NWO, grant 920-03-259 and 940-37-019).

087 EXPRESSION ANALYSIS OF CITRULLINATED PROTEINS IN SYNOVIAL TISSUE OF PATIENTS WITH RHEUMATOID ARTHRITIS, REACTIVE ARTHRITIS AND OSTEOARTHRITIS

C. Zimmermann, M. Tohidast-Akrad, P. Zenz, J. S. Smolen, G. Steiner.Second Dept. of Medicine, Hietzing Hospital, Vienna, L. Boltzmann-Institute for Rheumatology, Vienna, Dept. of Rheumatology, Medical Univ. of Vienna, Austria

Background: Autoantibodies against citrullinated proteins are highly specific for RA, while the specificity of citrullinated protein expression is still a matter of debate. To further investigate this issue, we performed immunohistochemical analyses in synovial membrane specimen from patients with RA, ReA and OA.

Methods: Staining of frozen sections obtained at the time of joint surgery or by biopsy, respectively, was performed using a rabbit polyclonal anti-citrulline antibody. To characterise citrulline expressing cells monoclonal antibodies against various cell lineage-specific antigens, including anti-CD68 (macrophages), ASO2 (fibroblasts), anti-CD34 (endothelial cells), anti CD3 (T cells) and anti-CD20 (B cells), were used.

Results: In specimens from all patients expression of citrullinated protein was found in macrophages and fibroblasts of the lining layer and sublining areas. Furthermore, pronounced staining was seen in endothelial cells of RA and ReA patients. Staining was more intense in specimens from RA and ReA patients than in sections from OA patients. Areas containing cellular infiltrates with an abundancy of T cells or B cells were commonly found in RA and ReA synovium. Interestingly, while only few CD3 positive cells were stained by the anti-citrulline antibody, approximately 20% of CD20 positive B cells were also citrulline positive.

Conclusions: Expression of citrullinated proteins was commonly detected in macrophages and fibroblasts of all patients as well as in endothelial cells and B cells of RA and ReA patients. Proteomic studies will be needed to elucidate if different proteins are citrullinated in RA synovial tissue thus eliciting the specific autoimmune response characteristic of RA.

088 IDENTIFICATION OF THE INTERLEUKIN-7 SIGNALLING PATHWAY IN RHEUMATOID ARTHRITIS SYNOVIAL TISSUES WITH LYMPHOID NEOGENESIS

T. C. G. Timmer1, B. Baltus1, M. Vondenhoff1, T. W. J. Huizinga2, P. P. Tak3, C. L. Verweij1, R. E. Mebius1, T. C. T. M. van der Pouw Kraan1.1Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands; 2Department of Rheumatology, LUMC, Leiden, The Netherlands; 3Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Objective: In approximately 25% of the synovial tissues of rheumatoid arthritis (RA) patients, infiltrates of T- and B cells and follicular dendritic cells (FDCs) are spatially organized into structures resembling lymph nodes with germinal centers. The remainder of the tissues lack FDCs and show either a diffuse or aggregated T- and B cell infiltrate.

Methods: We set out to identify the genes that are expressed in RA tissues with ectopic lymphoid structures by cDNA microarray analysis and real-time PCR to gain more insight into this specific disease process.

Results: Gene expression analysis confirmed earlier reports that tissues with lymphoid structures showed elevated expression of CXCL13, CCL21, CCR7, and Lymphotoxin alpha and beta mRNA. In addition, these tissues also showed enhanced expression of the chemokines CXCL12, CCL19 and the associated receptors CXCR4 and CXCR5, important for the attraction of T- and B cells and dendritic cells. Pathway analysis revealed increased expression of genes involved in Jak-STAT signalling, T- and B cell-specific pathways, Fc Epsilon Receptor I Signalling in Mast Cells, and IL-7 Signal Transduction in the tissues with ectopic lymphoid follicles accompanied by increased expression of the IL-7Ralpha/IL-2Rgamma chains and IL-7. Protein expression of IL-7 in RA tissues was localized within fibroblast-like synoviocytes, macrophages and blood vessels and co-localized with extracellular matrix structures around the B cell follicles.

Conclusion: Activation of the IL-7 pathway may play an important role in lymphoid neogenesis, analogous to its role in normal lymphoid tissue development.

089 A FUNCTIONAL NONINVASIVE METHOD FOR EARLY DETECTION THE DAMAGE OF CARTILAGE JOINT USING VIBROACOUSTIC AND THERMIC SPECTRUMS

S. Stanciu1, M. Cirmaci2, F. Berghea3, M. Bugaru4, L. Ciobica1, C. Jurcut1, T. Chereches2, S. Blaj2.1Central Military Hospital Bucharest “Carol Davila”; 2Military Technical Academy; 3University of Medicine and Pharmacy “Carol Davila”; 4Polytechnic University of Bucharest, Romania

Recently, our team reported on a project which has, as the principal objective, the study of the vibroacoustic mechanism in the normal knee joint compared with arthrosic joints. The primary aim of this project is to develop a precise non-invasive method to diagnose and monitor the pathologic elements on the diarthrodial articulation level based on registering and analysing vibroacoustic and thermic signals.

Method: The vibroacoustic signals emitted by the diarthroidal articulation during the normal movement differentiate a healthy case from a pathologic one, due to alteration in form and contact surfaces. The difference shows through various dynamics of the vibroacoustic spectra. In order to record, brief, decode and classify them it is necessary to use high-class apparatus and to apply advanced techniques for signal processing, such as: linear prediction, spectral analysis, time-frequency analysis, statistic determination, auto-regressive shaping techniques etc. Our method of acquiring the sound and vibration signals is achieved completely non-invasively, with a set of translators using Pulse—sounds and vibrations analyzers (a matrix of prepolarised microphones with measurement domain in infrasound scale and piezoelectric acceleration transducers) Our project proposes to research the evaluation of the vibroacoustic and thermic spectra using this method on the clinically and imagistically normal articulations, on a range of ages, and equally on afflicted articulations, especially on arthritic articulations, with the prospective and comparative study of the groups included in the study as in predefined including criteria. We expect that, in RMN normal-catalogued articulations, there should appear alterations of the vibroacoustic spectra, which analyzed, to raise a future morphological alterations, having the possibility of some prediction diagrams and an evolving trend on the joint morpho-functionality. This way, the proposed method will be able to decide in a precocious stage (subclinical stage) which articulation will be affected by degenerative alterations, and identify the best moment of the therapeutic intervention. The method could launch a new concept of the arthritic disease, the reversibility stage. Applying some new therapies in this stage should therefore lead to curing the disease and a visible improvement of the prognosis. Our comparative study (from January 2007 to November 2008) will help us to evaluate this functional method in terms of sensibility, specificity, negative and positive predictive value, indices that assure the diagnostic power to the method.

Conclusions: We are given the opportunity to propose a study of a new diagnostic method, to objectify and measure the precocious functional alterations in diverse arthritic articulations, that correspond to some certain morphological stages in the disease, during which the cartilage is very low affected, with a degree of complexity given by the diagnostic and prognostic power.

090 ENDOTHELIN-1 AND ESTROGENS INCREASE CELL PROLIFERATION OF CULTURED HUMAN SKIN FIBROBLASTS

S. Soldano1, M. Cutolo1, P. Montagna1, S. Shabanova2, A. Sulli1, S. Capellino1, C. Pizzorni1, M. Secchi1, B. Seriolo1, S. Paolino1, A. Parodi3, B. Villaggio4.1Division of Rheumatology - Dept Internal Medicine, University of Genova, Genova, Italy; 2Department of vascular disorders, Institute of Rheumatology, Moscow, Russia; 3Dept Dermatology, University of Genova, Genova, Italy; 4Division of Nephrology - Dept Internal Medicine, University of Genova, Genova, Italy

Background: Endothelin-1 (ET-1) exerts vasoconstrictive properties but also potent mitogenic effects on fibroblasts (Fb).1 Estrogens (E) are involved in autoimmunity as enhancers of the immune response and cell proliferation; opposite effects are exerted by androgens (A).2

Purpose: To evaluate the effects of mythogenic Endothelin-1 (ET-1) on cell proliferation of cultured human normal fibroblasts (Fb) in presence of Estrogens (E) or Androgens (A) in order to evaluate possible synergistic gender effects.

Methods: Cultures of normal Fb (obtained from skin biopsy of 6 volunteer subjects after informed consent) were characterised by immunocytochemistry (ICC, mAb anti-human Fb surface protein =  Fb-SP). Serum-starved Fb were cultured up to confluence in multiwells and treated for 24 hrs with the ET-1 concentration having mitogenic effect (100 nM). Further aliquots of Fb were treated with 17beta-estradiol (E2) or testosterone (T) (100 pM and 1 nM respectively) alone or plus ET-1 for 24 hrs. Cell proliferation was evaluated by the methyltetrazolium salt test (MTT) and confirmed by LDH test. Western blot analysis (WB) was performed for proliferating cell nuclear antigen (PCNA) expression as a marker of cell cycle progression. Experiments were done in triplicate.

Results: Both ET-1 and E induced significant cell proliferation of serum-starved human skin Fb (p<0.001). Opposite effects were found when Fb were treated with T (p>0.05). The expression of PCNA was increased in Fb treated with ET-1, E2 and in their combination. A significant reduction of PCNA expression was observed in Fb treated with T and with T plus ET-1. However, the comparison between E and T-treated Fb was still significant (p<0.001).

Conclusions: Results indicate that ET-1 is able to enhance the cell proliferation by normal human Fb. Interestingly, E per se and in combination with ET-1 was found to further enhance the proliferative activity of treated normal Fb. As a matter of fact, E are involved in autoimmunity as enhancers of the immune response and cell proliferation; opposite effects are exerted by A.

1

2

091 CD49B+ REGULATORY T CELLS ARE ABLE TO CURE ARTHRITIS

L. M. Charbonnier1, F. Apparailly1, W. Han2, L. M. Van Duivenvoorde2, R. E. M. Toes2, C. Jorgensen1, P. Louis Plence1.1INSERM U844, Hopital Saint Eloi, Montpellier, France; 2Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands

Objective: Dendritic cells (DCs) are specialized antigen presenting cells with an important role in the initiation and regulation of immune responses. We previously demonstrated that immature DCs (iDCs) can prevent arthritis by inducing a regulatory population of T lymphocytes (Treg). These Tregs were characterized by the expression of the CD49b molecule and by interleukin-10 (IL-10) secretion. In the present study we evaluated and compared the therapeutic potential of iDC versus Treg to cure established arthritis.

Methods: The Treg population was purified from the liver and spleen of DBA/1 mice that were repetitively injected with 5×105 iDCs by sorting the CD4+CD49b+ cells. These Tregs were adoptively transferred in DBA/1 mice (n = 7–8/group) with established collagen-induced arthritis (day 28). In parallel iDC were repetitively injected in arthritic mice in order to evaluate their therapeutic potential and compared with the therapeutic potential of T regs.

Results: We showed that the repetitive injections of iDC just after the boost in incomplete Freund adjuvant did not protect the mice from severe arthritis. However a single injection of less than 100 000 T reg cells was able to decrease the severity of arthritis and protect mice until day 43. The severity score of the Treg treated group was 0.2 versus 2.4 for the control group. We are currently working on the characterization of the Treg cells in order to better define this new regulatory population.

Conclusion: These results suggest that the use of iDC vaccination in an inflammatory setting has to be carefully addressed and the in vivo maturation of such iDC has to be blocked before their injection. Such strategy has been evaluated with IL-10 or dexamethasone modulated DC. More importantly, we demonstrated in this study the high therapeutic potential of the CD49b+ regulatory T cells on established arthritis in the CIA model.

092 T REGULATORY CELLS IN SJöGREN’S SYNDROME (SS): CORRELATION WITH THE PROGRESSION OF THE AUTOIMMUNE LESION

M. I. Christodoulou1, E. K. Kapsogeorgou1, N. M. Moutsopoulos2, H. M. Moutsopoulos1.1Pathophysiology Department, Medical School, University of Athens, Greece; 2Oral Infection & Immunity Branch, National Institute of Dental & Craniofacial Research, NIH, Bethesda, Maryland, USA

Introduction: SS is an autoimmune exocrinopathy associated with lymphocytic infiltration of the affected organs (mainly salivary and lacrimal glands). The etiopathogesis of the disorder remains largely unknown and the role of the regulatory T cells (Treg), a population implicated in the development of autoimmunity, has not been explored in depth for this disease. The goal of the present study was to investigate the presence of this population in the minor salivary gland (MSG) lesion of SS patients as well as potential associations of Tregs with markers of the autoimmune lesion progression [such as the grade of lymphocytic infiltration, the number of infiltrating mononuclear cells and the presence of specific types of immunocytes that are thought to characterize advanced lesions, such as macrophages and dendritic cells (DC)].

Materials and methods: Tregs were identified by the immunohistochemical detection for the specific marker FOXP3 in MSG biopsy specimens obtained from 27 SS patients and 9 controls [3 with negative biopsy score that did not fulfil the SS American-European classification criteria, 2 with sarcoidosis- and 4 with viral (3 HCV and 1 HIV)-related sialadenitis]. SS specimens were classified in three categories according to the MSG biopsy focus score (by Tarpley et al.). The first group (n = 8) included specimens with 1+ biopsy score, the second (n = 10) samples with 2+ score and the third (n = 10) biopsies of 3+ or 4+ score. In all SS patients, the biopsy focus score (lymphocytic foci/4-mm2 of tissue) was ⩾1. Immunohistochemical staining was applied to identify T cells (CD3), B lymphocytes (CD20), follicular DC (fascin), interdigitating DC (S100) and macrophages (CD68). Positively-stained cells, as well as the entire population of infiltrating mononuclear cells were counted field-by-field in each section. Statistical correlations were evaluated by non-parametric Spearman analysis.

Results: FOXP3+ cells were detected in the areas of infiltrating CD3+ T cells in all SS and sialadenitis samples, but not in the 3 controls with negative biopsy score. The mean of the FOXP3+/CD3+ cell percentage±SE in the first, second and third SS group was 2.52%±0.17, 13.21%±0.61 and 8.52%±0.27, respectively. In sarcoidosis- and virus-related sialadenitis samples, it was 7.18%±1.52 and 9.26%±2.10, respectively. The number of FOXP3+ cells was found to be significantly associated with the number of infiltrating mononuclear cells (r:0.8358, p<0.0001), fascin+ DC (r:0.8111, p = 0.0080), S100+ DC (r:0.7000, p = 0.0077) and CD68+ macrophages (r:0.5822, p = 0.0071). The FOXP3+ cell number was negatively correlated to the CD3+/CD20+ cell ratio (r:−0.7821, p = 0.0006).

Conclusion: Tregs are present in the autoimmune MSG lesions of SS patients and their frequency is associated with lesion progression, with the highest frequency detected in the intermediate and the lowest in the early lesions. The role of Tregs and the factors implicated in their recruitment/differentiation in SS lesions need further investigation.

093 A NOVEL B7.2 (CD86) SPLICE VARIANT WITH A PUTATIVE NEGATIVE REGULATORY ROLE

E. K. Kapsogeorgou, H. M. Moutsopoulos, M. N. Manoussakis.Dept Pathophysiology, School of Medicine, National University of Athens, Greece

Introduction: Human non-neoplastic salivary gland epithelial cell (SGEC) lines have been shown to express functional full-length and soluble protein forms of the B7.2 molecules (Kapsogeorgou et al, J Immunol 2001). The study of the B7.2 mRNA molecules that are expressed by SGEC revealed the existence of a novel human B7.2 mRNA splicing variant, designated B7.2C. In this study, the properties of B7.2C isoform are investigated.

Methods: The B7.2 mRNA molecules were analyzed by RT-PCR (specific for the entire coding region) and sequencing. The cell-surface expression of the B7.2 molecules was evaluated by the biotinylation of the cell-surface proteins followed by immunoprecipitation of the B7 proteins. B7.2C function was investigated by stable transfection of CHO cell lines and standard T cell costimulation assays.

Results: B7.2C splice isoform is characterized by the deletion of exon 4 that encodes the IgV-like CD28/CTLA4-binding domain of the B7.2 protein (full-length protein; B7.2A). Besides SGEC, it was also detected in peripheral blood monocytes, but not fibroblasts, peripheral blood T and B cells and certain epithelial tumor cells. B7.2C was found to encode a cell-surface protein, whose expression by monocytes is down-regulated during activation processes. CHO-B7.2C cell lines were found unable to provide T cell costimulation. The co-expression of B7.2C in B7.2A-expressing cells was found to associate with significantly reduced costimulatory activity, as indicated by analyses of double-transfected CHO cell lines [5 distinct cell lines, 3 independent experiments each; median reduction (range): 69% (58–82%), 39% (24–44%), 34% (31–41%), 32% (23–33%) and 23% (21–27%)]. Transfectants with reduced costimulatory activity were found to associate with significant lower cell-surface B7.2A to B7.2C ratios (1.0–3.5), compared to those with unaffected costimulatory function (10.0–19.5).

Conclusion: Our findings indicate that B7.2C splice isoform is expressed by classical antigen-presenting cells (monocytes), as well as non-immune cells with potential antigen-presenting capacity. This isoform appears to participate in the fine tuning of T cell responses by attenuating the positive costimulatory signals of B7.2A-expressing cells, possibly by interference with the formation of B7.2 clusters and network.

094 PLACENTA GROWTH FACTOR (PLGF) EXPRESSION IN INFLAMMATORY ARTHRITIS

M. Gogarty, E. P. Murphy1, U. Fearon, D. J. Veale, B. Bresnihan, O. FitzGerald.Department of Rheumatology, St. Vincent’s Hospital, Elm Park, Dublin 4; 1Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland

Introduction: Placenta growth factor (PlGF) binds only to the vascular endothelial growth factor (VEGF) receptor Flt-1 and not KDR. Inhibition of Flt-1 has been shown to be protective against joint destruction by reducing synovial inflammation and neovascularization in a mouse model of collagen-induced arthritis. Two of the four PlGF isoforms (PlGF-2 and -4) have heparin binding sites resulting in their being sequestered to the extracellular matrix; the other isoforms (PlGF-1 and -3) are soluble. When measuring VEGF and PlGF, their bioavailability must be taken into account, due to the presence of their natural antagonist soluble Flt-1 (sFlt-1), a Flt-1 splice variant.

Aims: To determine (1) serum and synovial fluid (SF) levels of PlGF and sFlt-1, (2) synovial tissue expression of the PlGF and Flt-1 isoforms, and (3) establish their cellular source.

Methods: PlGF and sFlt-1 levels were measured by ELISA in serum and matching SF samples obtained from patients with rheumatoid arthritis (RA) n = 17, spondyloarthropathy (SpA) n = 12 [comprising of psoriatic arthritis (PsA), seronegative arthritis and ankylosing spondylitis] and osteoarthritis (OA) n = 2. Synovial biopsy was obtained at knee arthroscopy. RNA was extracted from synovial tissue (n = 14), synoviocytes (PsA n = 2) and peripheral blood neutrophils (PsA n = 2) and monocytes (PsA n = 1). PlGF, Flt-1 and sFlt-1 mRNA were measured by RT-PCR.

Results: PlGF was detected in the serum of 64% of RA and 50% of SpA patients. All SF samples were PlGF positive. PlGF was significantly higher in SF when compared to serum (mean pg/ml ± s.e.m., RA 2730.9±426.9 vs 100.9±48.4, SpA 2665.9±469.1 vs 29.8±24.5 and OA 256.5 vs 0). The SpA group had an overall lower serum PlGF level. sFlt-1 was significantly higher in SF when compared to serum (mean pg/ml ± s.e.m., RA 944.9±273.1 vs 89.6±5.8, SpA 595.1±174.5 vs 76.38±4 and OA 326.9 vs 96.9). Both PlGF (343.8 pg/mg) and sFlt-1 (164.9 pg/mg) were spontaneously released from synovial biopsies when incubated in serum-free media for 48 hours, suggesting that synovial tissue is a source of SF levels. PlGF-1, -2 and -4 mRNA was detected in synovial tissue, synoviocytes and neutrophils. Monocytes express mRNA for PlGF-1, -2 and -3. The soluble isoform PlGF-1 was the most abundant isoform in the synovial tissue and synoviocytes. Both Flt-1 and sFlt-1 mRNA was detected in synovial tissue, neutrophils and monocytes.

Conclusion: PlGF is present at high levels in synovial tissue and SF, implicating it in the promotion of monocyte migration and angiogenesis in the arthritic joint. Therefore, Flt-1 and PlGF may represent novel therapeutic targets allowing down-regulation of both angiogenesis and inflammatory cell migration.

095 ANALYSIS OF MUTATIONS/SNPS IN JUN AND FOS PROTO-ONCOGENE PROMOTERS IN THE RHEUMATOID ARTHRITIS SYNOVIAL MEMBRANE

R. Huber, B. Lanick, B. Ukena, S. Dunger, D. Pohlers, R. W. Kinne.Experimental Rheumatology Unit, Department of Orthopedics, Friedrich Schiller University Jena, Waldkrankenhaus “Rudolf Elle”, Eisenberg/Thuringia, Germany

Objective: To identify relevant mutations and single nucleotide polymorphisms (SNPs) in the promoters of jun and fos proto-oncogenes in the synovial membrane (SM) of rheumatoid arthritis (RA), osteoarthritis (OA), and normal control (NC) samples.

Methods: Mutations/SNPs in the respective jun/fos promoters were identified in DNA samples isolated from RA (n = 10), OA (n = 10), and NC (n = 5) SM tissue and blood by Non-Isotopic RNase Cleavage Assay. Wildtype (wt) and mutated promoter fragments were cloned into the reporter gene expression vector pUBT-luc (expressing luciferase). Reporter gene expression of the respective promoter fragments was measured in NIH 3T3 cells one and two days after transfection (both constitutively and following stimulation with PMA) using a dual luciferase assay system.

Results: In the cjun promoter, a known insertion (at the position −617/−618, 2 RA samples) and a new mutation were identified (−823/−824, 1 OA sample). In addition, a new mutation was identified in the junB promoter (−484, 2 RA/2OA samples). In the cfos promoter, 2 known SNPs (−60, −135) were detected (both SNPs in 9 RA/8 OA/4 NC samples). The simultaneous presence of all detected nucleotide exchanges in SM tissue and blood indicates that they are germline mutations/SNPs. All detected mutations/SNPs caused a significant (P<0.005) decrease of constitutive reporter gene expression compared to the wt. Following stimulation with PMA, mutations/SNPs in the promoters of cjun and cfos caused a remarkable decrease of reporter gene expression (90% compared to the wt), whereas the mutation −484 in the junB promoter only caused a limited reduction (<50%) in reporter gene expression.

Conclusion: Different germline mutations/SNPs are located in proto-oncogene promoters of RA and OA patients. All detected mutations/SNPs result in a significant functional inhibition of the promoter, hinting at a possible role for the expression of disease-relevant genes in RA and/or OA. Frequency analysis of the mutations/SNPs in larger patient cohorts is ongoing.

096 ANTI-RO60 443-454 ANTIBODIES DO NOT CROSS WITH THE HOMOLOGOUS COGAN PEPTIDE

I. A. Tsonis, J. G. Routsias, H. M. Moutsopoulos, A. G. Tzioufas.Department of Pathophysiology, Medical School, University of Athens, Greece

Background: Cross-reactive antibodies against the epitope aa443-454 of Ro60 (Ro60pep) autoantigen and a synthetic peptide named Cogan’s peptide (CGpep) have been described to be associated with sensorineural hearing loss in patients with Cogan’s syndrome.1

Aim: To investigate this cross-reaction and its clinical relevance in various autoimmune diseases.

Materials and methods: Sera were obtained from patients with systemic lupus erythematosus (SLE, n = 14), primary Sjögren’s syndrome (pSS, n = 31), rheumatoid arthritis (RA, n = 31), Cogan’s syndrome (CG, n = 2) and 35 healthy blood donors, which served as controls. CGpep (SGRDTSIQILWI) and the Ro60pep (GGTDCSLPMIWA) were synthesized by solid phase peptide synthesis and ELISA assays were performed against both peptides. In addition, antibodies from a serum that recognized Ro60pep were purified by affinity chromatography and both the specific antibody (anti-Ro60pep) as well as the antibodies that remained in the unbound fraction (UnbAb) were tested for their capacity to recognize both peptides.

Results: The prevalence of anti-Ro60pep reactivity was 28.5% (4/14) for SLE, 35.4% (11/31) for pSS and 3.2% (1/31) for RA sera. Interestingly, CG peptide was not recognized by any of the autoimmune sera, even the anti-Ro60 specific ones. As far as the two CG sera are concerned, they did not appear to recognize either Ro60pep or CGpep. Although a preferential recognition of the latter peptide was noted it did not however precede the estimated cut-off values. Purified anti-Ro60pep from a pSS serum reacted significantly with Ro60pep but not with CGpep. More precisely, the unbound fractions contained the 2/3 of the initial anti-CGpep reactivity but only 1/9 of the initial anti-Ro60pep reactivity, indicating that different antibody subpopulations recognize the two peptides.

Conclusion: In conclusion, anti-CG pep reactivity was not detected in Greek patients with autoimmune diseases or even Cogan’s syndrome. In addition, no cross-reaction with the homologous Ro60pep was observed.

1

097 TOLL-LIKE RECEPTOR-3 TRIGGERING OF SALIVARY GLAND EPITHELIAL CELLS (SGEC) CAUSES DETACHMENT-INDUCED CELL DEATH (ANOIKIA) THAT IS MORE PRONOUNCED IN CELLS DERIVED FROM SJöGREN’S SYNDROME PATIENTS

M. P. Spachidou, E. K. Kapsogeorgou, H. M. Moutsopoulos, M. N. Manoussakis.Department of Pathophysiology, Medical School, University of Athens, Athens, Greece

Aim of the study: We have previously demonstrated that cultured SGEC derived from Sjögren’s syndrome patients (SS-SGEC) manifest higher constitutive Toll-like receptor-3 (TLR-3) mRNA expression than disease controls (control-SGEC), in support of their intrinsic activation. In preliminary experiments we have observed that TLR-3 triggering with the synthetic analogue of viral ds-RNA, poly-inosinic:cytidylic acid (polyI:C, TLR3-ligand) causes significant detachment of SGEC from matrix. Herein, we investigated whether such detachment corresponds to a well-described biologic process, whereby detached cells are viable but deprived of survival signals and subsequently undergo apoptosis (anoikia or detachment-induced apoptotic cell death). In this context, we have also assessed the induction of the pro-apoptotic molecule BimEL previously associated with anoikia.

Materials and methods: Cultured non-neoplastic SS-SGEC (n = 6) and control-SGEC lines (n = 6) were stimulated with polyI:C (50 ng/ml, 500 ng/ml, 5 μg/ml, 50 μg/ml) or peptidoglycan (PGN; TLR2-ligand; 100 μg/ml, as control) for 24–96 hours. Cell loss in cultures was examined by trypan-blue exclusion and the MTT survival-assay, whereas the occurrence of anoikia was assessed by monitoring the induction of apoptosis in detached cells using the Annexin-V/Propidium iodide (PI) assay. The implication of Fas, IFN-beta, IFN-gamma and TNF-alpha was also studied by the addition of neutralizing monoclonal antibodies. The expression of the pro-apoptotic molecule BimEL was also tested in treated SGEC, using quantitative real-time PCR (Q-PCR) analyses.

Results: SGEC stimulation by polyI:C (5 μg/ml; but not PGN) for 96-hrs resulted in significant cell detachment from the cell matrix (mean cell loss ±SE: 56.02%±11.4, compared to untreated cells, p<0.05). As judged by sequential Annexin-V/PI analyses, approximately 48.1% (±4.41) of the detached cells were initially viable, but subsequently (over 48-hrs) acquired phenotypic features of early apoptosis, late apoptosis and necrosis, compatible with anoikia. Prior to detachment, SGEC treated with polyI:C were found to express 1.9-folds (±0.3) higher levels of BimEL mRNA than untreated cells. On the other hand, the inhibition of Fas, IFN-beta, IFN-gamma or TNF-alpha failed to protect SGEC from polyI:C-induced anoikia. Compared to control-SGEC, SS-SGEC lines manifested significantly increased susceptibility to anoikia after treatment with low amounts of polyI:C (50 ng/ml and 500 ng/ml, p = 0.01 and p = 0.02, respectively).

Conclusion: TLR-3 triggering of cultured SGEC by polyI:C appears to induce significant anoikia associated with increased expression of the pro-apoptotic molecule BimEL. SS-SGEC were found to be more sensitive to polyI:C-induced anoikia than controls, possibly owing to the increased constitutive TLR-3 expression of these cells. These findings may be suggestive of increased vulnerability of salivary epithelia of SS patients to viral infection.

098 TRANSCRIPTIONAL REGULATION OF CONSTITUTIVE AND LPS-INDUCED TOLL-LIKE RECEPTOR-4 EXPRESSION IN THP-1 MONOCYTIC CELLS: ROLE OF INTERFERON REGULATORY FACTOR 1 (IRF-1) TRANSCRIPTION FACTOR

C. I. Maratheftis, M. P. Spachidou, H. M. Moutsopoulos, M. Voulgarelis.Pathophysiology Dept, Medical School, University of Athens, Greece

Aim: Toll-like receptors (TLRs) are thought to regulate various aspects of the pathological processes of chronic inflammatory diseases. TLRs recognize endogenous proteins released in response to stress or tissue damage such as heat shock proteins, necrotic cells and immune complexes that are found abundantly at sites of chronic inflammation. TLRs activation leads to the induction of pro-inflammatory gene expression; however, expression of many other genes, including the Tlr genes, has also been shown to be modulated following TLR triggering.

The transcription factors, IRFs, have been implicated in TLR signalling leading to pro-inflammatory gene expression. IRF-8, acting synergistically with IRF-1 and having suppressing transcriptional abilities, has been shown to recognize a binding site in the Tlr-4 gene promoter and suppress its expression. In this study, we investigated the plausible implication of IRF-1 in TLR-4 and ICAM.1 expression as well as in the level of apoptosis.

Material and methods: Small interfering RNA-based (siRNA) process was employed to silence IRF-1 gene expression in the leukemic monocytic cell line (THP-1). The mRNA of TLR-4 was measured using Real-time PCR, whereas the protein levels of TLR-4 and ICAM.1 were detected by flow cytometry. The apoptotic levels were estimated with Annexin-V binding assay either constitutively or after LPS-triggering.

Results: The silenced IRF-1 THP-1 cells (siIRF-1) presented elevated levels of TLR-4 mRNA and protein by 90±10% and 77±8% (p<0.01) respectively, in contrast to the control population (treated with a scrambled non-sense nucleotide, scrIRF-1). LPS addition increased the TLR-4 protein levels in siIRF-1 THP-1 populations (46±8%) compared to controls. LPS triggering did not alter the TLR-4 protein expression in the siIRF-1 cells. The siIRF-1 cells presented an 8.35 fold increase in ICAM.1 expression when compared with the control group (p<0.01). Following LPS-triggering, siIRF-1 cells revealed a 3.98 fold increase in ICAM.1 protein levels when compared to the LPS-treated controls and a 27.3 fold increase when compared to the untreated controls. In the scrIRF-1 cells, the Annexin-V+ cells were 7±2% of the total population. The siIRF-1 cells presented a significant increase in apoptosis (4.25 fold) with the Annexin-V+ cells reaching 29±3% [increased by 23% (p<0.01)]. LPS treatment of the control population led to a 0.55 fold increase in the Annexin-V+ positive cells (10.85±2%). In LPS treated siIRF-1 cells, the Annexin-V+ cells were 39.5±4%, revealing a 4.1 fold increase when compared with the LPS-control, and a 1.4 fold increase when compared with siIRF-1 cells before LPS addition.

Conclusion: IRF-1 is implicated in the expression of TLR-4 receptors and its silencing fully activates them on the surface of siIRF-1 cells. IRF-1 absence up-regulates ICAM.1 expression and prompts cell death.

099 CHARACTERIZATION OF CCL21 EXPRESSION IN HUMAN SECONDARY LYMPHOID ORGANS AND ECTOPIC LYMPHOID TISSUES

S. Bugatti1, A. Manzo1,2, R. Caporali1, R. Prevo3, D. G. Jackson3, M. Uguccioni4, C. D. Buckley5, C. Montecucco1, C. Pitzalis2.1Chair and Division of Rheumatology, University of Pavia, IRCCS Fondazione San Matteo, Pavia, Italy; 2Rheumatology Unit, Guy’s, King’s and St Thomas’ School of Medicine, Guy’s Campus, London, UK; 3Institute for Molecular Medicine, John Radcliffe Hospital, Oxford, UK; 4Institute for Research in Biomedicine, Bellinzona, Switzerland; 5Department of Rheumatology, Birmingham University, Birmingham, UK

Background: The chemokine (CK) CCL21 is required for T-cell and dendritic cell recruitment and organization into lymphoid T-areas. Alongside its constitutive function in secondary lymphoid organs (SLOs), CCL21 also plays a role in the constitution of ectopic lymphoid tissues (ELTs). An unresolved issue remains whether ectopic CCL21 is regulated by inflammation-specific processes or rather the same mechanisms account for CCL21 production in SLOs and ELTs. We thus systematically investigated the cellular sources of CCL21 in human SLOs and in lymphoid-neogenetic diseases.

Materials and methods: Twenty-seven synovial samples from rheumatoid arthritis (RA), 21 minor salivary glands from Sjogren’s syndrome (SS), 6 intestinal biopsies from Crohn’s disease (CD) and 10 from ulcerative colitis (UC) were analyzed. Control materials consisted of human tonsils and mesenteric lymph nodes (MLNs). CCL21-producing vessels were investigated by in situ hybridization and immunohistochemistry (IHC) for the pan-vascular markers CD31 and CD34, the high-endothelium venule (HEV) marker PNAd and the lymphatic markers LYVE-1, podoplanin and D6. CCL21-producing non-endothelial cells were characterized by combined IHC and immunofluorescence for CCL21, the haemopoietic marker CD45 and smooth muscle actin (SMA), which is expressed by myofibroblasts and fibroblastic reticular cells.

Results: All CCL21-producing vessels in RA synovium were CD31+LYVE-1+podoplanin+lymphatic vessels displaying variable levels of D6. Conversely, inflamed blood vessels lacked CCL21 mRNA. No PNAd expression was found in CCL21+ vascular structures. The exclusive lymphatic nature of CCL21+ vessels in ELTs was confirmed in different diseases (SS, CD, UC), and overlapped the vascular pattern of the CK in human SLOs. In some lymphoid aggregates in RA, SS, CD and UC, CCL21+ cells were found peri-vascularly, sometimes tightly adherent to PNAd+ HEVs. Such peri-HEV expression qualitatively overlapped the distribution of the CK in human SLOs. As in SLOs, CCL21-producing cells were also scattered within T-cell areas of ELTs. All CCL21+ cells adjacent to HEVs and those scattered in the T-zone both in human MLNs and in synovial ELT were CD45-negative stromal cells. Furthermore, CCL21-expressing cells in MLNs co-localized with a meshwork of SMA+ dendritiform cells disseminated throughout the node. Similar co-localization was observed in RA ELT where, despite most of CCL21+SMA+ cells were isolated or organized in small clusters, a more developed lymph node-like CCL21+SMA+ meshwork characterized a proportion of large-size aggregates.

Conclusions: Our results demonstrate that lymphatic vessels and myofibroblast-like cells account for CCL21 production not only in SLOs but also in ELTs. Furthermore, a vestige of the lymphoid fibroblastic reticular network can develop in ectopic sites. We conclude that ectopic CCL21 production is promoted by amplification of physiological mechanisms rather than by inflammation-specific pathways. The identification of SMA as a marker of enrichment for CCL21+ cells provides an important reference for future cell isolation and functional studies on the regulatory mechanisms of CCL21 production.

100 INTRA-ARTICULAR GENE THERAPY IS POTENTIALLY PREVENTED BY IMMUNITY AGAINST AAV SEROTYPES1, 2, 5 AND 8

M. C. Boissier1, D. Lemeiter1, C. Valvason1, T. Begue2, L. Laroche3, N. Bessis1.1INSERM ERI-18 and Rheum Dept, University Paris 13 and Avicenne hosp (AP-HP), Bobigny; 2Orthopaedics Dept, University Paris 13 and Avicenne hosp (APHP), Bobigny; 3Dermatology Dept, University Paris 13 and Avicenne hosp (APHP), Bobigny, France

Background: Intraarticular (i.a) gene transfer with AAV vectors may allow efficient therapeutic transgene expression within the joint in diseases such as rheumatoid arthritis (RA), allowing high expression of the protein within the joint, preventing both systemic diffusion and side effects. However, humans demonstrate antibodies against AAV virus, which can influence gene transfer. To better understand critical obstacles to i.a gene therapy with AAV, we have previously shown that synovial fluid (SF) contains IgG to AAV that neutralized chondrocyte infection in vitro. Our objective was therefore to compare neutralization exerted by SF for 4 different AAV serotypes (AAV1, 2, 5 and 8).

Methods: SF from 10 patients with RA were collected. Primary synovial cells were obtained from human synovium during surgical procedures. AAV with serotypes 1, 2, 5 and 8, encoding IL-4 cDNA, were produced (GTL, Nantes). Efficacy of synoviocytes infection by AAV interleukin IL-4 was assessed by ELISA within the culture supernatant. Neutralizing activity against AAV/IL-4 was determined by assessing the ability of SF to inhibit AAV/IL-4 transduction to synoviocytes

Results: Serotype 2 infected synoviocytes the most efficiently, and, in decreasing order, serotype 1, then 5, then 8. All SF partially inhibited infection on synoviocytes for at least one of the 4 serotypes. Infection with serotypes 1 and 2 were the most inhibited by SF, while inhibition was weak for serotype 5 and 8. Lastly, we have shown that the inhibition of AAV-1/IL-4 infection on synoviocytes cells by SF could be reversed by increasing the number of AAV-1/IL-4 particles, with a dose dependant effect.

Conclusion: The most infecting AAV serotypes (1 and 2) in synoviocytes are also the ones against whom neutralization exerted by the SF is the highest. Thus, serotype 5 seems to demonstrate the best infection efficiency/immunogenicity ratio for a local use in articular diseases. In the future, these data may be useful for tailoring intraarticular AAV-mediated gene therapy to individual patients.

101 WEGENER’S GRANULOMATOSIS IN WOMEN WITH ANKYLOSING SPONDYLITIS, HLA B 27 ANTIGEN POSITIVE FORM

S. Vladeva, I. Manolova, M. Galabova.Trakian University, Bulgaria

Summary: A case of Wegener’s granulomatosis in a 47-year-old female patient is described with pre-existing ankylosing spondylitis, HLA B 27antigen positive form. We present the problems in the diagnosis, treatment and evolution of systemic vasculitis. The stage of pulmonary involvement is diagnosed and treated like tuberculosis. The use of only antituberculosis therapy and small dose corticosteroid has reduced cavity infiltrations. We suppose retrospectively from the biopsy result these pulmonary lesions to be localized initial form of Wegener’s granulomatosis of the lower respiratory tract. Nasal involvement developed two years ago, with typical saddle-nose deformity. Antineutrophilic cytoplasmic antibodies with cytoplasmic pattern (cANCA) by indirect immunofluorescence were titre 1: 640 positive, PR3-25 U/ml positive, MPO – 2.9 U/ml negative.

102 AN ASSOCIATION STUDY OF AN INTERFERON-ALPHA RECEPTOR PROMOTER POLYMORPHISM IN RHEUMATIC DISEASES

G. B. N. Nordang1, A. Smerdel-Ramoya1, B. Flato2, A. M. Selvaag2, E. Thorsby1, T. K. Kvien3, O. Forre2, B. A. Lie1.1Inst. Of Immunology, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway; 2Dept. of Rheumatology, Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway; 3Dept. of Rheumatology, Diakonhjemmet Hospital, Oslo, Norway

Background: Interferons (IFNs) are known to be important mediators of immune responses. Binding of type I IFNs to the heterodimeric IFN-alpha receptor (IFNAR) trigger a signal cascade which therefore may result in the initiation or amplification of an autoimmune process and further tissue damage. Different type I IFNs has been linked to various autoimmune diseases. Elevated serum levels of IFN-alpha are correlated with disease severity or activity in systemic lupus erythematosus (SLE). In juvenile idiopathic arthritis (JIA) and rheumatoid arthritis (RA) accumulation of IFN producing cells in the inflamed joints have been observed.

Objectives: To study the association between a promoter polymorphism in the IFNAR1 gene and susceptibility to different rheumatic diseases in the Norwegian population.

Materials and method: 166 SLE patients, 474 JIA patients, 863 RA patients and 654 healthy controls were genotyped by allelic discrimination for the -408 C>T polymorphism (rs16997869, Taqman by demand, Applied Biosystems). Both patients and controls were in Hardy-Weinberg equilibrium for the polymorphism.

Results: No significant deviation in the allele frequencies (C-allele: SLE: 72%; JIA: 74%; RA: 70%: controls: 72%), nor in genotype frequencies, were observed between patients and controls. Conclusion: Even though our current data do not support an involvement of the IFNAR1 gene in these different rheumatic diseases, the tested polymorphism does not capture all the genetic variation within the IFNAR1 gene. Thus the next step will be to test other IFNAR1 polymorphisms.

103 COMPARATIVE ANALYSIS OF PERIPHERAL BLOOD GENE EXPRESSION PROFILES OF RHEUMATOID ARTHRITIS AND SYSTEMIC SCLEROSIS PATIENTS

C. L. Bos1, L. G. van Baarsen1, T. Timmer1, F. Rustenburg1, H. J. Thiesen2, B. A. C. Dijkmans2, T. C. T. M. van der Pouw Kraan1, A. E. Voskuyl3, C. L. Verweij1.1Department of Molecular Cell Biology and Immunology, VU Medical Center, Amsterdam, The Netherlands; 2Department of Immunology, University of Rostock, Germany; 3Department of Rheumatology, VU Medical Center, Amsterdam, The Netherlands

Objective: We set out to investigate whether there are differences in the peripheral blood gene expression profiles between rheumatoid arthritis (RA) and systemic sclerosis (SSc) patients.

Methods: Large-scale expression profiling by cDNA microarrays was performed on peripheral blood from 13 SSc patients, 28 RA patients and 6 healthy individuals. Differential gene expressions were obtained from Significance Analysis of Microarrays (SAM), subsequently analyzed by pathway analysis. Identification of SSc and RA subtypes was performed by unsupervised hierarchical clustering together with samples from healthy volunteers. Gene Set Enrichment Analysis (GSEA) was applied to identify pathways relevant to disease.

Results: A spectrum of genes involved in immune defence was remarkably elevated in peripheral blood of both SSc and RA patients compared to healthy individuals. SAM analysis revealed a highly significantly elevated expression of interferon (IFN) type 1 regulated genes in a subset of the RA and SSc patient, which was confirmed by Gene Ontology and Pathway analysis, suggesting a systemically activated pathway in both diseases. Preliminary comparative analysis revealed differences between the peripheral blood gene expression profiles of RA and SSc patients. Among these, we found that the mean expression levels of IFN-induced genes was significantly higher in SSc compared to RA.

Conclusions: Type 1 IFN regulated genes are highly upregulated in a subset of SSc and RA patients. Among the differences observed, the mean expression levels of type 1 IFN regulated genes were significantly increased in SSc compared to RA.

104 CLINICAL AND SEROLOGICAL FEATURES OF 29 PATIENTS WITH ANTI-KU ANTIBODIES

I. Cavazzana, A. Ceribelli, F. Franceschini, M. Quinzanini, L. Bettoni, R. Cattaneo.Rheumatology Unit and Chair, Spedali Civili- Università degli Studi di Brescia, Italy

Background: Anti-Ku antibodies, recognising a DNA-binding protein of 70/80 kD, are originally described as a marker of SSc-PM overlap syndrome. We previously described 14 patients with anti-Ku affected by different autoimmune diseases, showing mild articular and muscular involvement.

Aim: To analyse clinical and serological features associated to anti-Ku antibodies in 29 patients, followed in Italian rheumatologic outpatients clinic.

Patients and methods: 29 patients with anti-Ku antibodies were retrospectively analysed. Anti-Ku were detected by counterimmunoelectrophoresis (CIE) using rabbit thymus extract and confirmed by commercial immunoblot (IB).

Results: 29 patients (25 female and 4 male, mean age at onset: 52.7 y, SD: 17.6 y) were affected by different autoimmune diseases and followed for a mean of 7.7 years (SD: 6.5). An overlap syndrome was diagnosed in 8 patients (6 PM/SSc, 1 DM/SSc, 1 PM/SLE), while UCTD and SLE were diagnosed in 8 and 6 subjects, respectively. Three patients were affected by primary SS and two by PM. SSc and RA were diagnosed in one subject each. The main clinical features were represented by arthralgias (83%), arthritis (52%), myositis (34%), Raynaud’s phenomenon (69%), xerostomia (58.6%) and xerophthalmia (41.4%). Pulmonary interstitial involvement was detected in 12 cases (44.4%) with a severe reduction of CO diffusion in 8 patients, while two cases only developed pulmonary hypertension. Isolated anti-Ku antibodies were detected in 14 sera (48.3%): 7 affected by myositis, 6 by UCTD and 1 by SSc. The remaining 15 patients had anti-Ku antibodies associated with other antinuclear specificities: anti-Ku and anti-dsDNA antibodies in 6 sera, anti-Ku and anti-Ro/SSA in 4 sera, anti-Ku with multiple antinuclear antibodies in 5 sera (namely, anti-La, anti-DNA, anti-Ki). No difference was found comparing clinical and laboratory features of isolate anti-Ku positive patients and patients with anti-Ku associated with other anti-nuclear specificities. No sera showed anti-centromere, anti-Sm or anti-topoisomerase I antibodies, while anti-cardiolipin and anti-β2glycoprotein I antibodies were detected in 25% and 24% of sera, respectively.

Conclusions: Anti-Ku antibodies are detectable in different autoimmune diseases, frequently as overlap syndromes including SLE, SSc and myositis. Raynaud’s phenomenon, articular and muscular involvement, represent the main clinical features associated with anti-Ku. Twelve patients developed pulmonary involvement with severe diffusion reduction in 8 of them. Isolate anti-Ku antibodies were present in about 50% of sera, while specific markers of SLE or SSc (namely anti-Sm, anti-centromere or anti-Scl70) were not detectable.

105 ANALYSIS OF REGULATORY T CELL POPULATIONS IN PATIENTS WITH AUTOIMMUNE DISEASE

M. Bonelli1, A. Savitskaya1, K. v. Dalwigk1, A. Rapp1, J. S. Smolen1, C. Scheinecker1.1Department of Rheumatology, University of Vienna, Austria

Introduction: Regulatory T cells (Treg) specialize in the suppression of immune responses and can be identified among CD4+ T cells by the expression of the transcription factor Foxp3. Besides Foxp3, Treg express the IL-2R alpha chain CD25, which is also expressed on activated T cells and is therefore considered of minor specificity. Treg might be critically involved in the pathogenesis of autoimmune diseases but there is still only limited information concerning the phenotypic and functional characteristics of Treg, and moreover of Treg subsets, in autoimmune diseases. We therefore performed phenotypic analysis of CD4+Foxp3+ T cells in SLE patients (SLE), patients with Systemic Sclerosis (SSc) and with Rheumatoide Arthritis (RA) as compared to healthy controls (HC).

Methods: Peripheral blood mononuclear cells from SLE (n = 18), SSc (n = 3), RA (n = 7) patients and HC (n = 6) were analyzed by 6-color flow cytometry (FACS). Proportions of Treg and Treg subsets were correlated with clinical data, the daily cortisone dose and the European Consensus Lupus Activity Measurement (ECLAM), the SLE disease activity index (SLEDAI) and SLE index score (SIS). In addition, in vitro cultures of purified CD4+ T cells with different cytokine combinations were performed.

Results: In contrast to SSc, RA patients and HC, we consistently observed a population of CD4+Foxp3+ T cells in SLE patients, that virtually lacked expression of CD25. Similar as CD4+CD25+Foxp3+ T cells, CD4+CD25-Foxp3+ T cells contained low proportions of CD69+ and HLA-DR+ cells. On the other hand CD4+CD25-Foxp3+ T cells were found to be CTLA-4+, CD27+, CD45RBlow CD30low and negative for CD103. In vitro stimulation of purified CD4+CD25- T cells with IL-2, IL-10, TNF-alpha, INF-gamma and phytohaemagluttinin did not induce Foxp3 expression. A significant correlation of proportions of CD4+CD25-Foxp3+ T cells was observed for the SLEDAI and the ECLAM score as well as the daily cortisone dose.

Conclusion: A population of CD4+CD25-Foxp3+ T cells can be consistently observed in SLE patients compared to SSc and RA patients and HC. CD4+CD25-Foxp3+ T cells might arise either to counteract, or as consequence of, disease activity. Phenotypic and in vitro culture experiments, however, suggest that CD4+CD25-Foxp3+ T cells represent a regulatory T cell population rather than recently activated T cells. Functional analysis are currently performed to determine the suppressive capacity of CD4+CD25-Foxp3+ in order to clarify their functional role in SLE patients.

106 CLINICAL ASSOCIATIONS TO HUMAN CYTOMEGALOVIRUS-SPECIFIC CD28null T cells in patients with polymyositis and dermatomyositis

A. Fasth1, M. Dastmalchi1, A. Rahbar2, C. Söderberg-Naucler2, C. Trollmo1, I. E. Lundberg1, V. Malmström1.1Rheumatology Unit and 2Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Introduction: Dermato- and poly-myositis, DM and PM, are chronic autoimmune muscle disorders leading to muscle weakness, decreased muscle endurance and reduced life expectancy. We have previously shown increased frequencies of hyperresponsive and perforin-expressing CD4+ CD28null and CD8+ CD28null T cells in the circulation of patients with PM and DM compared to healthy controls. These data are in concordance with previous publications where patients with different autoimmune diseases and chronic virus infections also have shown increased frequencies of CD4+ CD28null and CD8+ CD28null T cells. In addition to accumulation of CD28null T cells in the circulation of patients with DM and PM we have also shown that they are the major cell type infiltrating the inflamed muscle tissue. In this study we have analyzed the association of clinical parameters and presence of human cytomegalovirus infection (HCMV) with the frequency of circulating CD28null T cells.

Patients and analyses: 65 patients diagnosed for DM (n = 22) or PM (n = 43) were included in a multivariate regression analysis, which allows control for interaction between the variables. Included parameters were: diagnosis, age, disease activity, disease duration, response to treatment and HCMV-IgG serotyping. To test CD28null T cells for HCMV reactivity PBMC from 5 patients positive for HCMV-IgG and 4 patients negative for HCMV-IgG with PM and DM were cultured with HCMV-pp65 and HCMV-IEA. Following 12h stimulation the PBMCs were analyzed by flow cytometry and intracellular levels of IFN-gamma was used as readout for activation.

Results: With multiple regression analysis we found that chronic HCMV infection strongly increased the frequency of CD4+CD28null T cells, while each year of disease decreased the frequency. None of the other tested parameters significantly influenced the frequency of CD4+CD28null T cells. The determination coefficient (R2) for prediction of the frequency of CD4+ CD28null T cells by this model was 0.39, p<0.05. The frequency of CD8+ CD28null T cells was as well strongly associated to HCMV infection and decreased with about one percentage point for each year of disease. Interestingly, the CD8+ CD28null T cells did in addition increase with age. The determination coefficient for the prediction of the frequency of CD8+ CD28null T cells was 0.25, p<0.05. Results from the reactivity assays confirmed the associations to HCMV since the CD28null T cells were activated by HCMV-peptide stimulation.

Conclusion: This study demonstrates a clear association of CD28null T cells to chronic HCMV infection and indicates a decrease of these cells with increasing disease duration in DM and PM. Hence we suggest a role for HCMV infection in the pathogenesis in subsets of DM and PM and that the effector T cells, CD28null T cells, are consumed during the progress of disease.

107 AUTOANTIBODIES AND ORGAN INVOLVEMENT IN SYSTEMIC SCLEROSIS

M. De Santis1, F. Pregnolato2, B. Tolusso1, S. Bosello1, G. Peluso1, S. Alivernini1, P. Meroni2, G. Ferraccioli1.1Department of Rheumatology, Catholic University, Rome; 2Unit of Immunology and Rheumatology, IRCCS, Auxological Institute, Milan, Italy

Aim: To investigate the non-organ-specific autoantibodies frequency in systemic sclerosis (SSc) patients and the possible association with internal organ involvement.

Patients and methods: 95 SSc patients (mean age 55.8 SD 11.9 years, mean disease duration 8.3 SD 7.9 years, 13 males, 33 with diffuse skin disease) were enrolled in a cross sectional study. All patients were screened for the following autoantibodies: anti-centromere (ACA) (immunofluorescence (IF)), anti-nucleolus (IF), anti-Scl70 (ELISA), anti-SSA 60 KDa and 52 Kda (ELISA), anti-ribonucleoprotein (ELISA), anti-single-strand-DNA (ELISA and IF on Crithidia luciliae), anti-histones (ELISA), anti-chromatin (IgG and IgM) (ELISA), anti-RNA-polymerase (ELISA), anti-IFI16 (ELISA), rheumatoid factor (RF IgM and IgA) (ELISA), p and c anti-neutrophil-cytoplasmic (ANCA) (ELISA), anti-mitochondrial (IF), anti-smooth-muscle (IF) and lupus anticoagulant (determined through the index of circulating anticoagulant, the tissue thromboplastin inhibition test and the dilute Russell viper venom test with confirming tests). Moreover the patients were classified based on skin involvement and internal organ involvement (restrictive lung diseases on pulmonary function tests, lung fibrosis on high resolution computed tomography, pulmonary arterial hypertension (PAH) isolated or associated with lung fibrosis, chronic renal failure based on serum creatinine value, myocardial ischaemia and arrhythmias, esophageal involvement on esophageal manometry, myositis, skin ulcers).

Results: ACA were found in 35% of patients, anti-Scl70 in 40%, anti-nucleolus in 5%, anti-SSA 60 KDa in 5%, anti-SSA 52 KDa in 3%, anti-ribonucleoprotein 7%, anti-single-strand-DNA in 8%, anti-histones in 31%, anti-chromatin IgG in 57%, anti-chromatin IgM in 19%, anti-RNA-polymerase 12%, anti-IFI16 27%, RF IgM in 18%, RF IgA in 15%, pANCA in 7%, cANCA in 0 patients, anti-mitochondrial in 4%, anti-smooth-muscle in 0 patients and lupus anticoagulant in 22%. Besides confirming the association between antiScl70 and either diffuse skin involvement or interstitial lung disease and between ACA and either limited skin involvement or isolated PAH, a significant association was found between antiScl70 and anti-chromatin IgG (p = 0.0003) and between ACA and anti-histones (p<0.0001). Anti-histones antibodies were also significantly associated with isolated PAH (p = 0.0009). Moreover renal failure was found to be associated with anti-RNA-polymerase (p = 0.005) and skin ulcers with antiScl70 (p = 0.02), antiSSA 52 KDa (p = 0.009), RF IgM and IgA (p = 0.04 and 0.02 respectively) and lupus anticoagulant (p = 0.004).

Conclusions: Different autoantibodies have been found in our SSc cohort and they associate with various organ involvements within the two major subsets of SSc.

108 SYSTEMIC SIRNA LIPOPLEXES SILENCING PRO-INFLAMMATORY CYTOKINES SUCCESSFULLY PREVENTS AND CURES ARTHRITIS

M. Khoury1,2, V. Escriou3,4,5,6, A. Galy7, R. Yao7, C. Largeau3,4,5,6, D. Scherman3,4,5,6, C. Jorgensen1,2,8, F. Apparailly1.1Inserm U844, Montpellier, France; 2Université Montpellier1, Montpellier, France; 3Inserm, U 640, Paris, France; 4CNRS, UMR8151, Paris, France; 5Université Paris Descartes, Faculté de Pharmacie, Laboratoire de Pharmacologie Chimique et Génétique, Paris, France; 6Ecole Nationale Supérieure de Chimie de Paris, Paris, France; 7Inserm, U 790, Stem cell group, Genethon, Evry, France; 8CHU Lapeyronie, service Immuno-Rhumatologie, Montpellier, France

Aim: TNF-alpha is one of the most prominent cytokines responsible of the physiopathology of rheumatoid arthritis (RA). We recently demonstrated that systemic non-viral delivery of a siRNA targeting TNF-alpha efficiently restored an immunological balance in an experiment model of RA. Yet, 30% of treated patients do not respond to anti-TNF biotherapies. Strong association of other pro-inflammatory cytokines with the pathogenesis of RA prompted us to seek which cytokine other than TNFalpha could be targeted for therapeutic benefit using RNA interference.

Methods: Two siRNA sequences were designed for IL-1beta, IL-6 and IL-18 pro-inflammatory cytokines and their efficacy and specificity was validated in vitro on J774.1 mouse macrophage cells, measuring both mRNA and protein levels following an LPS challenge. For in vivo administration, siRNAs were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA and 10 µg was injected intravenously per mouse in DBA/1 mice having collagen-induced arthritis (CIA). Clinical course of the disease was assessed by paw thickness over time, and radiological and histological scores were obtained at euthanasia. The cytokine profiles were measured by ELISA in sera and knee-joint conditioned media. The immunological balance was assessed using anti-type II collagen assays. The distribution of siRNAs was evaluated by fluorometry in GFP transgenic mice treated with anti-GFP siRNA-lipoplex injection.

Results: The designed siRNA sequences provoked a 70–75% decrease of the LPS-induced IL-1beta, IL-6 and IL-18 mRNA levels in macrophages in vitro compared with the control siRNA. Each siRNA affected the targeted cytokine specifically without modifying other pro-inflammatory cytokine mRNAs. In the CIA arthritis model, weekly injections of siRNA-lipoplexes significantly reduced incidence and severity of arthritis, abrogating joint swelling, destruction of cartilage and bone, in both preventive and curative settings. The most striking therapeutic effect was observed when combining the 3 siRNAs targeting IL-1beta/IL-6/IL-18 as once. Such tri-therapy was associated with down-regulation of both inflammatory and autoimmune components of the disease, and overall parameters were improved compared with the TNF-alpha siRNA lipoplex-based treatment. The siRNA formulation was widely distributed, delivering the siRNA to several organs with a strong efficacy in liver and spleen.

Conclusion: Tri-therapy targeting IL-1beta/IL-6/IL-18 seems highly effective to reduce all pathological features of RA including inflammation, joint destruction and Th1 response. These data show that cytokines other than TNF-? can be targeted to improve symptoms of RA and reveal novel potential drug development targets. The systemic administration of anti-cytokine siRNA cocktails as lipoplexes could represent a novel and promising anti-inflammatory therapy in RA.

109 ENDOTHELIN-1 AND ESTROGENS INCREASE EXTRA CELLULAR MATRIX EXPRESSION BY CULTURED HUMAN SKIN FIBROBLASTS

P. Montagna1, M. Cutolo1, S. Soldano1, S. Shabanova2, A. Sulli1, S. Capellino1, C. Pizzorni1, M. Secchi1, B. Seriolo1, S. Paolino1, A. Parodi3, B. Villaggio4.1Division of Rheumatology - Dept Internal Medicine, University of Genova, Genova, Italy; 2Department of vascular disorders, Institute of Rheumatology, Moscow, Russia; 3Dept Dermatology, University of Genova, Genova, Italy; 4Division of Nephrology - Dept Internal Medicine, University of Genova, Genova, Italy.

Background: Endothelin-1 (ET-1) exerts vasoconstrictive properties but also potent mitogenic effects on fibroblasts (Fb). Therefore ET-1 may have a role both in the initiation and in the maintenance of fibrosis by increasing extra cellular matrix (ECM) synthesis (i.e. Fibronectin-FN) and collagen metabolism (i.e, prolyl-4-hydrohylases - PrH).1234 Estrogens (E) are involved in autoimmunity as enhancers of the immune response and cell proliferation; opposite effects are exerted by androgens (A) (5).

Aim: To evaluate the effects of mitogenic Endothelin-1 (ET-1), on extra cellular matrix (ECM) synthesis and collagen metabolism (i.e. Fibronectin-FN and prolyl-4-hydrohylases-PrH) of cultured human normal fibroblasts (Fb) in presence of Estrogens (E) or Androgens (A) in order to evaluate possible synergistic gender effects.

Methods: Cultures of normal Fb (obtained from skin biopsy of 6 volunteer subjects after informed consent) were characterised by immunocytochemistry (ICC, mAb anti-human Fb surface protein =  Fb-SP). Serum-starved Fb (10.000) were cultured in Flexiperm chamber slides and treated for 24 hrs with the ET-1 concentration having mitogenic effect (100 nM). Further aliquots of Fb were treated with 17beta-estradiol (E2) or testosterone (T) (100 pM and 1 nM, respectively) alone or plus ET-1 for 24 hrs. Cell proliferation was evaluated by the methyltetrazolium salt test (MTT). Fb were immunostained with mAb anti-human FN and anti-human PrH (dilution 1:100). ICC staining was performed with the biotin-streptavidin-peroxidase method and image analysis by the Leica Q500 MC System. Experiments were done in triplicate.

Results: A significant increase of the FN synthesis was detected by ICC at 24 hrs in ET-1 and ET-1 plus E2 treated-Fb versus untreated control Fb (p<0.001 and p<0.01, respectively). Opposite effects were found when Fb were treated with T (p>0.05). However, the comparison between E2 and T-treated Fb was still significant (p<0.001). No significant changes were observed for PrH, excluding a significant reduction after T treatment (p<0.001). The addition of ET-1 to T treated cells did not determine a significant difference versus control.

Conclusions: Results indicate that ET-1 is able to enhance the ECM synthesis (i.e. FN) by normal human Fb. Interestingly, E2 per se and in combination with ET-1 was found to further enhance the proliferative and ECM-synthetic activity of treated normal Fb. In fact, estrogens are involved in autoimmunity as enhancers of the immune response and cell proliferation; opposite effects are exerted by androgens.5

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110. THE ROLE OF THE ADIPOKINES RESISTIN AND ADIPONECTIN AND OF SURFACTANT PROTEIN A AS MODULATORS OF THE PROINFLAMMATORY RESPONSE IN RA

I. H. Tarner1, A. Knedla1, S. Lefevre1, J. Steinmeyer2, H. Stuerz3, W. Seeger4, A. Guenther4, S. Gay5, U. Mueller-Ladner1, E. Neumann1.1Dept. Rheumatology, 2Dept. Exp. Orthopedics, 3Dept. Orthopedic Surgery, 4Dept. Int. Med. II, University of Giessen, Germany; 5Ctr. Exp Rheumatol, University Hospital Zurich, Switzerland

Objectives: Adipokines appear to play a specific role in inflammation in rheumatoid arthritis (RA). Resistin, a new adipokine, belongs to a family of cysteine-rich proteins. It accumulates in inflamed joints of RA patients, and intraarticular injection of resistin in mice induces synovitis and cartilage destruction. Surfactant protein A (SP-A) belongs to the collectin-family, is the main protein constituent of surfactant, is primarily expressed in the lung, and shows striking homologies to adiponectin. Patients with RA show increased concentrations of SP-A in synovial fluid and SP-A specific autoantibodies have been identified. As Resistin and SP-A may influence inflammation and cartilage destruction in RA, their effects in comparison to adiponectin on inflammation and matrix degradation were examined.

Methods: Synovial tissue specimens were obtained from patients with RA and osteoarthritis (OA) undergoing joint replacement. After enzymatic digestion, isolated RA synovial fibroblasts (RASF) and OASF were cultured under standard conditions for 4 passages. Cells were stimulated at 70% confluency with different concentrations of resistin (1 µg/ml), SP-A (5 µg/ml) and adiponectin (25 µg/ml). The effects on production of cytokines and matrix-degrading enzymes in RASF and OASF were measured by ELISA.

Results: Resistin induced a 1.7-fold increase of IL-6 and a 5.2-fold increase of IL-8 in OASF, whereas in RASF, the induction was weaker and not significant (IL-6: 1.1- fold, IL-8: 1.4-fold). In contrast, the induction of IL-6 and IL-8 in RASF was even stronger in response to Adiponectin (8.3- and 11-fold, respectively). Interestingly, resistin was able to reduce activin expression 2.5-fold in RASF, but no effect was seen in OASF. In addition, stimulation of RASF with resistin resulted in a 1.2-fold induction of Follistatin. In contrast to adiponectin, which showed a 2-fold induction of TIMP-1 in RASF, resistin had no effect. Moreover, adiponektin induced a 10-fold increase of MMP-3 and an 8.2-fold increase of proMMP-1 in RASF. MMP-3, TNF and IL-12 were not induced by resistin. No regulation of cytokines or MMPs in SF was seen after stimulation with SP-A.

Conclusion: These data suggest that resistin and adiponectin, but not SP-A, contribute to inflammatory responses in arthritis by inducing the production of pro-inflammatory cytokines and matrix degrading enzymes in SF.

111 CCR2-DEFICIENT MICE ARE OSTEOPETROTIC DUE TO DISTURBED OSTEOCLASTOGENESIS

N. B. Binder, B. Tuerk, O. Hoffmann, G. Schett, J. S. Smolen, K. Redlich.Department of Rheumatology, Medical University Vienna, Austria

Enhanced osteoclast (OC) activity or uncoupling of osteoclastic bone resorption from bone formation results in focal or generalized bone loss. This is a characteristic feature of several bone diseases such as osteoporosis or Paget disease. Beside the two major osteoclastogenic cytokines M-CSF and RANKL, other cytokines and chemokines are active contributors in OC formation. Among them monocyte chemotactic protein 1 (MCP-1), signalling through its receptor CCR2 might also play a role in osteoclastogenesis. We therefore analyzed the effects of CCR2 deficiency on osteoclastogenesis in vivo and in vitro. To this end histological sections from tibial bones of CCR2-/- and wild-type mice were used. CCR2-/- mice exhibited a 50% increased bone volume. Bone mineral density (BMD) measured by micro CT was also higher in CCR2 deficient mice. This was accompanied by a 40% decrease in OC numbers, indicating that CCR2 is indeed a mediator of osteoclast formation. To test if CCR2 has also an influence on the capacity of OCs to resorb bone, CCR2-/- and wild-type OCs, were cultured on bovine bone slices after stimulation with M-CSF and RANKL. A marked reduction of bone resorption by CCR2-/- OCs clearly points out that this receptor is not only important for OC development but also function.

112 IL-1-BETA, MMP-1, AND MMP-3 TRANSCRIPT LEVELS ARE ASSOCIATED WITH CD3-DELTA EXPRESSION IN THE OSTEOARTHRITIC SYNOVIAL MEMBRANE

C. R. Scanzello1, A. D. Pearle2, E. F. DiCarlo3, M. K. Crow1.1Departments of Rheumatology, 2Orthopedics, and 3Laboratory Medicine, Hospital for Special Surgery, New York, USA

It is known that many patients with osteoarthritis (OA) develop inflammatory infiltrates containing T-lymphocytes within the synovial membrane. Whether these infiltrates play an active role in propagation of disease is unclear, but they have the potential to augment cytokine (i.e. IL-1b and TNF-a) and degradative enzyme (i.e. MMPs) production by macrophages and synovial fibroblasts.

Aim: To determine if lymphocytic synovial infiltration in patients with OA was associated with increased expression of cytokines and enzymes implicated in cartilage catabolism.

Methods: Synovial membrane (SM) specimens from 37 OA patients undergoing total hip or knee arthroplasty (n = 31) or arthroscopy (n = 6) were obtained. RNA was extracted from SM specimens, and cDNA synthesized. Expression levels of CD3-d IL-1b, TNF-a, IL-15, IL-6, IL-10, TGF-b1 MMP-1, and MMP-3 were measured by quantitative real-time PCR. Synovial inflammation was graded histologically in H&E stained sections on a four-point scale representing increasing mononuclear cell infiltrate. Spearman correlation coefficients were calculated to determine if transcript levels of the cytokines and enzymes varied with level of CD3-d expression or histologic score.

Results: CD3-d transcript levels correlated most significantly with IL-1b (r = 0.677, p<0.0001), but were also associated with TNF-a (r = 0.487, p = 0.003), IL-15 (r = 0.524, p = 0.001), IL-10 (r = 0.570, p = 0.0004), and TGF-b1(r = 0.381, p = 0.022). CD3-d mRNA levels were also associated with MMP-1 (r = 0.432, p = 0.008) and MMP-3 (r = 0.506, r = 0.0014) mRNA levels. When utilizing our synovial inflammation grading scale, we found that MMP-1 expression was significantly associated with increasing histologic score (r = 0.412, p = 0.011). Furthermore, there were trends toward correlations between histologic score and IL-1b (r = 0.315, p = 0.057) as well as CD3-d (r = 0.303, p = 0.068).

Conclusions: mRNA expression levels of cytokines including IL-1b, as well as MMP-1 and MMP-3 correlated with levels of CD3-d transcripts within the SM of patients with both knee and hip OA. IL-1b is known to promote degradative enzyme synthesis in chondrocytes. The relationship between expression of IL-1b, MMP-1 and MMP-3 with CD3-d levels suggests that synovial infiltrating T-cells may contribute to disease pathogenesis in OA by promoting synthesis of these mediators.

113 ALTERED IL-1ß SECRETION IN A PATIENT AFFECTED BY SCHINTZLER’S SYNDROME

C. Pizzirani, A. Lo Monaco1, S. Falzoni, M. Govoni1, R. La Corte1, M. Bruschi1, F. Trotta1, F. Di Virgilio.Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara; 1Department of Clinical and experimental Medicine, Section of Rheumatology, University of Ferrara, Italy

Introduction: Schintzler’s syndrome (SS) is a rare disabling disorder characterised by a chronic urticarial rash and a monoclonal IgM gammopathy together with at least two of the following features: intermittent unexplained fever, arthralgia or arthritis, bone pain, lymphoadenopathy, hepato- or splenomegaly and an acute phase response. One of the clinical features of typical Schnitzler’s syndrome is IgM macroglobulinemia, but rare cases with IgG gammopathy have been observed. IL-1ß is considered to play a central role in the pathogenesis of this disease as well as for the other autoinflammatory syndrome, and purinergic P2 receptors (P2Rs) are implicated in its secretion.

Objective: To test the in vitro effects of extracellular nucleotide stimulation on IL-1ß/IL-18 secretion in order to verify the role of P2Rs in the cytokines release.

Patient and methods: The patient was a 42 year old woman with a three year history of chronic fever (>39°C) associated with polyarthritis, latero-cervical lymphadenopathy and daily recurrent episodes of pruritic urticarial lesions on the extremities, trunk and face. Laboratory tests showed an increased C-reactive protein (CRP 6.30 mg/dl), erythrocyte sedimentation rate (120 mm/h) and the presence of monoclonal IgG-kappa gammopathy. A complete battery of tests for autoimmunity, infection diseases, cryoglobulinaemia and malignancy did prove negatives, thus an atypical subset of SS was suspected.

Leucocytes were purified by Ficoll gradient from 20 ml of peripheral blood of both a healthy donor and the patient. Cells were left untreated or primed for 2 h with 1 µg/ml bacterial endotoxin (LPS). LPS-treated cells were then stimulated for 30 min with increasing concentrations of 2′,3′-(4-benzoyl-benzoyl)-ATP (Benzoyl ATP). The presence of proinflammatory cytokines in the culture supernatant were measured with the human IL-1β (R&D Systems, Minneapolis, MI, USA) and IL-18 ELISA kits (MBL, Woburn, MA, USA).

Results: Leukocytes isolated from the untreated patient showed a spontaneous high basal IL-1β release. Unlike controls, LPS—without any other additional stimuli—triggered IL-1β secretion. In contrast to leukocytes from healthy subjects, the addition of a P2X7 receptor agonist, Benzoyl ATP (100–300 µM), caused a very small increase in cytokine release. Treatment with prednisone (0.5 mg/Kg/die) normalized the IL-1β basal secretion, as well as the secretory response to Benzoyl ATP. IL-18 secretion followed a similar pattern. Concomitant clinical remission was noticed.

Conclusion: High basal secretion of IL-1β from leukocytes of patient affected by SS was observed. Leukocytes, following LPS stimulation without any other additional stimuli, enhanced IL-1β secretion but failed to increase significantly IL-1β secretion in response to subsequent BzATP stimulation, thus a condition of “secretory exhaustion” could be considered. Corticosteroid treatment restores the normal secretive pattern in response to LPS. On the basis of these observations it is conceivable that subsequent stimulation by ATP, as a secretory trigger, might be not necessary in the IL-1β process of secretion in SS. Thus, a non-specific stimuli alone (i.e. bacterial products) could be enough to induce a dramatic inflammatory cascade in these patients.

114 CHALLENGING THE ROLE OF PHOSPHOINOSITIDE 3-KINASE GAMMA IN ARTHRITIS

S. Hayer1, N. Pundt2, T. Pap2, G. Kollias3, J. Penninger4, G. Schett5.1Division of Rheumatology, Medical University of Vienna, Austria; 2Division of Molecular Medicine of Musculoskeletal Tissue, University Hospital Muenster, Germany; 3Alexander Fleming Biomedical Sciences Research Center, Vari, Greece; 4Institute of Molecular Biotechnology, Austrian Academic Science, Vienna, Austria; 5Department of Rheumatology, University Hospital Erlangen, Germany

Aim: To investigate whether phosphoinositide 3-kinase gamma (PI3Kγ), a class IB PI3K which is involved in neutrophil and macrophage migration to infection sites, plays a role in the signalling of tumor necrosis factor (TNF)- mediated arthritis.

Methods: We crossed human TNF transgenic (hTNFtg) mice with PI3Kγ-/- into the F2 generation and compared offsprings of all 4 genotypes (wild-type, PI3Kγ-/-, hTNFtg, PI3Kγ-/- hTNFtg) for articular changes using immunohistochemistry and histomorphometry. In addition, we investigated the mRNA and protein expression of PI3Kγ in synovial fibroblast from patients with osteoarthritis and rheumatoid arthritis by quantitative real time PCR and Western blot.

Results: PI3Kγ-/- hTNFtg mice developed slight reduced clinical signs of arthritis than hTNFtg littermates, suggesting that TNF can induce chronic inflammatory arthritis in the absence of PI3Kã although to a lesser extend. Wild-type and PI3Kγ-/- mice did not develop arthritic symptoms. PI3Kγ-/- hTNFtg mice demonstrate less local bone erosions and articular cartilage degradation. However, recruitment of hematopoietic cells at sites of inflammation was not altered, as evident from similar macrophage and granulocyte numbers in the synovium of hTNFtg and PI3Kγ-/-hTNFtg mice. In addition, histomorphometrical analysis show a reduction of TNF-mediated systemic bone loss in the absence of PI3Kγ. Studies on human synovial fibroblasts, showed upregulation of PI3Kγ in rheumatoid arthritis but not osteoarthritis, suggesting that PI3Kγ is not confined to the hematopoietic compartment.

Discussion: Our data suggest that PI3K-gamma is involved but not critical for TNFalpha-mediated joint inflammation. Macrophages as well as neutrophils can enter the joint even in the complete absence of PI3Kγ. These findings together with the mesenchymal expression of PI3Kγ in human rheumatoid arthritis raise concern whether pharmacological inhibition of PI3Kγ is a suitable tool to effectively and/or specifically target influx of inflammatory cell in arthritis.

115 TNF-ALPHA KINOID VACCINATION-INDUCED NEUTRALIZING ANTIBODIES TO TNF-ALPHA PROTECTS MICE FROM AUTOLOGOUS TNF-ALPHA-DRIVEN CHRONIC AND ACUTE INFLAMMATION

L. Delavallée1, N. Bessis1, H. Le Buanec2, B. Bizzini2, D. Zagury2,3, M-CBoissier4.1INSERM/ERI-18, University of Paris 13, Bobigny, France; 2Neovacs Inc, Paris, France; 3University of Paris 6, Paris, France; 4APHP, Avicenne Hospital, Dept. of Rheumotology, Bobigny, France

Aim: The major proinflammatory cytokine TNF-alpha is a potent mediator of septic shock and a therapeutic target for chronic inflammatory pathologies including rheumatoid arthritis (RA). Currently available treatments use passive immunization with anti-human TNF-alpha (hTNF-alpha) mAbs, which showed high efficacy, although some concerns remain such as the high cost and side effects. As an alternative to the hTNF-alpha blocker approved drugs, we developed an active anti-hTNF-alpha immunotherapy, based on a vaccine comprised of a KLH-hTNF-alpha heterocomplex immunogen (hTNF-alpha kinoid) adjuvanted in IFA.

Methods: Mice transgenic for hTNF-alpha (TTg) were purchased from Taconic (Germantown, USA). They develop spontaneous arthritis 8–10 weeks after birth. Treatments consisted of 3 IM injections of hTNF-alpha kinoid (at days 0, 7 and 28) from 7 weeks of age; control groups received KLH. Anti-hTNF-alpha Abs were measured by ELISA and their bioactivity by hTNF-alpha-induced L929 cells cytotoxicity assay.

Arthritides were blindly evaluated by long-term (4 months) clinical scoring of four paws and histology at sacrifice. In parallel lethal shock was induced in TTg mice by hTNF-alpha and D-galactosamine IP injection.

Results: In TTg mice, hTNF-alpha kinoid vaccination elicited high titres of Abs which neutralized hTNF-alpha bioactivities but did not result in a cellular response to hTNF-alpha. Arthritides were clinically observed in all mice. Treated mice exhibited low scores of arthritis vs control mice treated with KLH alone (maximal scores: 1.4±0.2 vs 8.6±0.6, p<0.01) with a 13.4 days delay of onset (p<0.05) with low histological scores of inflammation (0.1±0.1 vs 1.5±0.1, p<0.01) or destruction (0.1±0.1, vs 1.2±0.1, p<0.01). Arthritic TNF-alpha kinoid vaccinated mice recovered from their clinical inflammation, since at the end of the experiment (4 months after priming) the incidence of arthritis was 1/8 (vs 7/7 in control mice, p<0.001). Mice treated by hTNF-alpha-kinoid were protected against TNF-alpha-galactosamine induced lethal shock (survival: 6/6, vs 0/6 in control groups); this protection was transferable by anti-hTNF-alpha anti-sera.

Conclusion: These data are the first demonstration of a 4 months anticytokine induction of autoimmune protection against acute and chronic hTNF-alpha dependent pathologies.

Funds were received from Neovacs, Debiopharm, Association de Recherche sur la Polyarthrite (ARP), Société Française de Rhumatologie (SFR), Agence National de la Recherche (ANR).

116 T CELL RECEPTOR-MEDIATED INDUCTION OF THE B CELL CHEMO-ATTRACTANT CXCL13 (BCA-1) IN ANTIGEN EXPERIENCED SYNOVIAL T LYMPHOCYTES

A. Manzo1, B. Vitolo2, M. Uguccioni3, C. Montecucco2, C. Pitzalis1.1Rheumatology Unit, King’s College London, UK; 2Cattedra di Reumatologia, Universita’ degli Studi di Pavia, Italy; 3Institute for Research in Biomedicine, Bellinzona, Switzerland

Aim: CXCL13 is a lymphoid chemokine instrumental in the follicular localization of B cells in secondary lymphoid organs and in ectopic lymphoid tissues of chronic inflammatory diseases. Recent studies demonstrate that a subset of tonsil T cells, involved in B cell differentiation and germinal centre reactions (CXCR5+ follicular helper T cells), can synthesize CXCL13 following immunological activation, suggesting a role of CXCL13 as a direct mediator of the T-B immunological synapsis. Although T-B cross-cooperation has been shown to contribute to the inflammatory activity in rheumatoid synovitis, the mechanisms regulating the dynamic interaction between these cell populations in the periphery are still unclear. To investigate whether the T cell receptor (TCR)-CXCL13 pathway can be induced in infiltrating T cells in peripheral inflamed tissues, we tested the capacity of synovial T cells to produce CXCL13 in rheumatoid arthritis (RA).

Methods: Radioactive in situ hybridization (ISH) and double immunohistochemistry were used to determine CXCL13 expression pattern in RA synovial samples. RA synovial fluid mononuclear cells (SFMC) were isolated by density gradient centrifugation and T cells purified by positive selection. CXCL13 mRNA and protein were analysed by real time PCR and immunofluorescence (IF). Phenotypic characterization was performed by multicolour FACS analysis. Cell cultures were established with un-stimulated synovial T cells and with SFMC stimulated with anti CD3-CD28 or autologous synovial fluid (ASF). CXCL13 expression after cell culture was determined by ELISA on supernatants, by FACS and quantitative PCR.

Results: Tissue expression studies revealed CD3 co-localization with a variable percentage of CXCL13+ cells within and outside synovial lymphoid aggregates (mean = 36.3%, range 8.3–54.7%). CXCL13 expression was also recognised in CD68+ cells but not in CD20+ and CD83+ cells. ISH confirmed area co-localization between CXCL13 protein and mRNA. CXCL13 production was confirmed by PCR and IF in synovial fluid primary T cells and by ELISA in culture supernatants. Multi-colour phenotypic analyses revealed CXCL13 expression in a subset of helper-memory cells (CD3+ CD4+ CD45RO+). Chemokine receptor expression analysis of CXCL13+ T cells showed uniform positivity for CXCR4 (mean = 98.1% positive cells), partial expression of CCR7 (44%) and CCR6 (21.6%), but rare expression of CXCR5 (6.6%). Regulation studies by in vitro functional assays demonstrated the significant effect of CD3-CD28 stimulation (p<0.01 vs unstimulated) but not ASF in CXCL13 protein expression by SFMC. CXCL13 transcriptional regulation after TCR stimulation was confirmed at mRNA level in short term culture experiments.

Conclusions: These results provide evidence that cells of the adaptive immune system and T cells can produce the B cell chemo-attractant CXCL13 outside secondary lymphoid organs and in chronic synovitis. Our data define a new property of synovial T cells and suggest a candidate molecular mechanism participating in T-B cell cognate and/or bystander interactions in RA.

117 LYMPHOCYTE, MONOCYTE AND ENDOTHELIAL ACTIVATION IN EARLY RHEUMATOID ARTHRITIS PATIENTS

F. Atzeni1, P. Sarzi-Puttini1, S. Piconi2, D. Trabattoni2, M. Schenal2, A. Batticciotto1, M. Turiel3, M. Carrabba1, M. Clerici2.1Unità di Reumatologia, Ospedale L Sacco, Università di Milano; 2Cattedra di Immunologia, Ospedale L Sacco, Università di Milano; 3IRCCS Unità di Cardiologia, Istituto Galeazzi, Milano

Background: Rheumatoid arthritis (RA) is associated with increased cardiovascular morbidity and mortality, which is modulated by anti-inflammatory treatment. Endothelial inflammation is correlated to increase expression of endothelial adhesion molecules.

Objective: To investigate the correlation between endothelial inflammation and lymphocyte and monocyte activation in early rheumatoid arthritis (ERA) patients.

Material and methods: 33 ERA patients (28F and 5M, mean age 52.3, range 28–77 yrs) and 11 healthy controls (HC) were enrolled in this study. ERA patients were followed up at the rheumatology outpatients clinic at the University Hospital L.Sacco and were asked to participate in this study, if they met the study criteria. All patients fulfilled the 1987 American College of Rheumatology (ACR) classification criteria for RA and duration of disease was >6 weeks and <1 year. Patients were not eligible if they had received previous disease-modifying anti-rheumatic drug (DMARD) therapy, if they were allergic to or intolerant of Methotrexate, if they had received dosages of oral steroids, or if they had received intraarticular injections within the last few weeks. Clinical and laboratory assessment included number of tender and swollen joints, duration of morning stiffness, ESR, CRP, anti-cyclic citrullinated peptide antibodies (anti-CCP), and RF. Blood was drawn between 08:00 and 09:00. The blood was immediately stored on −20°C. ERA patients were divided into two groups: individuals with positive or negative anti-cyclic citrullinated peptide antibodies (ERA-CCP+ and ERA-CCP-). Markers of immune activation were evaluated in lymphocytes (CD25, DRII, CD44, CD49d and CD11a) and monocytes (CD80, CD86, CD36, TLR-2 and TLR-4). Endothelial adhesion molecules (CD44, CD49d, CD11a and CD62L) were evaluated as well by flow cytometry on the same cells.

Results: As compared to results obtained in HC, CD4+/CD25+ T lymphocytes (p = 0.01) and CD14+/CD86+ monocytes were significantly increased in ERA patients (p = 0.04); these differences were more evident in ERA-CCP+ individuals. CD80 expression on monocytes was decreased in both ERA groups (CCP+, p = 0.01; CCP-, p = 0.02). No significant differences were observed in endothelial adhesion molecule expression.

Conclusions: These data suggest that early rheumatoid arthritis is characterized by lymphocyte and monocyte activation. No signs of endothelial inflammation are observed in ERA patients, suggesting that the first stage of RA is limited to the synovial tissue. Immune activation is mostly evident in ERA-CCP+ individuals.

118 STATUS OF INFLUENZA AND PNEUMOCOCCAL VACCINATION IN RHEUMATOID ARTHRITIS PATIENTS AND CONFIRMATION OF DATA FROM GP SURGERIES

V. Vagadia, M. Bridges.University Hospital of North Tees and Hartlepool, UK

Background: It has been found that patients with rheumatoid arthritis are at approximately doubled risk of infection as compared to normal populations; this is partly due to ill-defined immunoregulatory abnormalities associated with rheumatic diseases, but mostly this is due to their regular immunosuppressive therapy. Department of Health United Kingdom guidelines 05-06 and British Society of Rheumatology guidelines recommend that patients on immunosuppressive disease modifying anti-rheumatic drugs( DMARDs) like methotrexate, leflunomide, azathioprine and Anti TNF blockers should have annual influenza vaccination and once only pneumococcal vaccination. Even the CDC (Centres for Disease Control and Prevention) also recommend vaccination as mentioned above. Department of Health guidelines suggests that all patients >65 should have influenza and pneumococcal vaccinations and for <65 years age group, to those who are ‘At Risk’ only. The ‘At Risk’ group includes those who are immunosuppressive either due to disease or treatment and also patient groups who have chronic respiratory conditions, chronic renal failure, or chronic heart disease. We performed an audit to analyse status and awareness of pneumococcal and influenza vaccination in patients treated with DMARDs or Anti TNF blockers.

Methods: This was a prospective audit of 80 patients with rheumatoid arthritis and on DMARDs or biologics therapy across both Trust Hospitals, University Hospital of Hartlepool and North Tees. It was carried out between Feb 2006 to July 2006 and all patients data were cross checked from GP surgeries in October 2006. It was questionnaire based in outpatient clinics and later confirmed with actual figures from GP surgeries record. Patients were randomly selected from outpatient clinics to answer questions concerning vaccination status, presence of other risk factors and indication for vaccination. Information was also collected on medications and disease duration and ‘At Risk’ category.

Results: Out of 80 RA patients 55 (67%) were females and 45 (33%) were male. Among them 31 (40%) were >65 years old. 28 (35%) had a RA duration of 1–5 years, 22 (28%) had 6–10 years and 30 (37%) had more than 10 years of disease duration. In <65 years age group (n = 49) the majority were on methotrexate 28(58%) and the rest were on other single DMARD or combination of DMARDs with biologics. In the >65 years age group 84% had received regular influenza vaccination but only 52% had received pneumococcal vaccination. From GP surgery records it was found 72% for influenza and 40% for Pneumococcal vaccination. In the <65 years age group 43% had received influenza vaccination while only 11% had received pneumococcal vaccination which again varied from actual GP surgery records and in <65 age group it was 40% for influenza and 20% for Pneumococcal vaccination. There was quite low uptake rate of vaccination in ‘At Risk’ immunocompromised group. 8 patients had diabetes and were on DMARDs but had not received vaccination against either influenza or pneumococcus although 2 of them were offered vaccination.

Conclusions: Uptake of influenza vaccination in patients >65 years old is almost equivalent to the national target of 70% which could be due to widespread campaigns and awareness for vaccination in this age group. Uptake of influenza vaccination in patients <65 years ‘At Risk’ group receiving DMARDs is suboptimal. Pneumococcal vaccination uptake is unsatisfactory in any age groups which need more widespread campaigns and Rheumatology team input in annual review clinics. The Rheumatology team need to take a more proactive stance regarding patient education for need for pneumococcal and influenza vaccination in ‘At Risk’ group. Vaccination status should not be left upon general practitioners only, especially for ‘At Risk’ RA group. Outpatient clinic based questionnaire patient’s data may vary from actual data and may need to verify it if in doubt. So, how reliable is a patient questionnaire based study?

119 REFINEMENT OF THE CONTRIBUTION OF SMOKING TO THE ACPA RESPONSE

A. H. M. van der Helm-van Mil, K. N. Verpoort, T. W. J. Huizinga, R. R. P. de Vries, R. E. M. Toes.Dept of Rheumatology, Leiden University Medical Center, The Netherlands

Objectives: Recently two studies revealed that smoking interacts with the HLA-DRB1 shared epitope (SE)-alleles for the presence of anti-citrullinated peptide antibodies (ACPA) and that smoking had no effect on the presence of ACPA in SE-negative rheumatoid arthritis patients. The present study aimed to further refine the contribution of smoking to the ACPA- response and determined: (1) whether smoking interacts differently with the various SE-subtypes for the presence and level of ACPA; and (2) whether smoking influences the ACPA isotype usage in both SE-positive and SE-negative ACPA-positive rheumatoid arthritis patients.

Methods: The effect of smoking and SE-subtypes on the presence and level of ACPA was assessed in 977 early arthritis patients that were included in the Leiden Early Arthritis Cohort. In 216 ACPA-positive rheumatoid arthritis patients, the baseline IgA, IgM and IgG subclasses of ACPA were measured by ELISA.

Results: Smoking interacted significantly with the HLA-DR1 and HLA-DR10 SE-alleles but not with the HLA-DR4 SE-alleles for the presence of ACPA. Conversely, in the absence of smoking the HLA-DR4 SE-alleles conferred the highest risk to develop ACPA (OR 5.0, compared to 2.0 and 1.7 for HLA-DR1 and -DR10 respectively). The effect of smoking on the levels of ACPA was larger in patients carrying HLA-DR1 or HLA-DR10 SE-alleles than in patients harbouring HLA-DR4 SE-alleles. The isotypes IgA and IgM ACPA were more frequent within smokers than within non-smokers (OR 2.8, 95% CI 1.6–5.0 for IgA and OR 1.8, 95% CI 1.03–3.1 for IgM). The levels of all isotypes of ACPA except IgG3 were higher in smokers as well. The number of ACPA isotypes was higher in smokers compared to non-smokers. Intriguingly, this effect was independent from the presence of SE-alleles as this finding was the most pronounced in the SE-negative subjects.

Conclusion: The present study further refines the contribution of smoking to the ACPA response. The effect of smoking on the presence and level of ACPA is not present in all SE-positive arthritis patients, but is confined to patients that carry the HLA-DR1 or HLA-DR10 SE-alleles. Smoking rheumatoid arthritis patients display a more extensive ACPA isotype usage compared to non-smokers. This influence of smoking on the constitution of the ACPA response is, in contrast to its effect on the incidence of ACPA-positivity, independent on the presence or absence of HLA-DRB1 SE-alleles.

120 THE HLA-DBR1 SHARED EPITOPE ALLELES INFLUENCE BOTH THE MAGNITUDE AND SPECIFICITY OF THE ACPA-RESPONSE IN ACPA-POSITIVE RHEUMATOID ARTHRITIS PATIENTS

A. H. M. van der Helm-van Mil, K. N. Verpoort, G. J. M. Pruijn, R. R. P. de Vries, C. Allaart, T. W. J. Huizinga, R. E. M. Toes.Department of Rheumatology, Leiden University Medical Center and Department of Biomolecular Chemistry, Radboud University Nijmegen, The Netherlands

Objective: In classic studies on the genetic background of antibody production, the MHC has been shown to act as the most prominent immune response gene that dominantly controls both the magnitude and the specificity of antibody response. The strongest genetic risk factor for rheumatoid arthritis (RA), the human MHC, HLA-DRB1 shared epitope (SE) alleles, predisposes for the presence of antibodies against anti-citrullinated proteins (ACPA). The present study determines whether SE influences the magnitude and/or the specificity of the ACPA response.

Methods: ACPA were determined by a CCP2 ELISA in two cohorts of RA-patients. In APCA positive RA patients from both cohort 1 (n = 206) and cohort 2 (n = 141), serum antibodies against a citrullinated peptide derived from vimentin (cVim) and antibodies against a citrullinated fibrinogen peptide (cFibr) were determined by ELISA. HLA-DRB1 genotyping was performed in all patients.

Results: In APCA positive RA-patients, the levels of ACPA were higher in SE-positive than in SE-negative patients (mean ± SEM 1032±72 AU versus 652±86 AU, p = 0.001). No difference in anti-CCP levels were observed between patients with 1 or with 2 SE alleles (mean ± SEM 1029±86 AU versus 1041±134 AU, p = 0.9). In both cohorts, the SE-alleles were significantly associated with the presence of antibodies against cVim (OR 4.55, 95% CI 1.78–12.9 and OR 4.13, 95% CI 1.68–10.34) and not with the presence of antibodies against cFibr (OR 1.81, 95% CI 0.78–4.13 and OR 1.08, 95% CI 0.34–3.23).

Conclusion: These data indicate that SE-alleles act as “classic” immune response genes in the ACPA response, as they influence both the magnitude and specificity of this RA specific antibody response.

121 STIMULATION OF THE NICOTINIC ACETYLCHOLINE RECEPTOR ALPHA7 SUBUNITS WITH AR-R17779 IMPROVES THE COLLAGEN-INDUCED ARTHRITIS MODEL OF RHEUMATOID ARTHRITIS

M. A. van Maanen1, C. L. Lebre1, G. J. LaRosa2, D. Elbaum2, M. J. Vervoordeldonk1,3, P. P. Tak1.1Div. of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam, The Netherlands; 2Critical Therapeutics, Inc, Lexington, MA, USA; 3Arthrogen BV, Amsterdam, The Netherlands

Objective: The parasympathetic nervous system, through the vagus nerve, may downregulate inflammation in vivo by decreasing the release of cytokines, among which tumor necrosis factor-alpha (TNFα), by activated macrophages. It has been suggested that the vagus nerve might exert its anti-inflammatory effects by a specific effect of its principal neurotransmitter acetylcholine on the α7 subunits of nicotinic acetylcholine receptors (nAchR) on macrophages. We have previously shown in murine collagen-induced arthritis (CIA) that stimulation of the nAchR by adding nicotine to the drinking water resulted in a significant reduction in clinical arthritis scores. In the present study we investigated whether we could reproduce this beneficial effects by specifically targeting the α7 subunit of the nAchR. Therefore, we investigated the effect of a specific α7 agonist, (-)-spiro[1-azabicyclo [2.2.2]octane-3,5′-oxazolidin-2′-one] (AR-R17779), in murine CIA after intraperitoneal (i.p.) injection. In contrast to nicotine, AR-R17779 only poorly crosses the blood-brain barrier.

Methods: CIA was induced in DBA/1 mice at day 0 by immunization with bovine collagen type II (bCII) followed by a booster injection with bCII on day 20. AR-R17779 was injected i.p. at three different dosages (5 mg/kg, 2.5 mg/kg and 1 mg/kg) b.i.d. from day 20 till sacrifice on day 26 (n = 15 per dose). Control mice (n = 15) received saline. Disease progression was monitored by visual clinical scoring and measurement of paw swelling with a caliper over time. The effect of AR-R17779 on bone degradation and histologic joint damage and inflammation was assessed as well.

Results: In the mice treated with the highest dose of AR-R17779 a 40% reduction in clinical arthritis and a significant delay in onset of disease was observed (p<0.05 compared to saline treated mice). This was accompanied by a decrease in synovial inflammation and local TNFα expression (p<0.05). Moreover, the treated animals had less cartilage and bone destruction, although the difference did not reach statistical significance. Treatment with lower doses of AR-R1777 showed comparable effects, albeit less pronounced.

Conclusions: These data show that AR-R17779 can reduce the severity of arthritis, indicating that the alpha7 subunit plays an important role in synovial inflammation. Because the effect of AR-R17779 on the central nervous system is negligible, the therapeutic effect is based on binding of AR-R17779 to peripheral α7 subunits of the nAchR, which may be a future target for the treatment of rheumatoid arthritis.

122 PHENOTYPICAL AND FUNCTIONAL CHARACTERIZATION OF PERIPHERAL BLOOD NATURAL KILLER CELLS IN SPONDYLOARTHROPATHY PATIENTS

R. Scrivo1, S. Morrone2, A. Spadaro1, A. Santoni2, G. Valesini1.1Cattedra e Divisione di Reumatologia/Policlinico Umberto I/Università La Sapienza; 2Dipartimento di Medicina Sperimentale/Policlinico Umberto I/Università La Sapienza – Roma, Italy

Background: Spondyloarthropathies (SpAs) comprise a cluster of interrelated chronic inflammatory rheumatic diseases characterized by axial and peripheral involvement and extra-articular features. The hypotheses underlying the pathogenesis are increasingly questioned and recently a prominent role for natural killer (NK) cells has been advanced. NK cells, a crucial component of the innate immune system, exhibit cytotoxic properties and provide immunoregulatory cytokines, particularly TNF-alpha and IFN-gamma. Their function is regulated by the integration of both activatory and inhibitory signals from a wide range of cell surface receptors.

Objective: To focus on phenotypical and functional characterization of peripheral blood (PB) NK cells from SpA patients to better comprehend their pathogenetic role.

Methods: We enrolled seven consecutive out-patients with SpAs [six with ankylosing spondylitis fulfilling the modified New York criteria and one with psoriatic arthritis with prominent axial involvement diagnosed by the presence of psoriasis and seronegative arthritis (4 males and 3 females; mean age 46.4 yrs, range 39–68)]. Patients’ PBMCs were isolated from 35 ml heparinized blood by Lymphoprep gradient centrifugation. A comparative analysis of NK and T cell surface antigen expression was performed by staining PB lymphocytes with fluorescence-conjugated monoclonal antibodies directed to MIC A/B, a ligand for an activator receptor, and KIR3DL1, which mediates inhibitory signals, and to subset cell markers such as CD56, CD3, CD4, CD8 and four-color flow cytometric analysis. A sequential gating strategy, based on forward, side scatter and fluorescence parameters was used to evaluate antigen expression on NK and T cells. NK cell cytotoxicity was assessed by using a degranulation assay which measures cell-surface expression of the lisosomal protein CD107a (LAMP-1). PBMCs were incubated with either K562, an NK cell-sensitive erythroleukemia cell line, and with the mouse mastocytoma cell line FcR* (P815) pre-incubated with antibodies directed against NK-cell activating receptors CD16 and NKG2D at an effector to target ratio of 1:1. The degranulation was detected analyzing the expression of CD107a, recently described as a sensitive marker of NK and CD8+ T cell degranulation in healthy subjects, using multi-parameter flow cytometry. The intra-cellular production of TNF-alpha and IFN-gamma following 6 hrs PBMCs stimulation with P815 cells pre-incubated with anti-CD16, anti-NKG2D or both antibodies, was quantified using multi-parameter flow cytometry. Analysis was performed using Wilcoxon’s test for matched samples; the significance of any correlation was determined by Spearman’s rank correlation coefficient, where p values less than 0.05 were deemed statistically significant.

Results: A relevant increase in the expression of KIR3DL1 on PB NK cells was found in respect to CD4+ and CD8+ T lymphocytes (p<0.02). Also, stimulation of NK cells with P815 pre-incubated with both anti-CD16 and anti-NKG2D antibodies appeared statistically significant in inducing cytotoxicity in respect to stimulation of P815 pre-incubated with anti-NKG2D only (p<0.014). Similarly, cytotoxicity was significantly appreciable when P815 were pre-incubated with anti-CD16 only in respect to anti-NKG2D (p<0.014). A positive correlation was found between cytotoxic properties and TNF-alpha production following stimulation of NK cells with P815 pre-incubated with anti-CD16 only (r = 0.8; p<0.04).

Conclusions: KIR3DL1 increased expression on NK cells in SpAs, together with the upregulation of CD107a which demonstrates cytotoxic activity, indicate an activated status of NK cells in these patients, supporting a pathogenetic role for this lymphocyte subset. These preliminary results will be integrated with further experiments to better define the functional characterization of NK population in SpAs.

123 IL-17 REGULATES MATRIX TURNOVER AND CARTILAGE DEGRADATION IN RA SYNOVIOCYTES AND HUMAN CARTILAGE EXPLANTS

E. M. Moran, B. Bresnihan, C. Walsh, O. FitzGerald, D. J. Veale, U. Fearon.Dept. of Rheumatology, St Vincent’s University Hospital and Conway; Institute of Biomedical & Biomolecular Research, Dublin, Ireland

Introduction: IL-17 and OSM are raised in the RA joint and correlate with markers of inflammation and cartilage destruction. This study explores the mechanistic role of TNFa, OSM and IL-17 alone and in combination on matrix turnover and cartilage degradation.

Methods: IL-17 was measured in paired serum/synovial fluids and in synovial tissue protein lysates from patients with inflammatory arthritis by ELISA. The neoepitope C2C was measured in paired serum/synovial fluids by ELISA. Primary RA synovial fibroblasts (SFC), human cartilage explants and primary chondrocytes were stimulated with TNFa (10 ng/ml), OSM (10 ng/ml) and IL-17 (50 ng/ml) alone and in combination over a time course of 0–21 days. MMP-1 and TIMP-1 expression in culture supernatants was measured by ELISA. Cartilage explants were histologically assessed and proteoglycan depletion was measured by SafraninO/Fast Green staining.

Results: We demonstrated high expression of IL-17 in synovial tissue protein lysates. IL-17 levels in paired serum and synovial fluids (n = 25) were 59±19 pg/ml and 60±22 pg/ml respectively. Level of IL-17 expression was associated with C2C levels in paired serum and synovial fluids. IL-17 and OSM significantly stimulated MMP-1 production in human cartilage explants (from basal of 34±19 to 123±36 and 91±59 respectively, p<0.05) and in RASFC (from basal of 28±12 to 53.4±39 and 55.3±36 respectively, p<0.05) at day 4, an effect that was increased to day 17. In cartilage, IL-17 potentiated the effects of OSM on MMP-1 production two fold (p<0.05), an effect that reached maximal levels on day 17, however had no further effect OSM in RASFCs, suggesting differential cytokine sensitivity in different cell types. The combination of IL-17 and TNFa had no further effect on MMP-1 production compared to either cytokine alone, whereas OSM potentiated the effect of TNFa on MMP-1 production in RASFC (two fold). While TNFa, OSM and IL-17 also increased TIMP-1 production in RASFC and cartilage explant cultures, a shift in the relative ratio of MMP-1 to TIMP- in favour of matrix degradation in both RASFC and cartilage explants was demonstrated. Cartilage sections stained with Safranin-O demonstrated minimal proteoglycan depletion in response to TNFa, OSM and IL-17 alone but in combination resulted in almost complete proteoglycan depletion following 4 weeks culture.

Conclusion: IL-17, OSM and TNFa play a critical role in matrix turnover and cartilage invasion. The ability of these cytokines to interact with each other suggests a novel role for these cytokine in RA. Furthermore, these data demonstrate the importance of studying cytokine combinations in human models of disease.

124 REGULATION OF VEGF AND ANGIOPOIETINS IN INFLAMMATORY ARTHRITIS

A. Kennedy1, C. Sweeney1, T. Walshe2, P. A. Cahill2, B. Bresnihan1, O. Fitzgerald1, U. Fearon1, D. J. Veale1.University College Dublin and Dublin City University, Ireland

Introduction: Angiogenesis is an early event in arthritis dependent on the interaction between VEGF and Angiopoietin (Ang). However, upstream triggers involved in regulating VEGF and Ang expression in the inflamed synovial membrane (SM) remains under-investigated.

Aim: This study investigates effects of increased mechanical stress, neuropeptides (CRH and Substance P) and hypoxia on VEGF and Ang2 expression in inflamed SM.

Methods: Oxidative damage was measured in paired synovial fluid and tissue by examining expression of 8-oxo-dG by ELISA and immunohistology. Blood vessel maturity in synovium was measured by dual fluorescent staining for Factor VIII and alpha SMA using confocal microscopy. Ang2 and VEGF protein expression was measured in whole tissue explants, primary synovial fibroblast (SFC) and microvascular endothelial cells (EC) by ELISA. SM explant cultures, SFC and EC were exposed to graded hypoxia (1–10%), SubP (100 ng/ml) and CRH (100 ng/ml) for 24 h. pO2 levels were confirmed using fluorescent quenching oximetry. EC were exposed to varying levels of shear stress using a novel perfused transcapillary system (low  = 0.3 dyn/cm2, high  = 25 dyn/cm2). VEGF and Ang2 expression were analysed by quantitative real time PCR and/or ELISA.

Results: We demonstrated nuclear expression of 8-oxodG in synovial tissue from patients with RA and PsA, with expression localised to the lining layer and perivascular region, associated with high expression of 8-oxo-dG in synovial fluid. We demonstrated immature and mature blood vessel in the joint with the majority of them having some level of pericyte recruitment. High levels of VEGF (300–1210 pg/ml) and Ang2 (293–1262 pg/ml) were spontaneously released within 24 h from RA explant cultures. Level of expression in primary SFC was (71–167 pg/ml: VEGF) and (159–319 pg/ml: Ang 2) and in EC high Ang2 (1400–1800 pg/ml) level in comparison to lower VEGF (230–488 pg/ml) levels was demonstrated. In explant cultures, Sub P induced a 3.3±1.1-fold increase in VEGF protein, with no change in Ang2 protein levels. Exposure of EC to Sub P caused no change in either VEGF/Ang2 secretion. Increased mechanical shear stress resulted in a 3.69±0.68-fold increase in Ang2 secretion, and a 1.85±0.15 increase in VEGF mRNA expression in EC. Exposure of SM explants and SFC to graded levels of hypoxia resulted in 1.9±0.1-fold and 3.98±0.12-fold increase in VEGF expression at 1% hypoxia. Similarly, Ang2 expression increased 3.7,±0.6-fold following exposure to 1% hypoxia in SFC. No alteration in VEGF/Ang2 expression was observed in EC following exposure to graded hypoxia.

Conclusion: These data demonstrate high spontaneous release of the key angiogenic factors VEGF and Ang2 from RA explants, SFC and EC. Furthermore it demonstrates key upstream triggers involved in regulating VEGF and Angiopoietins in the inflamed joint such as SubP, mechanical stress, and hypoxia. These factors represent attractive therapeutic targets in the future for angiogenesis in arthritis.

125 EXPANSION OF SYNOVIAL FLUID CD56BRIGHT NKT CELLS IN INFLAMMATORY ARTHRITIS ASSOCIATED WITH INCREASED CYTOTOXICITY AND INFLAMMATORY CYTOKINE PRODUCTION

C. A. E. Walsh, O. FitzGerald, D. J. Veale, L. Golden-Mason, B. Bresnihan, U. Fearon.University College Dublin, Ireland

Introduction: Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are autoimmune disorders of unknown aetiology. T cell activation is presumed to be the primary event in the pathogenesis of inflammatory arthritis. Natural killer receptor expressing cells are increasingly recognised as playing a role in the pathogenesis of autoimmunity.

Methods: Three-colour flow cytometry was used to characterise NK, NKR+T cells and T cell populations in peripheral blood mononuclear cells and synovial fluid mononuclear cells from 13 patients with inflammatory arthritis (RA and PsA). Mononuclear cells were incubated at 37°C and 5% CO2 “b PMA and ionomysin in the presence of Brefeldin A for four hours and intracellular expression of interferon-gamma (IFNgamma) and tumour necrosis factor-alpha (TNFalpha) was measured. CD3+ T cells were purified using magnetic separation and cytotoxicity against leukaemia cell line K562 was measured following culture “b IL-2 for 48 hours (37°C, 5% CO2).

Results: We have demonstrated that CD56+CD3- (NK) and CD56+CD3+ (NKT) cells are present in PBMCs and SFMCs at similar levels. However phenotypic characterisation reveals major differences between the periphery and the joint. Both NK and NKT cells show significant expansion in CD56 bright populations in SFMCs (median 2.43% of CD56+ cells) compared with PBMCs (0.62%) (p = 0.007). These cells are also activated as indicated by a significantly higher expression of CD69 (43.79% of CD56 bright cells vs 9.06%) (p<0.0001) and HLA-DR (52.28% vs 22.26%) (p<0.05). In addition inhibitory receptors CD158a and CD158b are down-regulated (p<0.05) in the synovial fluid compared to matched peripheral blood. A significant reduction is seen in expression of CD57 (p<0.05) suggesting that these cells are protected from apoptosis. Upon stimulation with PMA and Ionomycin, an increase in CD3+ release of IFNgamma is seen in the SFMCs compared with matched PBMCs. Preliminary results also show increased cytotoxicity against K562!s in CD3+ cells separated from SFMCs.

Conclusion: Significant phenotypic differences are seen in synovial fluid compared with peripheral blood CD56+CD3+ cells. Synovial fluid has an increase in activated CD56bright populations showing increased immunomodulatory function with increased expression of the inflammatory cytokine IFNgamma and subsequent increased cytotoxicity of CD3+ cells in synovial fluid.

126 EFFICACY AND SAFETY OF RITUXIMAB IN HCV-RELATED MIXED CRYOGLOBULINEMIA

F. Marchi1, A. Della Rossa2, A. Tavoni1, C. Baldini2, A. d’Ascanio2, S. Bombardieri2, P. Migliorini1.1Clinical Immunology, 2Rheumatology Units, Department of Internal Medicine, University of Pisa, Italy

Mixed cryoglobulinemia (MC) represents the most frequent among systemic vasculitides in southern Europe. More than 90% of MC patients carry a chronic hepatitis C virus (HCV) infection and immune complexes formation seem to represent the main pathogenetic mechanism in the disease. The traditional therapeutic approach includes glucocorticoids, plasmapheresis, and immunosuppressive drugs (such as cyclophosphamide). Considering the disease is a particular expression of the HCV infection that drives lymphoproliferation into malignancy, new therapeutic strategies have been proposed; of course viral eradication is a chance, but frequent failure and side effects of the drugs to be administered strongly limit this approach. Recently, anti-CD20 antibody (Rituximab, RTX) has been proposed for the treatment of MC. We report data on the safety and efficacy of RTX in the treatment of 7 HCV-related MC patients. Three patients received RTX as treatment of lymphoproliferative disease; 3 for skin ulcers refractory to traditional therapies; 1 for peripheral neuropathy. Two patients affected by lymphoproliferative disorders were treated with 375 mg/m2 weekly for 4 weeks and the cycle was repeated every third month (in one of them the treatment was stopped after the first infusion); the other patients received two infusions of 1 g over 2 weeks. No serious infection was observed in any patient during treatment or follow up; liver enzymes were unaffected. Hemoglobin concentration increased and serum IgM levels were reduced after treatment in all the patients; RF titre decreased in those affected by lymphoproliferative disorders. Lymphoma remission was obtained in the 2 patients that completed the treatment. Ulcer healing was obtained in the 3 patients and no recurrence was observed for 18 months; on the contrary, peripheral neuropathy was unaffected by treatment, but 10-month follow-up may be too short to draw any firm conclusion. These results confirm that RTX is a safe and effective therapy of MC, with less side effects than traditional immunosuppressive regimens. Of particular interest is the ability of RTX to control disease manifestation such as skin ulcers for a long time after stopping the treatment. This observation raises interesting questions on the pathogenetic mechanisms underlying skin vasculitis in MC.

127 RAPID MICROARRAY-BASED T-CELL RECEPTOR REPERTOIRE ANALYSIS AT SINGLE-CLONE RESOLUTION

H. P. J. Bonarius, F. Baas, E. B. Remmerswaal, R. A. W. Van Lier, P. Peter Tak, I. J. M. ten Berge, N. De Vries.Dept Clinical Immunology & Rheumatology, AMC/University of Amsterdam, Amsterdam, The Netherlands

Aim: The adaptive immune system recognizes billions of unique antigens using highly variable T-cell receptors and antibodies. During an immune response, antigen-specific T- and B-cells may proliferate, resulting in clonal expansion. During this process specific antigen recognition resides in the T- and B-cell receptors respectively. Within each expanding clone, each T- or B-cell encodes the same T- or B-cell receptor. Recently we developed a novel microarray based technology that is able to pick up and monitor expansion of these T- and B-cell clones. Recently we showed that using this T-array technology can screen (part of) the TCR and Ig repertoire for dominant clones and quantitative changes, yielding sequence information. Here we test and show its sensitivity, and show its validity and applicability in monitoring T-cell responses.

Methods and materials: In T-array technology mRNA is isolated and cDNA produced using reverse transcriptase. After generic or selective amplification the cDNA is hybridized to selected sets of fluorescently labelled oligonucleotides. Signals are read on 4000 spot microarrays. To test the sensitivity, specificity and quantitativeness of the technique Jurkat T-cells were diluted in CD4 cells in dilutions from 1 in 10E4 up to 1 in 10E7. In addition, to test the applicability of this technique we isolated PBMCs from a healthy HLA-A2+ healthy donor latently infected with CMV. Cells were stimulated in vitro with the CMV peptide NLVPMVATV, and expansion followed using FACS after tetramer staining, spectratyping and T-array analysis.

Results: T-array detected T cells up to a dilution of 1 in 10E6 to 1 in 10E7 CD4+ T-cells. This is 3 logs more sensitive than spectratyping, the current standard in repertoire analysis. Analysis was clearly quantitative and could be done in 2 days. In the second experiment, after stimulation with CMV-peptide, the fraction of tetramer-positive cells increased from ∼5% at day 0 to 60% at day 10. Both tetramer analysis and spectratyping detected an immune response against CMV at day 6. T-array already identified the dominantly responding clone at day 0, and could show a clear increase in frequency at day 3. The results were quantitatively validated by repeated cloning and sequencing.

Conclusion: T-array has superior sensitivity, resolution and comprehensiveness compared to spectratyping. It is fast, allows quantitative monitoring of selected clones and yields nucleotide sequence information. It is equally applicable to B-cell repertoire analysis (results not shown). This new technology allows identification and quantitative monitoring of expanding T- and B-cell clones specific for autoimmune disease, not feasible with other techniques.

128 RHEUMATOID FACTORS BIND TO B CELL STIMULATORY CPG “K” CLUSTER OLIGOS: POSSIBLE INVOLVEMENT OF TOLL-LIKE RECEPTORS

K. Skriner, F. Schumann, G. Naddaf, K. Adolph, G. R. Burmester.Charité University Medicine, Department of Rheumatology and Clinical Immunology, Humboldt University and Free University, Berlin, Germany

Background: Despite its moderate specificity the rheumatoid factor (RF) is still the only established marker antibody for RA among the classification criteria of the American College of Rheumatology. Recently, it was shown that the proliferative response of mouse autoreactive rheumatoid factor (RF) B cell clones to mammalian chromatin-containing immune complexes (ICs) results from the sequential engagement of the B cell receptor (BCR) and Toll-like receptor 9 (TLR9) (Marshak-Rothstein et al 2003). This observation prompted us to use hypomethylated DNA sequences derived from known B cell and dendritic cell activating sequences to define which specific hypomethylated DNA sequences are crossreactive with RF and necessary for an effective activation of human RF B cells.

Objective: To elucidate which DNA sequences RF bind and under which conditions RF producing B cells are generated and RF is produced, especially with regard to Toll-like receptors

Methods: Affinity purified IgM and IgA RF from IgG1 and IgG3 were used to identify the binding to different oligonucleotides The synthetic peptides and hypomethylated Oligos (1 mM) were incubated with PBMCs or B-cells from RA-patients and healthy controls for 9 days at 37°C. Supernatants (40 ml/well) were tested for the presence of rheumatoid factor at day 9 by ELISA. We used CpG “D” cluster oligos which stimulate INFg production by NK cells, and the CpG “K” cluster oligos that stimulate IL6 production by monocytes. After harvesting the supernatant, cells were incubated with 1mC 3H-Thymidin and cell proliferation, cytokine profile was determined after 6h. Anti-citrullin-antibodies and RF are tested by ELISA.

Results: Affinity purified RF specifically bound to K but not to D type of oligonucleotides. Using hypomethylated mitochondrial DNA and a specific peptide derived from the IgG3 heavy chain we could efficiently stimulate RF B cells for RF production, in contrast to using PHA or control peptides. The synthetic IgG peptide and different oligonucleotide sequences were incubated with PBMCs or B-cells from RA- RF positive as well as RF negative patients and healthy controls. There was no correlation found with in vitro proliferation and RF production. In addition we could demonstrate that RF, but not anti-citrulline antibodies were induced by non-methylated oligonucleotides or the IgG3 peptide in PBMCS or B cells from RF patients.

Conclusion: Our results document the crossreactivity of RF to K type B cell stimulatory sequences. RF B cells are stimulated for RF production with IgG peptides and specific hypomethylated DNA sequences. Thus, costimulation of the B cell receptor and TLR9 appears to represent an effective mechanism to induce RF-IgM in RA patients. This may explain why RF is specifically produced when high levels of certain hypomethylated ssDNA sequences are present in the synovial fluid as is the case in RA due to the high content of mitochondrial DNA.

129 POSSIBLE ROLE FOR NUCLEOSOME SERUM ACCUMULATION IN THE GENERATION OF AUTOANTIBODIES IN PATIENTS WITH RHEUMATOID ARTHRITIS TREATED WITH ADALIMUMAB

R. Priori, C. Alessandri, L. Magrini, F. Ceccarelli, E. A. M. Cassarà, M. G. Modesti, G. Valesini.Cattedra e Divisione di Reumatologia, Università La Sapienza, Roma, Italy

Background: The development of autoantibodies in rheumatoid arthritis (RA) patients during treatment with anti-TNFalpha agents could be linked to the exposition of nuclear antigens along the apoptotic response evoked by these drugs.

Objectives: To evaluate the relationship, if any, between autoantibodies production and serum nucleosome concentration in RA patients treated with Adalimumab.

Methods: We enrolled 47 patients with RA not responsive to conventional disease-modifying antirheumatic agents, treated with Adalimumab (40 mg/sc every other week). Clinical response was evaluated monthly by DAS28 activity index; before starting therapy and after six months all sera were blindly analyzed for IgG ANA and IgG/IgM anti-dsDNA by indirect immunofluorescence on Hep2 cells (cut off value: serum dilution 1:80) and Crithidia luciliae (cut off value: serum dilution 1:10), respectively. CRP (Behring; Germany), RF (Behring; Germany), anti-nucleosome antibodies (Orgentec; Germany), and nucleosome serum levels (Roche Diagnostics, Germany) were detected by commercial kits. The McNemar test was used for testing the significance of autoantibodies frequency from baseline to six months of therapy, and analysis for matched pairs was performed using Wilcoxon’s signed rank test. A p value <0.05 was considered statistically significant. A Principal Components Analysis for Categorical Data (CATPCA) was also performed to assess the behaviour of nucleosome and CPR.

Results: A significant reduction from baseline after six months of therapy with Adalimumab was observed for DAS28 score, for tender joint count, for swollen joint count, for ESR (median ± SD from 34±25 to 19±12 mm/h; p<0,0001), for CRP (median ± SD from 12±44 to 2.5±9.9 mg/L; p<0.0001) and for serum titre of RF (median ±SD 94.5±228.5 to 41.4±199.9, p<0.0001). The frequency (19.1% vs 44.6%) and the titre of ANA changed significantly from baseline to six months of therapy (p = 0.0015). Grouping RA patients according to ANA development at six months, the mean nucleosome concentration at baseline showed a significant progressive increase (median (95 CI) from 98 (81–133) to 120 (93–239) U; p = 0.008) whereas CRP showed a significant decrease (median (95 CI) from 15.8 (3.7–48) to 3.5 (3.5–11.2) mg/L, p = 0.002). CATPCA showed that nucleosome and CRP behaves differently. None of the patients developed clinical signs of autoimmune or infectious disease.

Conclusion: Adalimumab induces ANA in patients with RA in the first six months of treatment. This phenomenon is not associated with clinical manifestation of autoimmunity and could be linked to a dysregulation of apoptosis and the consequent accumulation of nucleosomes, possibly fostering the development of autoantibodies. An impaired clearance of nucleosomes could contribute to this series of events.

130 CELL CYCLE DEPENDENT SUSCEPTIBILITY OF RASF TO FASL AND TRAIL INDUCED APOPTOSIS

N. Pundt, M. Peters, C. Wunrau, K. Neugebauer, L-HMeyer, T. Pap.Div Mol Med Musculoskel Tissue, University Hospital Münster, Germany

The progressive destruction of articular cartilage and bone by a hyperplastic and inflamed synovial membrane is a hallmark of rheumatoid arthritis (RA). The RA synovium is characterized by the presence of an aggressive population of stably activated synovial fibroblasts (RASF), which are involved prominently in the degradation of cartilage matrix. Growing evidence suggests that RASF are relatively resistant to FasL induced apoptosis, but the data concerning TRAIL have been conflicting. Here we hypothesized that the susceptibility of RASF to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL and TRAIL induced programmed cell death of RASF in vitro. Synovial fibroblasts were isolated from 5 patients with RA by enzymatic digesting, cultured under standard conditions (DMEM with 10% FCS) and used at passage 4–8. Cell cycle analysis was performed using flow cytometry (FACSCalibur, BD Bioscience) by incubation of cells with propidium iodide (40 µg/ml PI, 100 µg/ml RNase in PBS) for 2 days. Apoptosis was induced at different confluency states by stimulation of 5×104 cells with 100 ng/ml FasL and 100 ng/ml TRAIL over 18 hours. The apoptotic response was measured by a cell death detection ELISA (Roche) as well as Apo-One Homogenous Caspase-3/7 Assay (Promega). Staurosporin (1 µg/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) were determined by FACS analysis. As seen previously, freshly isolated RASF exhibited only a very low rate of proliferation in vitro (9.5%). Proliferation rate decreased further over time, particularly when RASF became confluent. Early culture RASF (50% confluency) were less sensitive to FasL (+30% vs. unstim. controls) and TRAIL (+20% vs. unstim. controls) induced apoptosis than late culture RASF (80% confluent, FasL: 60% vs. unstim. controls and TRAIL: +50% vs. unstim. controls) and 100% confluent RASFs (FasL: (+80% vs. unstim. controls and TRAIL: +90% vs. unstim. controls). RASF were found to express Fas and TRAIL receptors at different levels. Our data demonstrate that although RASF express functional Fas- and TRAIL- receptors, they are relatively resistant to receptor-mediated apoptosis. We suggest that in addition to this low apoptotic response, the actual susceptibility of RASF to FasL and TRAIL induced apoptosis depends on the cell cycle. These data may explain conflicting data on the ability of RASF to undergo FasL and TRAIL-mediated cell death and suggest that strategies to sensitize RASF to apoptosis may include genes that regulate cell-cycle.

131 IMPLICATION OF IL-17RA AND IL-17RC RECEPTORS IN IL-17A- AND IL-17F-INDUCED PROINFLAMMATORY EFFECTS: DEMONSTRATION USING SILENCING RNA AND EXTRACELLULAR INHIBITORS

S. Zrioual, M-LToh, Y. Zhou, V. Miossec, P. Miossec.Mixed Unit Civil Hospital of Lyon-BioMérieux, Lyon, France

Aim: IL-17A is implicated in rheumatoid arthritis (RA) pathogenesis, however the contribution of IL-17F and associated receptor(s) remain unclear. We compared the effects of IL-17F to IL-17A in RA synoviocytes and examined the role of IL-17RA and IL-17RC receptors.

Materials and methods: RA synoviocytes were stimulated with IL-17A or IL-17F and TNFalpha. IL-6 and IL-8 expression were measured by RT-PCR and ELISA. IL-17RA and IL-17RC inhibition was achieved by short interfering RNA (siRNA) and specific extracellular inhibitors.

Results: IL-17A or IL-17F upregulated IL-6 mRNA and protein levels. IL-17A was more potent than IL-17F alone, but equally synergized with TNFalpha to induce IL-6 or IL-8. Using posttranscriptional inhibition by siRNA and extracellular blockade using specific inhibitors, we showed that both IL-17A and IL-17RC are implicated in IL-17A-induced IL-6 and IL-8 secretion, whereas in the presence of TNFalpha, the inhibition of both receptors is needed to significantly downregulate IL-17A- or IL-17F-induced IL-6 secretion. Finally, the combination of an anti-IL-17RA antibody or an anti-IL-17RC antibody and a soluble TNFRII receptor (etanercept) completely inhibited IL-17A or IL-17F plus TNFalpha-induced IL-6 secretion by RA synoviocytes.

Conclusion: IL-17F has important proinflammatory synergistic effects with TNFalpha in RA synoviocytes. IL-17A-induced IL-6 and IL-8 expression is dependent on both IL-17RA and IL-17RC. Alongside IL-17A, IL-17F may be considered as a potential therapeutic target and may be modulated by IL-17RA or IL-17RC antagonism.

132 IN VITRO EVIDENCE THAT TREATMENT WITH HIGH DOSE METHYLPREDNISOLONE PULSE THERAPY IS SUPERIOR TO HIGH DOSE PREDNISOLONE OR DEXAMETHASONE

P. H. J. Remans, D. M. Gerlag, C. A. Wijbrandts, K. A. Reedquist, P. P. Tak.Div. of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, The Netherlands

Background: Glucocorticosteroids (GC) remain a very potent therapy to treat auto-immune disorders. Although it is generally accepted that there are genomic and non-genomic mechanisms of glucocorticoid effects, a lot of controversy exists about the potency of the different GCs. In particular it has been suggested that MP could have superior anti-inflammatory effects over other GCs. High dose MP pulse therapy has historically been used to treat life-threatening manifestations of rheumatic disease and recent evidence suggests that MP pulse therapy can also be used with good success in therapy of refractory RA.

Objective: To compare the effects of MP to dexamethasone and prednisolone.

Materials and methods: Mononuclear cells were isolated following ficoll separation from peripheral blood (PBMC) from healthy donors and from PB and synovial fluid (SF) from RA patients. T cells were purified through negative selection. Cell proliferation was measured using CFSE 48h after stimulus with CD3 and CD28. For determination of cytokine production, Brefeldine A was added 48h after CD3 and CD28 stimulus. After 4h cells were washed, fixed with PFA 4%, and cytokines were on FACS after intracellular staining. The percentages of early and late apoptotic, as well as necrotic cells, were determined by FACS analysis following annexin V-PI staining.

Results: Inhibition of cell proliferation and TNF production were observed after in vitro treatment with low dose (<1 microM) of both MP, dexamethasone, and prednisolone. This inhibition was due to the genomic effect of the GCs as Ru483 (a known inhibitor of the Glucocorticoid Receptor (GR) prevented the GC-induced effects on cell proliferation and cytokine production). The genomic effects correlated with know potencies of the different GCS (MP = 4/5Pred  = 1/7dexa). Additionally, after in vitro incubation of continuous (48h) high doses MP, we observed apoptosis of macrophages (>2 microM), B cells(>3 microM) and T cells (>5 microM). Apoptosis was not observed after in vitro treatment with dexamethasone nor prednisolone (max dose tested: 50 microM). MP induced apoptosis can be attributed to its non-genomic effects as Ru 483 failed to inhibit MP induced apoptosis, and MP also induced apoptosis in a T CLL cell-line with known defective GR. MP induced apoptosis is regulated by the intracellular redox potential: Cells pre-treated with BSO (which depletes glutathione (GSH)), or sub-apoptotic concentrations H2O2, were sensitised to MP induced apoptosis. NAC treatment (which upregulates GSH) protected against MP induced apoptosis. Also, T cells isolated from the synovial joint (which are known to suffer from oxidative stress) were susceptible to MP induced apoptosis, and apoptosis was observed in SF T cells at 0.5 microM.

Conclusions: Whereas all GCs induced inhibition of cell proliferation and cytokine production regulated by the GR, only in vitro incubation of MP induced apoptosis. Although this apoptotic process is independent from the GR and therefore considered a non-genomic effect, it only occurs after 48h continuous incubation. Additionally, cells suffering from oxidative stress such as SF T cells were sensitised to MP induced apoptosis. Together these data support evidence that MP pulse therapy has superior clinical effects over high dose of other GCs in the treatment of rheumatic diseases.

133 ENDOGENOUS LINE-1 RETROVIRAL ELEMENTS IN OLDER PATIENTS WITH PRIMARY SJOGREN’S SYNDROME: TRIGGERS OF INTERFERON-ALPHA PRODUCTION AND B-CELL ACTIVATION?

C. P. Mavragani1, D. Pierce1, E. K. Kapsogeorgou2, H. M. Moutsopoulos2, M. K. Crow1.1Mary Kirkland Center for Lupus Research, Hospital for Special Surgery, Weill Medical College of Cornell University, New York, USA; 2Department of Pathophysiology, School of Medicine, University of Athens, Athens, Greece

Purpose: B-cell hyperactivity is the hallmark of Sjogren’s syndrome (SS), manifested as hypergammaglobulinemia and the production of autoantibodies. The mediators of B cell activation and differentiation in SS have not been fully elucidated, although activation of the type I IFN pathway and BAFF (B-cell activating factor) overexpression have been implicated in the pathogenesis of this disorder. Viral infection and endogenous retroviral elements—known to be tightly regulated by methylating mechanisms—have been proposed as candidate triggers of SS. Endogenous retroviral LINE-1 transposable elements, possibly regulated by epigenetic mechanisms (e.g. methylation), may provide an endogenous trigger for IFN-alpha production and B cell activation in SS through Toll-like receptor (TLR) activation pathways.

Methods: Twenty patients with primary SS and 14 controls presenting with sicca features not fulfilling diagnostic criteria for SS were studied. All patients were followed in the Department of Pathophysiology, School of Medicine, University of Athens, Greece and the SS patients fulfilled the European-American revised criteria for the diagnosis of primary SS (pSS). Clinical, laboratory and histologic data were recorded for all patients. Relative mRNA expression of full-length LINE-1; IFN-alpha2; BAFF; IL-21, a cytokine that promotes B cell differentiation; TNF-alpha; TLR 3, 7 and 9, sensors for nucleic acids; DNA methyltransferases DNMT3A, DNMT3B, DNMT-1; and methyl-CpG binding protein MCP-2 was determined by real-time PCR performed on cDNA derived from DNAse treated RNA from minor salivary gland (MSG) biopsies. Mean values between different groups were compared using non-parametric Mann-Whitney test. Correlations were performed by using non-parametric Spearman’s test.

Results: LINE-1 mRNA expression in MSG tissues did not differ between the pSS group and controls. However, relative expression of full-length LINE-1 transcripts was significantly increased in pSS patients older than 60 years old (n = 6) compared to their younger counterparts (n = 14; p = 0.0011) and controls (n = 14, p = 0.0057). No difference in LINE -1 mRNA expression was detected in the control MSG samples between older and younger age groups. In SS tissues, a strong correlation was observed between full-length LINE-1 mRNA expression, IFN-alpha2, BAFF, IL-21, Toll-like receptors 7 and 9 and methylating enzymes DNMT3B and DNMT-1 (r = 0.8316, p<0.0001; r = 0.7188, p = 0.0004; r = 0.5970, p = 0.0055; r = 0.4897, p = 0.0284; r = 0.6281, p = 0.0040; r = 7853, p = 0.0001 and r = 0.7632, p = 0.0001, respectively). No statistically significant correlation was found between LINE-1 and TNF-alpha, TLR-3, DNMT3A or MCP-2 mRNA expression.

Conclusions: Impaired epigenetic control of LINE-1 retrotransposon expression in older individuals may be one contributor to the immune system activation that characterizes primary SS and may be mechanistically linked to IFN-alpha induction and B-cell activation through TLR activation pathways. Coordinate mRNA expression of methylating enzymes DNMT3B and DNMT-1 along with LINE-1 may reflect a host response that regulates inappropriate expression of endogenous retroelements in patients with SS.

134 DIFFERENTIAL ROLES OF TNFR-1 AND -2 IN EXPERIMENTAL ARTHRITIS

S. Blüml, N. Binder, K. Polzer, B. Türk, C. Scheinecker, J. Smolen, K. Redlich.Department of Rheumatology, Medical University Vienna, Austria

The role of TNF in the induction and maintenance of human rheumatoid arthritis is well established. In contrast, the roles of its two receptors, TNFR1-p55 and TNFR2-p75, are less well understood. In this study, we analysed the relative contribution of the two TNF-receptors on the development of TNF-driven arthritis. To this end, we reconstituted lethally irradiated TNF-transgenic mice with bone marrow derived from animals lacking either one or both of the two receptors. Subsequently, we assessed the development of joint inflammation, as well as cartilage and bone destruction in these animals. Mice receiving bone marrow deficient for TNFR1 showed a marked reduction of bone erosions, paralleled by a reduced number of osteoclasts present in the inflamed joints. However, mice reconstituted with bone marrow lacking TNFR-1 and -2 or TNFR-2 alone were characterized by a markedly enhanced arthritis with higher numbers of osteoclasts. Our results suggest that TNFR-1 on hematopoetic cells is necessary for the development of erosive arthritis, whereas the presence of TNFR-2 seems to have a protective effect in arthritis. Therefore, selective blocking of TNFR-1 might be superior to blocking TNF in rheumatoid arthritis.

135 TNF PROMOTES REORGANIZATION OF CADHERIN-11 INTERCELLULAR JUNCTIONS AND CELLULAR CONDENSATION OF FIBROBLAST-LIKE SYNOVIOCYTES

V. Kallab, I. Radda, B. Tuerk, A. Rapp, C. Scheinecker, J. Smolen, H. Kiener.Medical University of Vienna, Department of Medicine III, Division of Rheumatology, Vienna, Austria

Cadherin-11 is a homophilic adhesion molecule that is expressed on fibroblast-like synoviocytes (FLS). Cadherins mediate tissue morphogenesis during development and tissue architecture in adults. To investigate a role for cadherin-11 in mediating mesenchymal tissue reorganization and pannus formation in arthritis, we have analyzed the expression of cadherin-11 in diarthrodial joints of patients with rheumatoid arthritis. Immunohistochemistry revealed cadherin-11 expression in the synovial lining layer and in some cells of the sublining area. Strikingly, cadherin-11 was also expressed in condensed fibroblast-like cells at the erosive interface between pannus tissue and cartilage. To test the inflammatory response of FLS in a simplified model of the complex synovial microenvironment, we stimulated 3-dimensional micromass synovial organ cultures with tumor necrosis factor (TNF) or platelet derived growth factor (PDGF). When unstimulated or stimulated with PDGF, the FLS formed a lining layer at the micromass surface whereas the sublining FLS were loosely organized. In contrast, TNF stimulation induced pronounced FLS condensation not only at the surface lining layer but also in the sublining area. Thus, TNF stimulation had a profound impact on the cellular organization in this model system. Since cadherin-11 mediates homophilic adhesion of FLS, we explored the structure of intercellular junctions in TNF or PDGF stimulated cultured FLS. Using indirect immunofluorescence and confocal microscopy, we found that unstimulated or PDGF stimulated FLS connected to one another via filopodia-like processes that interdigitated to form a cadherin-11 adhesion zipper. In TNF stimulated FLS, the cells were intimately connected and the anti-cadherin-11 antibody labelled a mostly linear or jagged line of intercellular junctions. These studies suggest a role for cadherin-11 in TNF induced reorganization of FLS and provide insight into a critical pathway that determines the mesenchymal tissue response to inflammation in arthritis, especially rheumatoid arthritis.

136 STAT5 SIGNALLING VERSUS APOPTOSIS: EXPLAINING DIMINISHED RESPONSE OF SLE LYMPHOCYTES TO GAMMA CHAIN CYTOKINES

E. Feierl, T. Karonitsch, A. Hinek, C. W. Steiner, G. Steiner, J. Smolen, M. Aringer.Department of Rheumatology, Medical University of Vienna, Austria

Purpose: To investigate the diminished response of SLE lymphocytes upon stimulation with interleukin-2 (IL-2) and other alpha-chain cytokines (IL-7, IL-15), and the ability of these cytokines to induce Stat5 (Signal transducer and activator of transcription 5) phosphorylation, in particular.

Methods: PBMC of 63 SLE patients and 48 healthy controls (HC) were prepared. The surface expression of the IL-2 receptor alpha (CD 25), beta (CD 122), and gamma (CD 132) chains was determined by fluorocytometry. Phosphorylated Stat5 (pStat5) in lymphocytes both ex vivo and after 30 minutes in medium with or without rhIL-2, IL-7, or IL-15 was likewise measured by fluorocytometry after intracellular staining. CD25 upregulation after 24 hours of cytokine exposure was also determined by fluorocytometry. Gel shift assay showed specific DNA-binding of phosphorylated Stat5. Proliferation of lymphocytes with and without IL-2 supplementation was investigated after three day cultures, using 3H-thymidine uptake. Cell counts and AnnexinV/Propidium Iodide (PI) staining were also carried out. The percentage of regulatory T-cells (CD25/Foxp3/CD4+ and CD4+/CD25 high) was determined by fluorocytometry. Disease activity was measured using SIS, ECLAM, and SLEDAI scores.

Results: The expression of the three components of the IL-2 receptor was similar between SLE patients and HC. Likewise, Stat5 phosphorylation was not significantly different ex vivo, and not associated with disease activity. Moreover, determined by Stat5 phosphorylation, lymphocytes of SLE patients showed a normal early response to IL-2, IL-7, and IL-15. Binding of pStat5 to DNA appeared normal in gel shift assay. Lymphocytes also showed significant CD25 upregulation after 24 hours of IL-7 or IL-15 exposure, but again no difference between SLE and HC. However, the proliferative response upon IL-2 was clearly diminished three day cultures of SLE lymphocytes (cpm: SLE 2 947±2 490; HC 10 040±6 659; p<0.0001). Viable cell counts (Annexin/PI negative) were also significantly lower in SLE (0.51±0.20 ×106 cells/ml) vs HC (0.80±0.28 ×106 cells/ml, p<0.0006). Time course experiments showed a drastic decrease in viable cell counts for SLE PBMC within the first 24h, accounting for 80% of all cells dying in culture. Percentages of regulatory T-cells were similar between SLE and HC.

Conclusions: In contrast to their altered proliferative and anti-apoptotic response, SLE patients’ lymphocytes rather unexpectedly show a normal immediate response to IL-2 and other alpha-chain cytokines, as measured by Stat5 phosphorylation. There was also no hyporesponsiveness in CD25 upregulation. The reduced proliferation seems to be due to the increased in vitro apoptosis of activated SLE lymphocytes.

137 IS NIP3 THE CRITICAL MEDIATOR IN METHYLPREDNISOLONE INDUCED APOPTOSIS?

P. H. J. Remans, D. M. Gerlag, C. A. Wijbrandts, P. Reinders-Blankert, E. Eldering, K. A. Reedquist, P. P. Tak.Div. of Clinical Immunology and Rheumatology, University of Amsterdam, The Netherlands

Background: Glucocorticoid-induced apoptosis is a well known phenomenon. However, cell death phenotypes and their molecular mechanisms are highly diverse. In apoptotic processes, there is an interplay of a multitude of pro-and anti-apoptotic molecules, and to get a clear insight in the complex network of apoptosis one needs to gain information about each of the molecules involved.

Objective: To elucidate the molecular mechanisms of glucocorticoid-induced cell death.

Methods: T cells were purified from peripheral blood through negative selection. Total RNA was prepared before, 6, 12 and 24h after treatment with 50 μM methylprednisolone (MP) in vitro by the Trizol method, and analysed after multiplex ligation-dependent probe amplification (MLPA). MLPA has provided a new efficient and reliable assay for dosage screening of multiple loci in a single reaction. Probes used in this study were designed to hybridize with cDNA of 36 human apoptosis-related genes. The percentages of early and late apoptotic, as well as necrotic cells after in vitro treatment with corticosteroids were determined by microscopy and annexin V-PI staining. Intracellular colocalisation of Bax and cytochrome C were visualised using fluorescent microscopy. For detection of the mitochondrial transmembrane potential, cells were stained with JC-1 and oxidation of the dye DCF was used to measure reactive oxygen species (ROS) production. FACS was used for measurement of cytochrome C release and determination of cleaved caspase-3.

Results: After in vitro MP treatment we found a specific upregulation of the mRNA of the pro-apoptotic Nip3 and its related protein Nip3L ( = Nix) both in SF and PB T lymphocytes. No other significant changes were detected in any of the other apoptosis related genes. The upregulation of the Nip3 expression was subsequently confirmed by Western blotting and fax staining. Introduction of RNAi blocking Nip3 expression prevented 63% (mean) ± 14% (SD) of MP induced apoptosis in lymphocytes. MP induced apoptosis leads to an aponecrotic cell death via activation of the intrinsic apoptotic pathway. We found that Bax translocated to the mitochondria concomitant with mitochondrial depolarisation and subsequent cytochrome C and ROS release. Addition of the pan-caspase inhibitor zVAD did not alter loss of mitochondrial membrane potential or cytochrome C release, indicative for caspase-independent initiation of the (intrinsic) mitochondrial apoptosis pathway.

Conclusion: The pro-apoptotic BH3-like protein Nip3 plays a critical role in MP induced apoptosis. In vitro treatment of T lymphocytes with MP leads to upregulation of Nip3, and blocking of Nip3 expression with RNAi prevented MP induced apoptosis. Insight into these mechanisms might help to enhance the therapeutic efficacy of corticosteroid treatment in RA patients.

138 PEPTIDYLARGININE DEIMINASE 6 DOES NOT CONTRIBUTE TO THE GENERATION OF THE SYNOVIAL TARGETS OF THE RHEUMATOID ARTHRITIS-SPECIFIC AUTOANTIBODIES TO CITRULLINATED PROTEINS

C. Foulquier1, C. Clavel1, SChapuy-Regaud1, M-C. Méchin1, M. Yamada2, H. Takahara3, M. Simon1, M. Guerrin1, G. Serre1, M. Sebbag1.1UMR5165; CNRS-Toulouse III Univ., France; 2Int. Grad. School of Arts and Sciences, Yokohama Univ., Japan; 3Dpt of Appl. Biol. Resource Sciences, School of Agriculture, Ibaraki Univ., Japan

Autoantibodies to citrullinated proteins (ACPA) are highly specific for rheumatoid arthritis (RA) and probably involved in its pathophysiology. They are synthesised in the rheumatoid synovium where they recognize citrullinated proteins among which citrullinated fibrin is a major target. Citrullyl residues, generated by posttranslational deimination of arginyl residues by a peptidylarginine deiminase (PAD), are essential components of the epitopes recognized by ACPA. Five PAD isotypes (PAD1-4, and 6) encoded by five paralogous genes (PADI1-4 and PADI6, respectively) have been described in humans. At the mRNA level, the recently cloned PADI6 gene was shown to be mainly expressed in ovary, testis and peripheral blood leukocytes but no data have yet been published concerning tissular or cellular expression of the corresponding protein. Considering that the PAD6 enzyme could be present in mononuclear leukocytes infiltrating the ST of RA patients and therein involved in the generation of ACPA targets, we set to evaluate its expression in blood-derived mononuclear leukocytes and in the rheumatoid synovium. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and separated into purified monocytes (CD14+ PBMCs) and a lymphocyte-enriched fraction (CD14- PBMCs). Macrophages were generated by 7 days-culture of the monocytes in the presence of M-CSF and activated macrophages were obtained by 24 h-stimulation with LPS. Transcription of the PADI6 gene was assessed by RT-PCR using isotype-specific primers. PADI6 transcripts were present in the lymphocyte-enriched and monocyte fractions as well as in macrophages where their levels did not vary significantly upon LPS stimulation. To test expression of the corresponding protein, antibodies were generated by immunising rabbits with peptides chosen in isotype-specific regions of the predicted amino-acid sequence of human PAD6 determined by multi-alignment of all human, mouse and rat PAD sequences. After affinity-purification on the reactive peptides, the PAD6-specificity of the antibodies was confirmed as they recognized a recombinant human PAD6 and did not cross-react with purified rabbit Pad2 or with recombinant human PAD1, 3 or 4. Use of these antibodies in immunoblotting analyses of low salt-extracted proteins from the different blood-derived cell fractions at a detection sensitivity adjusted to ∼1 fmole, showed that neither lymphocytes nor monocytes nor resting or LPS-stimulated macrophages exhibited significant expression of the PAD6 protein. Similarly, immunoblotting analysis of proteins from low-salt extracts of synovial tissue samples from 16 RA patients demonstrated absence of the PAD6 protein in all samples. In contrast and in line with observations in mononuclear leukocytes, RT-PCR analysis in a subset of synovial tissue samples from 3 RA patients revealed the presence of PADI6 transcripts in 2 samples. In conclusion, even if transcription of the PADI6 gene can be evidenced in cells of the lymphocyte and monocyte lineages, and in the rheumatoid synovium, the expression of the corresponding protein appears as tightly posttranscriptionally downregulated. In the rheumatoid synovium, PAD6 clearly does not contribute to generation of the citrullinated targets of ACPA.

139 LIPOSOMAL ENCAPSULATION OF WATER-SOLUBLE DEXAMETHASONE (MICROMETHASON) ENHANCES AND PROLONGS THE ANTI-INFLAMMATORY EFFECTS IN RAT ADJUVANT ARTHRITIS

R. Anderson1,3, A. Franch3, M. Castell3, FranciscoPérez-Cano3, U. Rauchhaus2, S. Panzner2, DPohlers1, RBraeuer4, R. W. Kinne1.1Experimental Rheumatology Unit, Friedrich Schiller University Jena, Jena, 2novosom AG, Halle, Germany; 3Unit of Physiology, Faculty of Pharmacy, University of Barcelona, Spain; 4Institute of Pathology, Friedrich Schiller University Jena, Jena, Germany

Background: Glucocorticoids (GC) are well-known, efficacious anti-arthritic drugs. At high concentrations, however, their usage is limited by adverse effects. The present study therefore aimed at developing novel liposomal formulations of GC with improved targeting to activated phagocytic cells and increased efficacy. Serum-stable, unilamellar liposomes reliably encapsulated a water-soluble derivative of dexamethasone. The liposomes were free of polyethylene-glycol in order to avoid the immunological complications characteristic of StealthR liposomes.

Objective: Encapsulation of water-soluble dexamethasone-phosphate (DxM-P) in unilamellar anionic liposomes to avoid adverse effects and to enhance the therapeutic efficacy.

Methods: DxM-P was encapsulated into unilamellar liposomes (Micromethason) and injected intravenously (i.v.; 1, 0.1, or 0.01 mg/kg DxM each) into rats with established adjuvant arthritis (AA) on days 14, 15, and 16 following induction of arthritis. Controls were buffer, drug-free liposomes, and corresponding concentrations of non-encapsulated DxM-P. The treatment effects were monitored by assessment of arthritis score (total of 16 for 4 paws), joint swelling (paw volume), and body weight.

Results: Short-term i.v. treatment with Micromethason (total of 3, 0.3, or 0.03 mg DxM/kg body weight) resulted in strong, significant, dose-dependent, and long-lasting suppression to almost normal levels of arthritis score (days 17–23; p<0.01; minimum mean score of 0.25 on day 19) and joint swelling (days 19–28; p<0.01 or 0.05; minimum swelling of 0.01 ml on day 23) in comparison to buffer-treated AA controls. After an initial drop on days 19 and 20, the body weight of Micromethason-treated AA rats returned to that of buffer-treated AA controls. In contrast, treatment with the respective dose of free DxM-P only transiently and less clearly reduced the arthritis score (days 16 and 17; p<0.05; minimum mean score 1.5 on day 17), with a strong rebound and overshoot effect starting 2 days after the end of treatment. On day 23, Micromethason was significantly more effective in suppressing the arthritis score than free DxM-P. Drug-free liposomes remained completely ineffective. In vitro assays with human peripheral blood monocytes demonstrated optimal tolerability of the liposomes (<5% dead cells over 6 hours).

Conclusion: Systemic i.v. application of liposome-encapsulated, water-soluble DxM-P (Micromethason) is highly efficacious in suppressing rat AA. Whereas free drug is only transiently effective, Micromethason leads to long-lasting, dose-dependent suppression of arthritis (total of 12 days in the highly severe AA model) to almost normal levels. This new formulation of potent anti-inflammatory steroids may therefore allow to enhance the therapeutic efficacy and to limit potential side effects of treatment in inflammatory disorders like human rheumatoid arthritis.

140 CXCL13, CCL21 AND CXCL12 ARE UPREGULATED IN MUCOSAL ASSOCIATED LYMPHOID TISSUE LYMPHOMAS (MALT-L) IN PATIENTS WITH SJOGREN’S SYNDROME AND COOPERATE IN THE MAINTENANCE OF THE IMMUNE RESPONSE AND MALIGNANT B CELL PROLIFERATION

F. Barone1, M. Bombardieri1, J. Spencer2, P. Isacsoon3, F. Humby1, P. Morgan4, S. Challacombe4, G. Valesini5, C. Pitzalis1.1Rheumatology, 2Immunology, GKT School of Medicine; 3Immunology, UCL; 4Oral Pathology and Medicine, GKT School of Medicine, London, UK; 5Reumatologia, La Sapienza, Roma, Italy

Background: We previously demonstrated that in minor salivary glands of patients with Sjogren’s syndrome (SS-mSGs) a true phenomenon of ectopic lymphoneogenesis takes places, with the formation of germinal centres (GCs), in association with the ectopic expression of the lymphoid chemockines (CKs) CXCL13 and CCL21. In SS major SGs (SS-MSGs) these structures are known to support clonal immunoglobulin heavy chain gene rearrangements and somatic hypermutation, favouring expansion and selection of autoreactive B cell clones, clonally related to lymphomatous B cell clones (responsible for lymphoma development in SS). The involvement of lymphoid CKs and CXCL12 in the development of lymphoid malignancies has been demonstrated. However, at present no data on the expression of these CKs nor their cellular source within SS-MSGs and MALT-Ls from patients with SS have been described.

Objectives: To assess the specific contribution of lymphoid CKs in the organization of lymphoid proliferation within SS-MSGs and SS-MALT-Ls.

Methods: We studied 12 SS-mSGs, 4 SS-MSGs with lymphoepithelial lesion (LESA) and 20 SS-MSGs with non-Hodgkin B-cell MALT-Ls. In order to define the histological organization of the lymphoid infiltrate (reactive vs. malignant areas) and identify the B cell subpopulations infiltrating the glands, immunohistochemistry was carried out for the following cellular (CD21, bcl-2, bcl-6, IgD, CD20, CD3, CD68) and vascular markers (CD31, PNAd). On sequential sections, digital images for CXCL13, CCL21 and CXCL12 were analyzed by thresholding positive staining in detected areas of interest. Digital image analysis estimated CK’s volume fraction areas as a ratio between CXCL13, CCL21 and CXCL12 positive area over malignant or reactive areas. CK’s producing cells were identified by double staining on sequential sections. Mononuclear cells (MCs) were isolated from two SS-MALT-L parotids following enzymatic digestion and stained for CD19, CD38, CD27, CD24, IgM, IgD, CD10, CD5, CD3, CD4, CD69, CD45RO, CXCR5, CXCR4, CCR7. Finally mRNA from total SS-MSGs and MALT-Ls and isolated B and T cells from MALT-Ls were analyzed by RT-PCR for the transcript levels of CXCL13, CCL21 and CXCL12.

Results: Reactive areas, characterized by T/B cell segregation, CD20+IgD+bcl-2+ follicular B cells, presence of follicular dendritic cell (FDC) networks in GCs and high endothelial venule formation were detected in 100% of MALT-L. A B-cell population (CD20+IgD-bcl-2+), characterized by nuclear abnormalities and monocytoid appearance, was consistently observed within the proliferating ducts and identified as the B cell malignant component. FACS analysis on isolated MALT-L MCs showed B cells in diverse maturative stages (transitional, mature and memory B cells) and a B-cell population CD19posIgDlowCD24negCD27low/negCD5negCD10neg not detectable in the controls. Ectopic expression of CXCL13 and CCL21 was observed in 100% of SS-MSGs and MALT-Ls. A significant increase in CXCL13’s volume fraction analysis was observed both in SS-MSGs and SS-MALT-L reactive areas, as compared to SS-mSG follicular areas (p<0.05 and p<0.05, respectively). Strong difference in CXCL13 within MALT reactive area compared to malignant areas was detected (p<0.001). CCL21 was significantly increased in MALT-Ls compared to both SS-mSGs and MSGs and mainly confined to the T cell area. CXCL12 was strongly expressed by ductal epithelial cells, vessels and reactive areas both in 100% of the SS-mSG, SS-MSGs and MALT-Ls. Interestingly a significant increase in CXCL12 expression on MALT-L malignant areas as compared to reactive areas in mSGs and MSGs was detected (p<0.01 and p<0.05). RT-PCR analysis showed increased CXCL13 and CCL21 level in SS-MSG compared to MALT-Ls and mSGs, while strong up-regulation in CXCL12 transcript in MALT-Ls as compared to mSGs and MSGs was detected. CXCL13, CCL21 and CXCL12 were detected on CD68+ cells by immunohistochemistry, while CD20 and CD3, CXCL13 and CCL21 double staining and mRNA analysis on MALT extracted T and B lymphocytes, showed negligible expression of the two CKs in MALT-L extracted lymphocytes. Interestingly we identified strong CXCL12 expression on CD19+ MALT-L isolated cells both at protein and mRNA level and on ductal epithelial cells in close contact with the malignant B cell infiltration. In agreement with this increase, we demonstrated a significant down regulation of CXCR4 on MALT-L-isolated B cells, likely to be the result of CXCR4 internalization upon CXCL12 ligation.

Conclusion: These results imply that CXCL12/CXCR4 axis is functional in MALT-L malignant cells and suggests that the glandular microenvironment, BCR signalling upon Ag engagement and autocrine signals from the same malignant population concur in MALT lymphomagenesis, triggering local activation of malignant B cells and favouring their survival and expansion. The selective implication of CXCL13, CCL21 and CXCL12 in the maintenance and organization of diverse functional microenvironments within parotid SS-MALT-Ls advocate a role for these molecules in the continuous production of the malignant B clones and in the survival and spreading of these clones within the glands.

141 INDUCTION OF HIF-1ALPHA EXPRESSION DURING MONOCYTE-MACROPHAGE DIFFERENTIATION AND EFFECT ON PRODUCTION OF PRO-ANGIOGENIC FACTORS

J. Westra, E. Brouwer, M. D. Posthumus, B. Doornbos-van der Meer, C. G. M. Kallenberg, P. C. Limburg.Rheumatology and Clinical Immunology, University Medical Center Groningen, The Netherlands

Aim: The transcription factor hypoxia-inducible factor (HIF)-1 is activated during hypoxia by stabilisation of the subunit HIF-1alpha. This pro-angiogenic transcription factor may also be induced by non-hypoxic, inflammatory factors, and during differentiation of monocytes to macrophages. In rheumatoid arthritis (RA) macrophages expressing HIF-1alpha have been demonstrated in the synovial tissue. Aim of the study was to investigate whether HIF-1alpha expression was induced during differentiation of peripheral blood monocytes to macrophages. Furthermore it was investigated which pro-angiogenic mediators were produced during differentiation by LPS-stimulated monocytes or monocyte-derived macrophages (MDM).

Materials and methods: Human peripheral blood monocytes were left to differentiate with MCSF and 1% FCS for 5 days. Cells were stimulated with 50 ng/ml LPS at day 1, 2, 3, 4, and 5, and HIF-1alpha mRNA expression was measured by real-time RTPCR after 4 hours. Moreover, production of pro-angiogenic factors such as VEGF, MMP-9, IL-8 and COX-2 were measured at mRNA level by real-time RTPCR and at protein level by ELISA.

Results: During differentiation HIF-1alpha mRNA expression was induced after LPS stimulation and was maximal at day 4 with an 8.2 fold increase. LPS stimulation induced IL-8 and COX-2 mRNA expression during differentiation, while VEGF and MMP-9 mRNA levels were elevated at day 2 and 3, and returned to monocyte levels at day 4 and 5. Monocytes produced high levels of IL-8 after LPS stimulation, which was reduced twofold at day 2. Monocyte-derived macrophages produced higher levels of MMP-9 than monocytes (30 times in unstimulated cells, 3.3 times more in stimulated cells). Also LPS-induced production of VEGF was higher in macrophages than in monocytes.

Conclusions: The process of differentiation from monocyte to macrophage induces HIF-1alpha mRNA and has an effect on the production level of the pro-angiogenic factors. Although monocytes release higher levels of IL-8, the production of MMP-9 and VEGF was higher in monocyte-derived macrophages after LPS stimulation. Here we demonstrate that under non-hypoxic conditions HIF-1 is induced by differentiation, and that production of pro-angiogenic factors is increased in macrophages compared to monocytes.

142 LOCAL DELIVERY OF IFN-BETA USING AN ADENO-ASSOCIATED VIRUS TYPE 5 EFFECTIVELY INHIBITS ADJUVANT ARTHRITIS IN RATS

J. Adriaansen1, F. J. Fallaux2, C. J. de Cortie2, M. J. Vervoordeldonk1,2, P. P. Tak1.1Div. of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam; 2Arthrogen BV, Amsterdam, The Netherlands

Background: Interferon-beta (IFN-beta) is a cytokine with potent immunomodulatory properties, being a promising therapeutic molecule for the local treatment of rheumatoid arthritis (RA). Recently, it has been shown that the absence of IFN-beta exacerbates arthritis in mice by activation of osteoclasts and stromal cells. In a proof of concept study, we previously over-expressed IFN-beta intra-articularly using an adenoviral vector in rats with adjuvant arthritis (AA). This effect was powerful, albeit transient due to the vector chosen. Therefore, in the context of pre-clinical development, we selected another delivery vector, derived from adeno-associated virus (AAV). Recombinant AAV vectors are low immunogenic, non-toxic and are able to transduce dividing and non-dividing cells. Previously we have shown that rAAV5 is an optimal vector for intra-articular gene transfer. The goal of this study was to investigate the effects of intra-articular delivery of an optimized gene construct expressing the IFN-beta gene.

Methods: Three rAAV5 vectors containing the rat IFN-beta gene were constructed; rAAV5.CMV-IFN-beta, rAAV5.NF-kappaB-IFN-beta, and rAAV5.CMV-IFN-beta-intron; an empty vector served as a control. To exert a maximal effect, protein production should parallel the course of the disease. For this reason, we also placed the gene for IFN-beta under control of an inflammation responsive (NF-kappaB) promoter. In addition, we attempted to boost IFN-beta production by inclusion of an intron. The vectors (5×10e10 vm/joint) were intra-articularly injected 12 days after adjuvant immunization in rats (total n = 7/group). Paw swelling was measured daily by water displacement plethysmometry. Three weeks later, the animals were sacrificed and hind joints were collected for analysis of transgene and protein expression, inflammation, cartilage, and bone destruction.

Results: Intra-articular injection of the rAAV5 constructs resulted in local transcription of the transgene and production of the IFN-beta protein. There was a 50% reduction in paw swelling (P<0.05) after a single intra-articular injection of rAAV5.NF-kappaB-IFN-beta, resulting in sustained amelioration of arthritis until the end of the experiment. In contrast, neither rAAV5.CMV-IFN-beta nor rAAV5.CMV-IFN-beta-intron led to a significant improvement in arthritis compared to control animals. Additionally, a significant beneficial effect was observed on proteoglycan depletion and cartilage erosions in animals injected with either rAAV5.NF-kappaB-IFN-beta or rAAV5.CMV-IFN-beta compared to control (P<0.05).

Conclusion: Taken together, the combined use of a low-immunogenic rAAV5 vector and a disease inducible NF-kappaB-responsive promoter allows prolonged therapeutic effects in an animal model for RA. Thus, IFN-beta gene therapy using this innovative gene construct represents a promising approach for the intra-articular treatment of RA.

143 THE ASSOCIATION BETWEEN IL10 AND JOINT DESTRUCTION IN RA CAN BE ATTRIBUTED TO ENHANCED AUTOANTIBODY RESPONSES

F. A. S. Kurreeman, K. N. Verpoort, A. Ioan-Facsinay, J. J. M. Schonkeren, H. W. Uh, E. Slagboom, R. G. Westendorp, R. E. M. Toes, T. W. J. Huizinga.Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands

Introduction: Rheumatoid arthritis (RA) is a heterogeneous autoimmune disease. Recent data suggest that anti-citrullinated protein antibody (ACPA) positive and ACPA negative disease are genetically distinct. Previous research shows that IL10 polymorphisms (SNPs) associate with severity. Since IL10 affects B cell responses, we investigated whether the effect of IL10 SNPs can be attributed to antibodies.

Methods: To determine the level of linkage disequilibrium in the IL10 gene, 20 snps spanning 27 kb were genotyped in 60 healthy unrelated Dutch Caucasian. Nine tagging snps which capture the most information about the gene were genotyped in 2 independent cohorts of healthy individuals from which whole blood LPS-induced IL10 levels were available. DNA and serum from RA patients (N = 522) were available for IL10-2849 genotyping and ELISA measurement of different isotypes of ACPA levels.

Results: Individuals carrying the IL10-2849G allele produced significantly higher levels of IL10 as compared to non-G carriers in 2 independent cohorts (N = 100, N = 135). In the RA patients (N = 522), this SNP was associated with higher levels of joint destruction but intriguingly, this genetic effect of IL10-2849G is confined to the ACPA+ subgroup of RA patients. These data confirm the distinct genetic background of autoantibody positive and negative disease and indicate that IL10-2849G may affect autoantibody response. Indeed, our data show that high IL10 producers, as defined by IL10-2849G, displayed higher levels of ACPA and a more extensive isotype-usage as compared to non-carriers.

Conclusion: We therefore propose that the association between genetic polymorphisms in the IL10 region and RA is attributed to a broader and stronger autoantibody response.

144 HIGH LEVELS OF ANTIBODY-BOUND CIRCULATORY CELL FREE DNA IN RHEUMATOID ARTHRITIS

X-YZhong1, I. von Mühlenen2, Y. Li1, A. Kang1, A. Kumar Gupta1, A. Tyndall2, W. Holzgreve1, S. Hahn1, P. Hasler2.1Laboratory for Prenatal Medicine, 2Department of Rheumatology, University Hospital, Basel, Switzerland

Elevated concentrations of cell free DNA have been noted in a number of disorders, and have been interpreted as evidence of increased levels of cell death or turn-over associated with these pathologies. Immune complexes containing DNA and antibodies against DNA have been implicated in the etiology of rheumatoid arthritis (RA). In a DNAse II knockout mouse, erosive arthritis has been attributed to the inability of macrophages to digest engulfed DNA. We hypothesized that cell free DNA could be elevated in autoimmune diseases, in which anti-DNA antibodies are present. Cell free DNA was measured in plasma and serum from patients with RA and from healthy controls by glyceraldehyde-3-phosphate dehydrogenase quantitative PCR. SepharoseTM bead adsorption of plasma and elution were used to determine antibody-bound DNA. Initially we analyzed archived serum samples from 28 rheumatoid arthritis (RA) patients and 28 normal controls. Serum cell free DNA was significantly higher in the RA group than in the control group (median 69’300 GE/ml plasma vs. 36’653 GE/ml plasma; p = 0.009). In freshly obtained paired plasma and serum samples of 16 normal controls the median GADPH copies were 4524, range 319–20,974 and in 26 RA patients 17,205 (21,263–2,375,075, P = 0.0001). In the serum from normal controls the median copies were 34,949 (1705–239,237) and from RA patients 222,468 (21,263–2,375,075, P = 0.004). The serum: plasma ratio was 7.7 in the normal controls and 12.9 in RA patients. Sepharose bead precipitation of antibodies showed that the vast proportion of cell free DNA in cases with RA was antibody associated (median  = 81%). Cell free DNA in RA patients was subject to change when measured before, and 1 hour after completion of the infusion of the monoclonal anti-TNF antibody infliximab at 3 mg/kg over 2 hours. In 10 cases, elevations in the serum occurred in five, decreases in two, while plasma levels showed similar changes, though at a far lower level. Apoptosis of circulating lymphocytes in RA patients after infliximab remained unchanged and did not differ significantly from normal controls. Our results show high levels of cell free DNA and antibody binding of this DNA in patients with RA. Furthermore, they show the presence of large amounts of complexes of DNA and antibodies in RA, providing support for the concept that DNA and associated antibodies are crucial in the pathogenesis of RA. It will be of great interest to extend our findings at the levels of basic research, diagnosis and assessment of therapy in RA patients.

145 INTRA ARTICULAR ANTI TNF GENE THERAPY IN COLLAGEN INDUCED ARTHRITIS

A. Denys, A. Noël, K. Chekroud, K. Benihoud, M. C. Boissier, N. Bessis.INSERM Eri-18, Bobigny, France

Objective: Gene therapy using non viral vectors is a promising strategy which could allow an efficient, specific and long expression for therapeutic protein delivery. We previously demonstrated the relevance of electrotransfer (ET) of plasmid using a systemic (intra-muscular) administration. Local intra articular (i.a.) ET could also be of interest in organ-targeted diseases such as RA. It could permit us to use lower quantities of plasmid vector, avoid adverse effects such as systemic toxicity, and allow us to get higher transgenic protein within the joint. Our objective was to define in healthy mice the parameters for direct i.a. ET and to evaluate in arthritic mice its therapeutic potential using a plasmid encoding the fusion protein formed with the soluble type I TNF receptor (sTNFRI) and the heavy chain of IgG1 (sTNFRI/IgG1).

Methods: DBA/1 mice (6–8 weeks old) were used. To induce arthritis, DBA/1 mice were injected with native collagen type II emulsified in complete Freund’s adjuvant. Then, the plasmid pVax1 mTNFR-Is/mIgG1 was injected into the right and left knee at the time of onset of clinical symptoms. After plasmid injection, an electric field was applied with electrodes placed apart on either side of the joint. We evaluated the local effect of ET by clinical observation of the electrotransferred knees. The systemic effect was evaluated by clinical observation of the non electrotransferred paws. Plasmid localisation was evaluated within the joint after i.a. injection of the plasmid pVax/LacZ.

Results: After i.a. ET of various plasmid doses, a dose dependent expression was observed locally in knee-conditioned media and maximal secretion was observed after injection of 20 µg chimeric plasmid. Local treatment of CIA with pVax mTNFR/mIgG1 electrotransfer induced significant decrease in the percentage of arthritic knees as compared to controls. On the other hand, it is interesting to note that the systemic effect is transitory. Moreover, i.a. ET of pVax LacZ showed that plasmid expression could be detected in the synovial tissue or within the surrounding muscles.

Conclusion: Intra articular gene therapy opens perspectives for non viral gene therapy applications to rheumatoid arthritis. Local effect of TNF soluble receptor is significant since it diminished joint inflammation. The systemic effect is transitory. This strategy demonstrated the feasibility of local non viral gene therapy in arthritis.

146 SUPPRESSOR ACTIVITY AMONG CD4+CD25++ T CELLS IS DISCRIMINATED BY MEMBRANE-BOUND TNF

J. Wang, H. van Dongen, H. Ulrich Scherer, T. W. J. Huizinga, R. E. M. Toes.Dept. of Rheumatology, Leiden University Medical Center, The Netherlands

Objective: CD4+CD25+ T regulatory (Treg) cells are crucial in immune tolerance and have great therapeutic potential in the treatment of transplant rejection and autoimmune diseases. However, efforts in this regard have been hampered by the lack of specific cell surface markers to define and isolate Treg cells with highly suppressive capability as CD4+CD25++ T cells in humans represent a heterogeneous T cell population. This study aimed to define surface makers for identifying and enriching Treg cells with enhanced regulatory ability within the CD4+CD25++ T cell compartment.

Methods: Based on the observation that infliximab but not etanercept can restore regulatory T cell activity, we speculated that membrane-bound TNF-alpha (mTNF) is expressed on activated non-regulatory CD4+CD25++ T cells. Therefore, the expression of mTNF in human peripheral blood CD4+ T cells was analyzed by flow cytometry in both healthy individuals (n = 19) and RA patients (n = 40). Frequencies of mTNF-expressing cells were compared among different CD4+ T cell populations, between age-matched healthy controls and RA patients, and in anti-TNF treated RA patients (n = 10, before vs. 3 months after the first administration of TNF-blocking agents). To determine the functional capacity of mTNF positive and negative T cells within the CD4+CD25++ compartment, mTNF-CD4+CD25++ and mTNF+CD4+CD25++ T cells were purified by FACS-sorting. Cytokine profiles were examined and suppressive abilities were assessed by 3H-thymidine incorporation as well as by analyzing cytokine levels in supernatants.

Results: A significant amount of CD4+CD25++ T cells in human peripheral blood express mTNF, which increases with age in healthy individuals. Although mTNF negative and positive CD4+CD25++ T cells express comparable levels of Foxp3 and are both hyporesponsive to TCR stimulation, mTNF-CD4+CD25++ T cells produce more anti-inflammatory cytokines and suppress the proliferation and cytokines production of responder T cells in a more profound manner as compared to their mTNF+ counterparts. Moreover, the expression of mTNF on CD4+CD25++ T cells in RA patients is elevated, while treatment with anti-TNF Abs results in a reduced frequency of mTNF+ T cells in the CD4+CD25++ T cell compartment.

Conclusion: Expression of mTNF correlates with functional activities within CD4+CD25++ T cells and lack of this molecular on the cell surface could be used to identify and enrich Treg cells with maximal suppressor potency in human peripheral blood. In addition to blocking soluble circulating TNF, in vivo depletion of less suppressive mTNF+CD4+CD25++ T cells by anti-TNF Abs might contribute to the reversal of the compromised suppressive ability of CD4+CD25++ T cells in RA patients.

147 CITRULLINATED PROTEINS ARE ASSOCIATED WITH SERUM EXOSOMES IN NORMAL AND ARTHRITIC MICE AFTER COLLAGEN INDUCTION

K. Skriner1, K. Adolph1, P. Jungblut2, G. R. Burmester1.1Charité University Medicine, Department of Rheumatology and Clinical Immunology, Humboldt University and Free University, Berlin, Germany; 2Max-Planck-Institute for Infection Biology, Berlin, Germany

Background: In addition to soluble proteins and mediators, cells also release membrane vesicles into the extracellular environment—exosomes and apoptotic plebs. Apoptotic blebs have been studied in detail and been shown to contain multiple autoantigens. Recent studies demonstrated that exosomes can stimulate T-cells directly or bind to dendritic cells, suggesting a general function for exosomes in the immune response. Synovial exosomes have been studied by our group and been shown to contain specific citrullinated antigens. The exosomal protein content and its functions are only starting to be revealed.

Objective: To study the induction and distribution process of citrullinated proteins. Serum exosomes of collagen induced arthritis (CIA) mice were prepared to investigate the differences in protein content and the presence of autoantigens in serum exosomes. These may be important means for the distribution of autoantigens.

Methods: In the present study, 4 ml serum pool from each group of CIA and normal mice were used to purify serum exosomes (according to the protocol of Thery et al.) and analyzed for specific autoantigenic content by immunoblotting. To investigate the presence of citrullinated proteins in serum exosomes, an immunoblot with an anti-citrullinated protein antibody was performed. Immunoblots were analyzed with RA and control sera and were used to investigate immunoreactive proteins in the preparation.

Results: Identical citrullinated protein patterns were found in both healthy and CIA mouse exosomal preparations, and even the total protein patterns were identical. Four different proteins (70, 77, 140, 150 kD) in the mouse serum exosomal preparations were citrullinated and also highly reactive with citrulline positive human RA sera, but not with control sera. These proteins were separated and have been sequenced and not found to be identical to citrullinated synovial exosomal proteins. These observations also demonstrated that the serum exosomes contain specific citrullinated autoantigens different in size than in human synovial exosome preparations, even though these proteins reacted with RA sera.

Conclusion: Citrullinated proteins are present and recognized by human RA sera in both murine serum exosomal fractions in both healthy and disease situations. Since, therefore, citrullinated proteins can be distributed via the blood stream in both instances, a specific, altered immune recognition mechanism must play a role in the induction of an autoimmune response. The analysis of exosomal protein extracts may lead to the identification of novel protein autoantigens and help to understand how autoantigens may lead to pathogenesis in systemic rheumatic diseases.

148 TRANSIENT EXPRESSION OF FOXP3 IN HUMAN ACTIVATED NONREGULATORY CD4+ T CELLS

J. Wang, A. Ioan-Facsinay, H. Ulrich Scherer, E. I. H. van der Voort, T. W. J. Huizinga, R. E. M. Toes.Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands

Objective: Foxp3 plays a key role in CD4+CD25+ T regulatory (Treg) cell function and represents a specific marker for these cells in mice. Despite the strong association between FOXP3 expression and regulatory function in fresh human T cells, little is known about the dynamics of FOXP3 expression and its relation to the suppressive function in activated human T cells. Therefore, this study was undertaken to investigate the dynamics of FOXP3 expression during human CD4+ T cell activation as well as the relationship between its expression and the regulatory function at single-cell level.

Methods: FACS-purified human CD4+CD25- T cells were activated in vitro by anti-CD3/28 in the presence or absence of exogenous IL-2. The dynamics of endogenous FOXP3 expression induced by activation were measured at the single-cell level by flow cytometry. Cytokine profiles of the induced FOXP3+ conventional T cells were examined and compared to activated naturally occurring CD4+CD25++ Treg cells. Suppressive capabilities were assessed by 3H-thymidine incorporation, CFSE-dilution and IFN-gamma levels in supernatants.

Results: FOXP3 is rapidly induced and expressed in a high percentage of activated T cells after in vitro stimulation of human CD4+CD25- cells by plate-bound anti-CD3 and soluble anti-CD28 in the presence of exogenous IL-2. Additional TGF-beta is not required for the induction of high FOXP3 expression. FOXP3-expressing activated conventional T cells are hyporesponsive, but secret more pro-inflammatory and less anti-inflammatory cytokines as compared to stimulated naturally occurring CD4+CD25++ Treg cells. Furthermore, FOXP3 expression in activated conventional T cells is not directly correlated to the suppressive capabilities, as it is also expressed in activated nonsuppressive T cells. However, in this nonsuppressive T cell population, FOXP3 expression is transient, while it is stably expressed in activated T cells that do display suppressive function.

Conclusion: Expression of endogenous FOXP3 as such is not sufficient to induce regulatory T cell activity or to identify Treg cells in humans, especially in conditions in which T cells are thought to be constantly activated such as in many rheumatic disorders.

149 INTERACTION BETWEEN ORAL Streptococcus mutans colonisation, TLR responses to streptococci and low serum mannose-binding lectin levels in BehCet’s disease

G. Mumcu, N. Inanc, Y. Elbir, T. Ergun, S. Yavuz, H. Direskeneli.Marmara University Medical School, Istanbul, Turkey

Aim:Streptococcus mutans (S mutans) causes dental caries in the human oral cavity and might be associated with Behçet’s disease (BD). Streptococci activate the innate immune system through toll-like receptors (TLR). Serum levels of mannose-binding lectin (MBL), a complement-like protein of innate immunity associated with infections in immunocompromised patients, is shown previously to be decreased in patients with BD (Inanc et al). The aim of this study was to evaluate the relationship between S mutans presence, disease severity, MBL levels and TLR responses to streptococci in patients with BD.

Material and methods: Ninety patients with BD (M/F: 53/37, mean age: 37.9+11.8 years) were investigated. S mutans levels were investigated in whole paraffin-stimulated saliva by CRF Bacteria kits (Ivoclar, Vivadent). S mutans colonies were categorized as high (>100,000 CFU/ml of saliva) or low levels (<100.000 CFU/ml) of colony growth. Oral health was assessed by using dental and periodontal indices. Total BD clinical severity score was calculated as described by Krause et al. Serum MBL levels were measured by ELISA. PB cells cultured with S sanguis (StS) (10 microg/ml) for 3 hrs were flow-cytometrically analysed for TLRs 1, 2, 4 and 6 on granulocyte and monocytes.

Results: A high level of S mutans in saliva was observed in 51.1% of BD patients and 43.4% of HC. In patients with high S mutans level, BD severity score (6.2+2.7) significantly correlated with scores of gingival index (1.7+1.03) and periodontal pocket depth (2.7+0.5) (r = 0.34 p = 0.021 and r = 0.44 p = 0.009, respectively), showing a poor oral health. A significant correlation was also observed between the number of oral ulcers per month (2.1+1.1) and score of sulcus bleeding index (1.6+1.1) in these patients (r = 0,45 p = 0.022). In patients with low S mutans levels, only score of plaque index (1.4+1.1) correlated with the number of oral ulcers (r = 0.45 p = 0.04). Mean serum MBL level was lower in patients with high S mutans (1603+1846 ng/ml vs. 2396+1741 ng/ml), however without reaching statistical significance (p = 0.41). 68.8% of patients with low MBL levels (<500 ng/ml) had high levels of S mutans colonies in saliva. TLR-6 expressing granulocytes increased significantly in BD patients compared to HC after incubation with StS (BD: 12.8 + 0.6 vs HC: 8.3 + 0.7, p<0.05). However, no significant difference in TLR responses were observed on monocytes after in vitro stimulation.

Conclusion: The relationship between poor oral health and higher disease severity in BD patients with high S mutans colonisation suggest that oral streptococci might influence the clinical course of BD. Increased TLR-6 responses of BD granulocytes to streptococci might be among the pathogenic mechanisms between the environmental stimuli and the observed innate hyperreactivity in BD. Whether low MBL is associated with a predisposition to oral streptococcal colonisation requires a larger study.

150 A-SAA UPREGULATES CHEMOKINE EXPRESSION, MMP PRODUCTION AND GAG RELEASE IN INFLAMMATORY ARTHRITIS

M. Connolly, J. McCormick, R. Mullan, O. Fitzgerald, B. Bresnihan, D. Veale, U. Fearon.University College Dublin, Ireland

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory arthritis (IA) characterised by progressive joint destruction. Serum amyloid A (A-SAA) is an acute phase protein with cytokine-like properties that is increased up to 1000-fold during inflammation. Furthermore A-SAA induces cell adhesion and angiogenesis in vitro, processes that enhance leucocyte migration, essential for persistent tissue and cartilage invasion.

Aim: To examine the mechanisms involved in A-SAA-induced cell migration and invasion in RA.

Methods: Synovial fibroblasts (SFCs) and explant tissue were obtained from patients with RA at arthroscopy. Paired serum and synovial fluid was obtained from patients with IA (n = 11). Serum was obtained from RA patients at baseline and 3 months post anti-TNF treatment (n = 36). Neutrophil migration and monocyte adhesion was measured using transwell migration and adhesion assays. In whole tissue synovial explant cultures and HDECs, an NFkappaB inhibitor (PDTC) and Western Blotting were used to examine if A-SAA-induced IL-8 and MCP-1 production is mediated by NFkappaB signalling. Matrix metalloproteinases (MMP)-2 and -9 activity was examined by gelatin zymography. A-SAA, MCP-1, IL-8, MMP-1, MMP-13 and GAG were measured by ELISA.

Results: High levels of SAA were found in paired serum (range 14.82–314 ug/ml) and synovial fluid (range 7.34–173.43 ug/ml) from patients with IA. A-SAA was spontaneously released from RA whole tissue synovial explant cultures (200–1000 ug/mg tissue). We have previously shown that A-SAA significantly induced expression of IL-8 and MCP-1 (p<0.05) in primary RASFC, HMVECs and synovial tissue. Here we show SAA-induced transendothelial cell migration and monocyte adhesion is inhibited by pre-incubation with anti-IL-8 and anti-MCP-1 respectively. We demonstrated in RA synovial explants that addition of PDTC blocked A-SAA induction of IL-8 and MCP-1 (p<0.05), and using western blot demonstrated that A-SAA significantly induced NFkappaB expression in RA synovial explant cultures and HDECs, an effect similar to TNFalpha. In vivo A-SAA levels strongly correlate with MCP-1 and IL-8 in serum pre and post biologic therapy (p<0.01). To explore the role of A-SAA in cell migration and invasion we demonstrated that A-SAA induces MMP-2 and MMP-9. Furthermore, using human primary chondrocytes, we have shown that A-SAA can induce MMP-1, -13 and GAG release (p<0.05). Finally, while TNFalpha stimulates A-SAA production from RA synovial explant cultures, conversely we have also demonstrated that A-SAA upregulates TNFalpha.

Conclusion: A-SAA at physiological levels upregulates chemokine expression, neutrophil transmigration and cell adhesion through the NFkappaB pathway. Furthermore A-SAA is involved in matrix degradation, allowing advancement of blood vessels with subsequent cartilage invasion. We demonstrated high expression of A-SAA locally in the joint and that A-SAA induces TNFalpha expression in RA synovial tissue, supporting a pathophysiological role for A-SAA in RA. These results suggest A-SAA promotes cell migration, angiogenesis and subsequent cartilage invasion and therefore represents a new therapeutic approach in the treatment of RA.

151 EXPRESSION OF THE FOUR AND A HALF LIM ONLY PROTEIN FHL2 IN RHEUMATOID ARTHRITIS AND INVOLVEMENT IN MIGRATORY AND INVASIVE BEHAVIOUR IN RASF

L. H. Meyer1, K. Neugebauer1, E-MSchnäker2, B. Dankbar1, C. Wunrau1, N. Pundt1, V. Wixler3, T. Pap1.1Division of Molecular Medicine of Musculoskeletal Tissue, University Hospital Munster, Germany; 2Department of Dermatology, University of Muenster, Germany; 3Institut of Molecular Virology, ZMBE

The four and a half LIM-domain protein 2 (FHL2) is an interacting partner of the beta-subunit of integrins as well as focal adhesion- and mitogen-activated protein kinases. FHL2 also carries an intrinsic activation domain and acts as a co-transcription factor with AP-1. Furthermore, FHL2 has been shown to interfere with mesenchymal cell activation. Here, we studied the expression of FHL2 in RA and OA synovium and analyzed its expression and function in RA and OA synovial fibroblasts (RASF and OASF). Using FHL2 specific antibodies, the expression of FHL2 was studied in synovial tissue samples obtained from 4 RA and 3 OA patients by immunohistochemistry. SF were isolated from these tissues and the expression of FHL2 was analyzed by Western blot analysis to compare the total amount of FHL2 in these cells. Migration assays on collagen and fibronectin matrices were performed to investigate the migratory behaviour of these cells. siRNA was used to knock down FHL2-expression in RASF, and FHL2-silencing was analysed by Western blot and real-time PCR. To investigate the influence of FHL2 on the invasive behaviour of RASF, FHL2 was silenced and invasion of the RASF was measured in the recently established TEER assay with Vitrogen 100-coating of the epithelia cell layer. The invasion assay was performed in presence and absence of TNFalpha (100 ng/ml). We found a significant up-regulation of FHL2-expression in all RA synovial tissues with most prominent staining in the synovial lining and at sites of joint destruction. OA synovium exhibited only weak staining for FHL2. As seen in Western-blot analysis, RASF showed a marked upregulation of FHL2 as compared to OASF, and transfection of RASF with siRNA against FHL2 resulted in nearly complete (75%) knock down of the protein. Migration and invasion analysis of fibroblasts from RA and OA patients revealed higher mobility and invasiveness of RASF compared to OASF. Interestingly, silencing of FHL2 by siRNA significantly increased the invasiveness of RASF compared to controls. This was particularly true when RASF were stimulated with TNFalpha. Hence, our data suggest a disease-specific up-regulation of FHL2 in patients with RA. Moreover, the data imply that FHL2 is involved in balancing the invasiveness and migratory behaviour of RASF. Further studies are needed to determine the functional role and the molecular basis of increased FHL2 expression in RASF.

152 DIFFERENTIAL GENE EXPRESSION IN THE BONE MARROW AND PERIPHERAL BLOOD OF SYSTEMIC LUPUS ERYTHEMATOSUS (SLE): BONE MARROW BETTER SEPARATES ACTIVE FROM INACTIVE LUPUS PATIENTS

M. Nakou, N. Knowlton, H. Papadaki, A. Raptopoulou, P. Sidiropoulos, H. Kritikos, M. Centola, D. T. Boumpas.Rheumatology, Immunology and Allergy, and Hematology, University of Crete, Medical School, Heraklion, Greece and OMRF Microarray Core Facility, Oklahoma City, USA

Aim: The bone marrow has an important role in the development of B cell tolerance, the presentation of antigens to autoreactive lymphocytes and in homing the plasma cells that produce antibodies. Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by the production of antibodies with specificity for a wide range of self-antigens. In view of the importance of bone marrow in B cell biology, we examined the microenvironment of the bone marrow in these patients by the use of DNA microarrays and multiplex cytokine assay.

Patients and methods: We isolated: a) bone marrow mononuclear cells (BMMCs) from 20 SLE patients, 12 with active (SLEDAI >8) and 8 with inactive (SLEDAI<8) disease and b) peripheral blood mononuclear cells (PBMCs) from 27 patients, 16 with active and 11 with inactive disease. We used in-house arrays representing 21,325 genes. We also performed 23-multiplex cytokine assay using serum from 50 SLE patients.

Results: Our analysis (unpaired student t-test) resulted in ∼100 genes which are differentially expressed between bone marrow patients’ samples and controls. These genes represent several important biologic processes. 54 out of 100 differentially expressed genes are involved in 4 major networks including cell death, cancer, cell signalling and cellular growth and proliferation. Moreover, when the highly expressed genes were clustered, we identified 2 major SLE clusters in the bone marrow analysis but not in the peripheral blood. The first cluster represents patients with active disease and the second patients in remission. The upregulated genes in the bone marrow of active patients included genes involved in cell death and granulopoiesis. Comparative analysis of the bone marrow with the peripheral blood of SLE patients resulted in 88 genes; 61 out of 88 participate in various mechanisms that include cancer, cellular movement and morphology, immune response as well as functions of the hematopoietic system. In the multiplex cytokine assay, six cytokines were elevated in patients’ serum relative to controls: IL-1Ra (p = 0.0046), IP-10 (p = 0.03), IL-8 (0.0026), TNF-alpha (0.048), IL-15 (0.0097), and MCP-1 (0.0034). Furthermore some of the cytokines associate with clinical manifestations of lupus. We found a positive association of nephritis and IL-2, negative association of arthritis with IP10 and IL-12 and a negative association of CNS lupus and MIP1alpha. The BM serum cytokine analysis is in progress.

Conclusion: The microarray analysis of the bone marrow differentiated better the active from the inactive lupus patients and the patients from the controls. Further investigation of the bone marrow gene expression may reveal patient subgroups with different prognosis/response to therapy.

153 OVER-EXPRESSION OF INTERFERON ALPHA IN LUPUS FAMILIES: EVIDENCE FOR A COMPLEX HERITABLE TRAIT

T. B. Niewold1, J. Hua1, T. J. A. Lehman1, J. B. Harley2, M. K. Crow1.1Mary Kirkland Center for Lupus Research, Hospital for Special Surgery, New York; 2Oklahoma Medical Research Foundation, Oklahoma City, USA

Background: Interferon alpha (IFN-a) levels are elevated in many patients with systemic lupus erythematosus (SLE), however it is not known whether increased IFN-a is a cause or a result of SLE. Increased endogenous IFN-a expression could be heritable and represent some of the genetic risk of SLE. A haplotype of the IFN-a pathway gene IRF5 has been associated with risk of SLE.

Patients and methods: Serum samples from the Hospital for Special Surgery (HSS) Family Lupus Registry (n = 222, 105 SLE) and the Lupus Multiplex Registry and Repository (n = 341, 104 SLE) were assayed for IFN-a activity using a functional reporter cell assay and normalized to unrelated healthy donor samples (n = 86). The rs2004640 and rs2070197 SNPs in the IFN-á pathway gene IRF5 were genotyped in the HSS registry samples using ABI Taqman probes.

Results: 56% of SLE patients had high serum IFN-a activity, and the following healthy family members also had abnormally high IFN-a activity compared to healthy unrelated donors: 25% of SLE mothers (p = 0.0031), 20% of SLE siblings (p = 0.0145), and 17% of SLE fathers (p = 0.0086). High IFN-a expression is clustered in certain families. 61% of SLE patients with high IFN-a expression have a nuclear family member with high IFN-a expression, compared to 21% of SLE patients with low IFN-a expression (p = 0.00005), and concordant SLE sib pairs were concordant for IFN status 67% of the time (p = 0.023). Anti-RNA binding protein (anti-RBP) and anti-dsDNA auto-antibodies were both independently and additively associated with high IFN-a in SLE patients (p<0.0001 for each). Anti-RBP antibodies were present in only 2% of healthy family members, and no healthy family member had anti-dsDNA antibodies. Auto-antibodies did not account for the frequent over-expression of IFN-a in healthy family members. There was a trend toward higher IFN-a production in SLE patients with the IRF5 SLE risk haplotype (T:C at the rs2004640:rs2070197 SNPs), however the effect was modest. There were no racial differences in IFN-a expression, but the IRF5 risk haplotype was much more common in White and Hispanic populations than in Black or Asian populations (p = 0.0018).

Conclusions: Many SLE families demonstrate abnormally high IFN-a expression in both healthy and affected individuals compared to healthy unrelated donors, suggesting that high endogenous IFN-a expression is a heritable SLE risk factor. There is a trend towards increased IFN-a production in the presence of the IRF5 haplotype that is associated with SLE risk, however this haplotype is found predominantly in certain ethnic backgrounds. High IFN-a expression is frequently found in SLE patients regardless of ethnic background, and it is likely that many genetic factors underlie this complex trait.

154 GENETIC ASSOCIATION OF PROGRESSIVE SYSTEMIC SCLEROSIS (SSC) WITH PTPN22 POLYMORPHISMS

H. Kirsten1, P. Ahnert1, J. Dümmler2, N. Hunzelmann3, P. Vaith4, I. Melchers2.1University Leipzig, IKIT/BBZ; 2Clinical Research Unit for Rheumatology, Freiburg; 3Dept. of Dermatology, University Medical Center, Cologne; 4Dept of Rheumatology and Clin Immunol, University Medical Center, Freiburg, Germany

Recently, associations between type I diabetes, rheumatoid arthritis as well as several other autoimmune diseases (AIDs) and the PTPN22 single nucleotide polymorphism (SNP) 1858C→T were discovered. PTPN22 (encoding protein tyrosine phosphatase, non-receptor type 22) located on chromosome 1p13 has 21 exons spanning 58 kb. The variant 1858C→T in codon 620 results in the exchange of Arg to Trp (R620W). PTPN22 (also known as Lyp or Pep) is expressed primarily in lymphoid tissue, and most probably involved in the negative regulation of T cell activation via interaction with the protein tyrosine kinase Csk. It was suggested that the mutation 620W may interfere with the interaction between Lyp and Csk. However functional data comparing homozygous and heterozygous 620W carriers are not yet available. We collected DNA from 177 patients with SSc and 184 healthy blood donors (HD). Samples were analyzed for 13 SNPs covering PTPN22, including 1858C→T (rs2476601). SNPs were selected to represent the most common haplotypes. The analysis was performed by PCR, single base extension and MALDI-TOF mass spectrometry. Among HD, the allele frequency of 1858T was 9.0%, similar to published data. Genotype frequency of 1858T/T was 0.5%. Among SSc patients, the allele frequency of 1858T was 12.4%, genotype frequency of 1858T/T was 2.8%. The difference in allele frequencies did not reach statistical significance, however, the difference in genotype frequencies was significant (p = 0.01, genotype relative risk test: Lathrop. Tissue Antigens 1983;22:160–6). Data concerning subgroups of patients, additional polymorphisms and the other 4 major haplotypes of PTPN22, together accounting for about 98.5% of detectable variants, will also be discussed. So far, associations observed in AIDs with PTPN22 always only concerned a subpopulation of patients since only a subset of the patients carried the disease associated variant. For PTPN22, maximally 20% of AID patients were shown to carry the allele 1858T and even less carried the homozygous 1858T/T genotype. Therefore, only a small subpopulation of patients may be influenced by functional effects due to PTPN22. In SSc patients, this subpopulation may be quite small, but it does exist.

Supported by grants of the BMBF (“German Network for Systemic Scleroderma” to IM and NH, “University and Science” to PA), the Sächsische Aufbaubank (PA) and the EFRE (PA). H. Kirsten and P. Ahnert contributed equally.

155 ASSOCIATION OF ANTI-CCP ANTIBODIES AND YKL-40 LEVELS WITH RADIOGRAPHIC OUTCOME OF ETANERCEPT TREATED PATIENTS WITH RHEUMATOID ARTHRITIS

S. Drynda, A-KRossbach, J. Kekow.Clinic of Rheumatology, University of Magdeburg, Vogelsang, Germany

Radiographic progression is a main outcome measure in rheumatoid arthritis (RA). Anti-CCP antibodies which are highly specific to RA have been described as being a good predictive marker for distinguishing between erosive and non-erosive RA. YKL-40, the human cartilage glycoprotein 39, has been identified as a biomarker for articular cartilage degradation. We conducted this study to monitor radiographic changes in etanercept (ETN) treated RA patients in relation to the CCP antibody levels and serum concentrations of YKL-40 and to evaluate the diagnostic importance of these serological parameters for therapy outcome. 54 patients (11 male, 43 female, mean age 51.8±11.5 years) who met the ACR criteria for rheumatoid arthritis with a mean disease duration of 10.3±8.5 years and who had received between 1–4 different DMARDs were included in the study. Patients were treated with ETN twice weekly at 25 mg. At study entry and at the one year follow up, the modified total Sharp scores (TSS), RA-associated joint space narrowing and erosions were determined on hand and foot radiographs. To compare the radiographic progression of patients with different disease duration the mean yearly progression rates (TSS/year, erosion score/year, JSN score/year) were calculated. Serum levels of anti-CCP antibodies and YKL-40 at the start of ETN treatment were analysed by ELISA (Immunoscan RA Mark 2, Euro-Diagnostika, Netherlands and METRA YKL-40 EIA Kit, Quidel, USA, respectively) according to the manufacturer’s instructions. At the start of treatment a mean TSS of 53.9±6.2, a mean erosion score of 17.7±2.9, and a mean JSN score of 36.5±4.0 were determined. In the course of ETN treatment the mean values increased slightly without reaching statistical significance. Calculated for disease duration the mean yearly progression rate (TSS/year) in 54 patients at study entry was 6.30+0.84, the erosion score/year 2.18±0.41 and JSN score/year 4.17+0.57. In 40 out of 53 patients (75.5%) anti-CCP antibodies were detected, 13 patients were negative. The group of patients with anti-CCP antibody levels >1000 U/ml (n = 20) had a higher mean yearly progression rate at baseline (mean 8.80±1.35) compared with the CCP antibody negative group (n = 13, mean 3.25±1.24, p = 0.008) and the group of CCP antibody positive patients who had CCP antibody levels between 25–1000 U/ml (n = 20, mean 5.78±1.39, p = 0.130). The mean disease duration was similar in the three groups (10.15, 10.62 and 10.00 years, resp.) The mean YKL-40 levels were significantly elevated (168.73+18.97 ng/ml) in the patient group compared to normal range (25–100 ng/ml). YKL-40 levels were not associated with the average yearly progression rates. However, YKL-40 levels were correlated with the DAS28, CRP, ESR. Changes in the progression rate during the course of the ETN treatment were not associated with CCP antibody levels at the start of treatment, the progression rate decreased by an average of 53% in all patients. However, in patients with YKL-40 levels in the normal range (<100 ng/ml), the yearly progression improved by an average of 75% in contrast to patients with elevated YKL-40 levels (>200 ng/ml) who only improved by 27%. YKL-40 serum levels correlated significantly with the radiographic progression seen in the first year of ETN treatment. In conclusion, our results confirm that CCP antibodies are a predictor for the radiographic progression but not a predictive marker for radiographic changes in the course of ETN treatment. Serum YKL-40 levels, however, reflect the actual joint destruction activity in RA. Our results suggest that RA patients with high YKL-40 levels treated with ETN have a poor radiographic prognosis.

156 CD4+FOXP3+ REGULATORY T CELLS ARE PRESENT IN BONE MARROW OF RA AND OA PATIENTS

M. Chorazy-Massalska1, T. Burakowski1, A. Radzikowska1, P. Maldyk2, W. Maslinski.1Dept of Pathophysiology and Immunology, 2Clinic of Orthopaedy, Institute of Rheumatology, Warsaw, Poland

Aim: It is well documented that regulatory T (Treg) cells are critical regulators of the immune response that contribute to maintenance of tolerance. Recent data suggest that bone marrow (BM) is a major secondary lymphoid organ, where efficient antigen presentation and lymphocyte activation (especially during chronic inflammation) takes place. The aim of this study was to investigate whether regulatory CD4+Foxp3+ T cells are present in bone marrow of RA and OA patients, and to identify stimuli that can modulate Foxp3 expression in bone marrow cells cultured in vitro.

Materials and methods: Bone marrow mononuclear cells (BMMC) were isolated from bone marrow of 4 RA and 4 OA patients, undergoing hip replacement surgery, by density gradient centrifugation. Cells were stained with anti-CD4-PerCP, anti-CD-25-FITC and anti-Foxp3-APC either directly after the isolation, or cultured with indicated stimulus (LPS, IL-15, anti-IL-6 or their combinations) at 37°C for 72 hours, followed by staining. Stained cells were analyzed by multiparameter flow cytometry.

Results: CD4+Foxp3+ regulatory T cells are present in bone marrow of RA and OA patients. Their phenotype was confirmed as CD4+Foxp3+CD25+ cells. As shown by flow cytometric analysis, after 72 h in culture there was higher proportion of regulatory CD4+Foxp3+ T cells in RA than in OA BMMC among lymphocytes gated on FSC/SSC (0.94% vs. 0.61%). The percentage of regulatory CD4+Foxp3+ T cells present in OA, but not RA bone marrow, increased upon LPS stimulation (1.02% vs. 0.61%; p = 0.03). In contrast, regulatory CD4+Foxp3+ cells from both RA and OA BMMC can be propagated in vitro by exogenous IL-15: for RA patients from 0.94% to 2.75% (p = 0.01) and for OA patients from 0.61% to 1.97% (p = 0.0027). Interestingly, neutralization of IL-6, a cytokine considered as suppressing factor of regulatory T cells, did not affect the number of CD4+Foxp3+, neither in OA nor RA BMMC.

Conclusion: Our data support the concept that bone marrow is an active participant not only in the activation of the immune response, but also in suppression/tolerance that can be maintained in bone marrow environment (at least partially) by the development of regulatory T cells. Lower plasticity in Foxp3 induction in BMMC from RA (lack of response to LPS) in comparison to OA, suggest that threshold of Treg induction in RA patients may be already overstep.

157 EVALUATION OF OXIDATIVE STRESS MARKERS AFTER SIMVASTATIN SHORT-THERAPY IN SCLERODERMA

L. Bazzichi, F. De Feo, I. Mencaroni, M. Doveri, L. Ghiadoni, S. Favilla, S. Taddei, S. Bombardieri.Department of Internal Medicine, University of Pisa, Italy

Aim: The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used in first line management of cardiovascular diseases because of their efficacy in improving lipid profiles and direct vascular actions. Recent studies demonstrated that statins also possess pleiotrophic immunomodulatory and anti-inflammatory capabilities, such as downregulation of the expression of adhesion molecules, of monocyte chemotactic protein-1 (MAC-1) on leucocytes and endothelial cells, and the ingress of leucocytes into inflammation sites. Statins also scavenge free radicals and block the induction of nitric oxide synthase. They are thought to be beneficial against autoimmune disorders, especially those characterized by ingress of activated leucocytes into the skin such as SSc. The aim of the present study was the evaluation of endothelial response of a 2 months treatment with simvastatin in patients affected by SSc.

Materials and methods: 15 patients (all female; median age: 59) affected by SSc according to ACR criteria (1980) were enrolled at the division of Rheumatology of the University of Pisa. They were treated with simvastatin (10–20 mg/day PO according to their cholesterol LDL serum level) for 2 months. Fasting blood samples were drawn in the morning at baseline and after 2 months treatment. Lipid profile markers were determined: total cholesterol and HDL-cholesterol with an enzymatic colorimetric assay, triglycerides, apolipoprotein A1 and apolipoprotein B with a nephelometric assay, homocysteine with a polarized light immunofluorescent assay (FPIA). Oxidative stress markers were determined: the lipid peroxide (LPO) with a tiobarbituric acid reaction, the malondialdehyde (MDA) with a colorimetric assay for lipid peroxidation, the ferric reducing ability of plasma (FRAP). We also evaluated basal biochemical markers to assess therapy tolerability (hemocrome, plaques, CPK, transaminases, nitrogen, creatinine). Clinical parameters (total skin thickness score (modified Rodnan TSS), activity score, severity score, nail capillaroscopy) were measured in SSc patients.

Results: At baseline SSc patients showed altered serum levels of the following parameters: total cholesterol, LDL cholesterol, HDL cholesterol, LPO, MDA, FRAP. The following correlations were found at baseline: severity score vs MDA (**p = 0.0014), activity score vs LPO (**p = 0.0049) and vs MDA (*p = 0.017). After two months treatment we observed significantly reduced levels of total cholesterol (**p = 0.0012). After therapy we also observed slightly decreased levels of LPO (3.299±0.554 with respect to 5.55±1.532), MDA (4.471±0.914 with respect to 5.504±0.953), and increased levels FRAP (661.5±30.67 with respect to 610.3±25.89).

Conclusion: The statistically significant reduction of total cholesterol values has confirmed the efficacy of simvastatin therapy. The positive correlations between severity score and MDA and between activity score and LPO and MDA suggest that the oxidative stress is related to the development of SSc. So statins with their antioxidant properties should be useful in SSc.

158 SKIN ULCERS IN SYSTEMIC SCLEROSIS: POSSIBLE ROLE OF INFLAMMATORY AND ANGIOGENETIC FACTORS

S. Alivernini, B. Tolusso, M. De Santis, S. Bosello, G. Peluso, M. Pinnelli, G. Ferraccioli.Department of Rheumatology, Catholic University, Rome, Italy

Aim: Skin ulcers represent one of the disabling organ manifestation in systemic sclerosis patients (SSc). In this study we addressed the issue of some possible factors that can have a role in the development of skin ulcers in a large cohort of patients with SSc.

Patients and methods: 119 patients with SSc (mean age 56.9 SD 12.0 years, mean disease duration 8.5 SD 8.0 years, 14 males and 105 females), 29 with diffuse (dcSSc) and 90 with limited (lcSSc) skin involvement were enrolled in a cross-sectional study. 31 (26%) patients had ulcers at baseline, 6 (19%) of them had major ulcers. The same therapeutic approach was used for all the patients with skin ulcers. The plasma levels of sTNFRII, IL-6, HGF, IL-13, VEGF, VEGF-RI and VEGF-RII were determined by an ELISA procedure in all SSc patients at baseline.

Results: Patients with ulcers differed from those without for having diffuse skin involvement (p = 0.0012) and having higher systemic inflammation as shown by CRP (p = 0.03), fibrinogen (p = 0.02), sTNFRII (p = 0.001) and IL-6 (p = 0.013). Patients with ulcers did not differ from patients without ulcers for VEGF, VEGF-RI and VEGF-RII plasma levels. Similarly, no difference was seen between patients with diffuse and limited skin involvement. Analysing the subgroup of patients with ulcers, we found higher plasma levels of VEGF in patients with major lesions (156.8 SD 51.2 pg/ml) versus patients with minor ulcers (76.7 SD 68.7 pg/ml, p = 0.02) and without (101.1 SD 75.9 pg/ml, p = 0.01 vs major ulcers). There was no difference in plasma levels of VEGF-RI and VEGF-RII between patients with major and minor ulcers. Patients with major lesions differed from patients with minor ulcers for higher VEGF/VEGF-RI (52.6 SD 62.3 vs 3.5 SD 6.8, p = 0.03) and VEGF/VEGF-RII (0.03 SD 0.006 vs 0.013 SD 0.011, p = 0.005) ratios. Patients without skin ulcers had similar VEGF/VEGF-RI and VEGF/VEGF-RII (10.4 SD 20.0 and 0.017 SD 0.014, respectively) ratios compared to patients with minor ones. Concerning IL-6 and sTNF-RII, there was no difference between their plasma levels in patients with major and minor ulcers. Considering the inflammatory parameters, we found a positive correlation between VEGF-RI and IL-6 plasma levels (r = 0.32, p = 0.01) and a negative correlation between VEGF-RII and both IL-6 and TNF-RII plasma levels (r = −0.44, p = 0.0005 and r = −0.27, p = 0.04, respectively). There were no differences in the plasma levels of HGF and IL-13 between the two groups of patients with and without ulcers and in the two subgroups with limited and diffuse skin involvement.

Conclusions: The aetiology of skin ulcers in SSc is multifactorial. Inflammatory status and deficiency in angiogenetic factors may both contribute to skin ulcerative lesions appearance. Higher ulcerative process seems to be characterized by a defective angiogenetic stimulus rather than an inflammatory stress.

159 LYMPHANGIO-GENESIS IN RHEUMATOID SYNOVITIS: HISTOPATHOLOGICAL AND CLINICAL CORRELATES

V. Codullo1, S. Bugatti1, A. Manzo1,2, C. Alberto Sciré1, B. Vitolo1, O. Epis1, R. Caporali1, C. Montecucco1.1Chair and Division of Rheumatology, University of Pavia, IRCCS Fondazione San Matteo, Pavia, Italy; 2Rheumatology Unit, Guy’s, King’s and St Thomas’ School of Medicine, Guy’s Campus, London, UK

Background: Lymphatic vessels are involved in the regulation of inflammatory responses by transporting leukocytes from peripheral tissues to draining lymph-nodes. Lymphatic vessels can proliferate in response to inflammation, conditioning the degree of tissue flogosis and the magnitude of T-cell responses. Yet, whether lymphatic vessel amplification occurs within the inflamed synovium in rheumatoid arthritis (RA) and the functional consequences of this phenomenon are unknown.

Materials and methods: Synovial samples were obtained from 60 RA patients undergoing synovial biopsy, synoviectomy or joint replacement. Clinical and laboratory parameters were recorded. Control materials consisted of 2 normal and 14 osteoarthritic synovial samples. Lymphatic vessels were investigated by immunohistochemistry and immunofluorescence for the specific markers LYVE-1 and podoplanin, and the median lymphatic vessel density (MLVD) was quantified as number of LYVE-1+ podoplanin+ structures per medium-power field. Ki-67 served as proliferation marker. Expression of the lymphatic decoy receptor D6 was explored. Endothelial expression of lymphoid chemokines, characterization of infiltrating immune-cells and lymphoid neogenetic features were assessed by specific immunostainings. The inflammatory infiltrate was classified as follicular/diffuse and quantified with a semiquantitative score (0–3).

Results: In the majority of RA tissues, the MLVD was very low (MLVD 0.1±0.2), and lymphatic vessels were predominantly located in the fibrous synovium of the joint capsule. Control tissues exhibited this same lymphatic density and distribution. By contrast, significant lymphatic amplification was observed in the superficial and deep sublining in 25% of RA samples (MLVD 3.1±1.5), where numerous lymphatics were proliferating (Ki-67+). In samples exhibiting lymphangio-genesis, most of LYVE-1+podoplanin+ lymphatics were also D6+, whilst D6 immunoreactivity was weak/absent in tissues with low MLVD. Lymphatic vessels constitutively expressed CCL21 and CXCL12, and CCL21 appeared up-regulated in samples with lymphangio-genesis. In areas of dense cellular infiltration, some lymphatics contained mature dendritic cells, T and B lymphocytes. High MLVD tissues were characterized by higher inflammatory scores (2.5±0.8 vs 1.7±0.8; p = 0.002). Lymphoid aggregates in tissue areas with densely distributed lymphatics tended to be less organized and lacked typical lymphoid-neogenetic features. Patients exhibiting synovial lymphangio-genesis presented longer disease duration (185±88.5 vs 107.1±90.1 months; p = 0.02) and higher median values of C-reactive protein (3.6 vs 1.1 mg/dl; p = 0.019).

Conclusions: Our results demonstrate that: 1) the lymphatic network can proliferate in the inflammatory environment of RA synovitis, at least in specific disease subsets/stages or tissue areas; 2) through up-regulation of the scavenger receptor D6, synovial lymphatics may contribute to the control of local inflammation; 3) synovial lymphatics can mediate immune-cell recirculation through the expression of specific chemokines; 4) the development of a synovial lymphatic network may condition the topographical arrangement of immune-infiltrating cells. Lymphangio-genesis regulation thus appears an attractive target of future pharmacological research in RA.

160 REDUCTION OF HAEMOSTATIC AND INFLAMMATORY BIOMARKERS BY TUMOR NECROSIS FACTOR-ALPHA BLOCKADE IN PATIENTS WITH RHEUMATOID ARTHRITIS

F. Ingegnoli1, M. Cugno2, E. Favalli1, A. Soldi1, S. Griffini2, E. Bonanni2, F. Fantini1.1Department of Rheumatology, University of Milan, Istituto G. Pini, Milan, Italy; 2Department of Internal Medicine, University of Milan, IRCCS Ospedale Maggiore, Milan, Italy

Objective: It has long been recognized that rheumatoid arthritis (RA) is associated with increased cardiovascular risk, activation of coagulation and inflammation pathways. Treatment with Infliximab, a chimeric monoclonal antibody (mAb) to tumor necrosis factor-alpha (TNF-a) has been shown to reduce inflammation and improve clinical and laboratory measures of disease activity in RA. To date during this treatment the relationship between coagulation and inflammation has not been explored. The aim of this study was to investigate whether the haemostatic and inflammatory biomarkers were modified by administration of Infliximab in RA patients.

Methods: Eleven patients with active RA and 50 healthy controls (HC) were included. RA patients had been taking a stable dose of methotrexate of at least 10 mg/week and Infliximab (3 mg/kg) at week 0, 2, 6 and 14. At baseline and prior to the infusion at week 14 the following parameters were determined: disease activity score (DAS28), General Health Questionnaire (GHQ), visual analogue scale (VAS) pain, hemoglobin concentration, erythrocyte sedimentation rate (ESR), plasma levels of C-reactive protein (CRP), TNF-a, IL-6, prothrombin fragment 1+2 (F1+2) and D-dimer.

Results: At baseline, ESR, levels of CRP and IL-6 were significantly higher in RA patients than in HC (p = 0.0001), and levels of TNF-a tended to be increased; also F1+2 and D-dimer levels were significantly higher (p = 0.0001). After 14 weeks of Infliximab treatment, there was a significant clinical and laboratory improvement as judged by a decrease in the DAS28 (p = 0.0001), GHQ (p = 0.001), VAS pain (p = 0.002), number of swollen (p = 0.003) and tender joints (p = 0.001). Similarly, an increase of hemoglobin concentration (p = 0.02), and a decrease of ESR (p = 0.03), CRP (p = 0.02) and IL-6 (p = 0.05) were detected. F1+2 and D-dimer levels significantly decreased during the course of the study (p = 0.05).

Conclusions: We show that administration of anti-TNF-a mAb in RA patients results in a rapid clinical improvement and a decrease of both acute-phase proteins and haemostatic biomarkers. The reduction of prothrombotic biomarkers indicates that Infliximab treatment may reduce the thrombotic risk in RA patients.

161 INFLAMMATION-INDUCIBLE ANTI-TNF GENE EXPRESSION BY INTRA-ARTICULAR INJECTION OF SEROTYPE 5 ADENO-ASSOCIATED VIRUS REDUCES ARTHRITIS

M. Khoury1,2, J. Adriaansen3, M. J. B. M. Vervoordeldonk3,4, D. Gould5, Y. Chernajovsky5, P. Bigey6,7,8,9, C. Bloquel6,7,8,9, D. Scherman6,7,8,9, P. P. Tak3,4, C. Jorgensen1,2,10, F. Apparailly1.1Inserm U844, Hopital Saint Eloi, Montpellier, France; 2Université Montpellier, Montpellier, France; 3Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands; 4Arthrogen BV, Amsterdam, Netherlands; 5Bone and Joint Research Unit, Barts and The London Queen Mary’s School of Medicine and Dentistry, University of London, London, UK; 6Inserm U 640, Paris, France; 7CNRS, UMR8151, Paris, France; 8Université Paris Descartes, Faculté de Pharmacie, Laboratoire de Pharmacologie Chimique et Génétique, Paris, France; 9Ecole Nationale Supérieure de Chimie de Paris, Paris, France; 10CHU Lapeyronie, service clinique d’Immuno-Rhumatologie, Montpellier, France

Aim: Tumor necrosis factor (TNF)-alpha plays a central role in rheumatoid arthritis (RA) and current biotherapies targeting TNF-alpha have a major impact on RA treatment. The long-term safety concerns associated with the sustained systemic immunosuppression due to the TNF blockade prompted us to optimize therapeutic anti-TNF approaches. We recently demonstrated that intra-articular gene transfer using a recombinant adeno-associated virus serotype 5 (rAAV5) is far more efficient than the other serotypes for transgene expression in arthritic joints. In the present study we aimed at combining our optimized vector for local delivery to arthritic joints and regulated therapeutic gene expression to address the concern of safe and local gene therapy in RA.

Methods: A recombinant adeno-associated virus serotype 5 (rAAV5) encoding either the TNFR1-mIgG1 fusion protein (TR1) or a dimeric sTNFR2 (TR2) under a CMV or a NF-kappaÊB-responsive promoter was used to neutralize TNF-alpha in both human primary RA synovial fibroblasts and collagen-induced arthritic (CIA) mice. DBA/1 mice were immunized with collagen type II, boosted 21 days after and 1.5×109 particles of rAAV5 were injected in both knee joints 2 days after. Clinical course of the disease was assessed by paw thickness measuring over time, radiological and histological scores were obtained at euthanasia. The cytokine profiles were measured by ELISA in sera and knee-joint conditioned media. The immunological balance was also assessed using anti-type II collagen assays.

Results: The intra-articular gene transfer of both rAAV5-CMV-TR1 and rAAV5-CMV-TR2 vectors resulted in delayed disease onset (46.5 ± 0.7 and 48 ± 0 vs 38.2 ± 2.7), decreased incidence (44.5 and 12.5 vs 100%) and severity of joint damage compared with control group, the TR1-expressing vector being therapeutically more efficient (p<0.05). Importantly, when TR1 was expressed under the NF-kappaB-responsive promoter, the therapeutic effect was associated with a transient expression of the anti-TNF molecule, only detectable during disease flares, while the antagonist was rapidly, highly and stably expressed for nine weeks when delivered by a CMV-driven vector.

Conclusion: These findings strongly support the feasibility of improving the anti-TNF approach for the treatment of arthritis by local rAAV5-mediated gene expression under an inflammation-responsive promoter, able to provide a regulated, transient and therapeutically efficient dose of an immunomodulatory molecule.

162 ANTIBODIES AGAINST TYPE II COLLAGEN (CII) ARE ASSOCIATED WITH DESTRUCTION AT RA ONSET, WHEREAS RF AND ANT-CCP RELATE TO LATE ACCELERATION OF THE DESTRUCTIVE PROCESS

J. Rönnelid1,2, M. Mullazehi1, M. Wick3, L. Klareskog2, R. v. Vollenhoven2.1Unit of Clinical Immunology, Akademiska Hospital, Uppsala, Sweden; 2Unit of Rheumatology, Karolinska Hospital Solna, Stockholm, Sweden; 3Department of Radiology, Innsbruck Med Univ, Austria

Aims: We have recently shown that anti-CCP are associated with accelerated destruction during the first two years of RA, as well as with late appearance of extended inflammation. Anti-CII antibodies on the other hand associate with acute inflammation at disease onset (Mullazehi et al Ann Rheum Dis in press), and this is probably due to cytokine stimulation by solid phase immune complexes at the cartilage surface (Mullazehi et al, Arthritis Rheum 2006;54:1759). Both anti-CII and anti-CCP define phenotypically distinct populations of RA patients that are inversely related. We therefore hypothesized that these two autoantibodies also might characterize patient groups with divergent radiological outcome.

Materials and methods: 265 early RA patients (189 women and 76 men; mean age 56 years, range 18–85) were enrolled. Identical radiographs of the hands and feet from inclusion, 1 and 2 years were scored blindly according to Larsen. Rate of change was expressed as Larsen score between the investigated time points. RF was measured with nephelometry, and anti-CCP and anti-CII with ELISA.

Results: As we have earlier shown, anti-CCP positive patients had significantly higher Larsen score between baseline and 2 years compared to anti-CCP negative patients (p = 0.0080). The difference was even stronger during the second year (p<0.0001). Figures for RF were comparable but somewhat weaker (p = 0.0192 and 0.0010, respectively). Anti-CII positive patients showed increased baseline Larsen score (p = 0.0472) when only the very high anti-CII positive sera associated with immune complex-induced cytokine production in vitro were considered.

Conclusions: Serologically defined groups of early RA patients have divergent prognosis concerning future and present radiological destruction. Patients with very high anti-CII levels present both with acute inflammation and more radiological destruction at the time of diagnosis, probably due to local immune complex stimulation at the cartilage surface.

163 P38MAPK ISOFORM EXPRESSION AND ACTIVATION IN ANCA-ASSOCIATED GLOMERULONEPHRITIS

K. Polzer1, A. Soleiman2, K. Redlich3, A. Kilian1, G. Schett1, J. Zwerina1.1Department of Internal Medicine III, University of Erlangen, Germany; 2Department of Clinical Pathology and 3Department of Internal Medicine 3, Medical University of Vienna, Austria

Introduction: Anti-neutrophil cytoplasmatic antibody (ANCA)-associated vasculitis (AASV) is a life-threatening autoimmune disease and renal involvement presenting as crescentic glomerulonephritis is a major predictor for poor outcome in patients with AASV. Activation of pro-inflammatory cytokines such as TNF potentially contributes to renal damage by activation of pro-inflammatory signal transduction pathways and perpetuation of inflammation. Indeed, TNF-blockade by anti-TNF antibodies can be effective in inducing remission in patients with ANCA-associated glomerulonephritis. p38MAPKs and its downstream mediators play a major role in the regulation of inflammation and are activated by pro-inflammatory cytokines such as TNF. Additionally, p38MAPK inhibitors have been shown to inhibit renal inflammation in animal models of crescentic glomerulonephritis. However, it is unclear which of the four known p38MAPK isoforms (alpha, beta, gamma and delta) are expressed, activated and hence of major importance in ANCA-associated glomerulonephritis.

Methods: Histological sections of renal biopsy specimens of patients with ANCA-associated glomerulonephritis and healthy controls (living kidney donors) were investigated for the glomerular expression and phosphorylation of p38MAPK isoforms and MAPKAP2 protein by immunohistochemistry. Further, we analysed the mRNA and protein expression of p38MAPK isoforms in a human podocyte cell line and determined p38MAPK isoform activation in podocytes upon activation with TNF.

Results: Immunohistochemical stainings revealed expression of p38 alpha, beta and gamma isoforms in glomerular podocytes, whereas glomerular epithelial cells were predominantly positive for the p38 gamma isoform. Activation of both p38MAPK and its downstream mediator MAPKAP2 were seen in AASV but not control kidneys. Phosphorylation was localized to glomerular podocytes, inflammatory infiltrates and glomerular crescents but not glomerular epithelial cells. Interestingly, kidney biopsies of pre-treated vs. untreated AASV patients showed significantly less p38MAPK activation. Double immunohistochemistry showed glomerular co-localization of phosphorylated p38 protein with p38 alpha, beta and gamma isoforms. Next, we determined p38MAPK expression in a podocyte cell line and found expression of all 4 p38MAPK isoforms by RT-PCR and immunoblotting. However, upon activation with TNF, we found predominant activation of p38 alpha, weak activation of p38 gamma and delta and no activation of the beta isoform in vitro.

Discussion: This study shows, that p38 alpha, beta and gamma isoforms are predominantly expressed in vivo in glomeruli of patients with ANCA-associated glomerulonephritis and co-localize with phosphorylated p38MAPK protein. In vitro, activated podocytes predominantly show phosphorylation of p38 alpha.

164 COMPARATIVE ANALYSIS OF FOXP3 AND CD25 IN TARGET ORGAN AND CIRCULATION OF PATIENTS WITH CHRONIC ARTHRITIS

S. Raghavan, J. Herrath, P. Larsson, C. Trollmo, V. Malmström.Rheumatology Research Unit, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden

Background: Thymus derived regulatory T cells constitute a unique lineage of immune cells that are fundamental in control of autoimmunity in normal hosts. Co-expression of CD4 and CD25 (IL-2Ra) on the cell surface has allowed for isolation and functional characterization of regulatory T cells in vitro. In humans regulatory T cells are defined as CD4+ T cells with the highest surface density of CD25 (CD4+CD25bright). However, at the site of inflammation, e.g. in the rheumatic joint, CD25 is also up regulated on activated T cells making it difficult to clearly distinguish activated from regulatory T cells. The identification of the forkhead transcription factor scurfin (encoded by FOXP3 in humans) expressed specifically in regulatory T cells has made it possible to distinguish them from activated CD25+ T cells. Here, we study the longitudinal pattern of FOXP3+ expression in regulatory T cells in paired peripheral blood and synovial fluid of patients with inflammatory arthritis.

Methods: Mononuclear cells were isolated from paired peripheral blood and synovial fluid from 9 patients at several relapses. Intranuclear FOXP3 staining was carried out and analyzed by flow cytometry.

Results: Our data demonstrate that FOXP3 expression is most abundant in the CD4+CD25bright T cell population independent of site of sampling (blood or synovial fluid). Interestingly, the frequency and the amount of FOXP3 among the CD4+CD25- and CD4+CD25+ T cells was significantly elevated in the synovial fluid compared to paired peripheral blood (p<0.0001 and p<0.001 respectively). Our results indicate that at the site of inflammation either FOXP3 is induced on CD25- T cells or CD25 is down regulated on natural regulatory T cells, while still retaining FOXP3 expression. To support the latter hypothesis we could detect significantly increased amounts of sCD25 in the serum of patients with arthritis compared to healthy controls. A striking observation was that the frequency of CD4+CD25bright expressing FOXP3 in synovial fluid T cells varied greatly between individual patients (range 41–91%) compared to the counterparts in circulation (range 74–96%). Furthermore, low/high phenotype of FOXP3 expression among the synovial fluid mononuclear CD4+CD25bright T cells was consistent over time.

Conclusions: In summary, FOXP3+ T cells were detected at the site of inflammation of patients at each relapse. The question still remains why in spite of the presence of these potent suppressor T cells at the site of inflammation, symptomatic RA still persists in the patients. Further studies are in progress to correlate the level of FOXP3 to the disease activity in the patients at the time of sampling.

165 CD4+ CD28null T cells accumulate in blood but not in the joint of patients with rheumatoid arthritis

A. E. R. Fasth, A. Johansson, O. Snir, B. Nordmark, E. af Klint, R. Van Vollenhoven, K-JMalmberg, A-KUlfgren, C. Trollmo, V. Malmström.Rheumatology Unit, Department of Medicine, Karolinska Institutet; 2Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

Background: CD4+ CD28null T cells have in several studies been reported to be increased in the circulation of patients with rheumatoid arthritis (RA) and other autoimmune diseases, where they can comprise more than 50% of the CD4+ T cells. These CD4+ CD28null are differentiated effector T cells with a proinflammatory profile, including rapid secretion of TNF and IFN-gamma and a restricted TCR Vbeta usage. However, despite associations between increased CD4+ CD28null T cells and a more severe disease including extra-articular manifestations in patients with RA, the presence and clinical relevance of CD4+ CD28null T cells in the inflamed joint of patients with chronic joint inflammation is not clear.

Aim: To investigate if CD4+ CD28null T cells accumulate in inflamed synovial membrane and synovial fluid of patients with RA; to determine the association between the presence of CD4+ CD28null T cells in the joint and joint destruction.

Methods: Mononuclear cells were isolated from peripheral blood (PB) and synovial fluid (SF) of 115 patients with RA. The frequencies of CD4+ CD28null T cells were investigated by FACS and T cells were further analyzed for TCR Vb restrictions. The presence of CD4+ CD28null T cells in the inflamed synovial tissue was analysed by three-colour immunofluorescence microscopy by antibodies to CD3, CD4 and CD244, which in combination specifically detect CD4+ CD28null T cells. The local effects of the CD4+ CD28null T cell in the SF were evaluated by assessment of radiographic progression based on x-ray analyses.

Results: When present in peripheral blood, CD4+ CD28null T cells could often also be found in SF, however always in lower frequencies than in the circulation. Interestingly when subgrouping the CD4+ CD28null T cells into different TCR Vb families, we found in approximately half of the investigated patients that one or several of the dominant CD4+ CD28null “clones” were absent in the synovial fluid as if restricted to migrate there. Functionally, CD4+ CD28null T cells isolated from SF were similar to those from isolated from PB. Furthermore, immunofluorescent stainings of synovial membrane biopsies showed scattered CD4+ CD28null T cells within the T cell infiltrates, but these only represented a few percent of the total T cell population. When subgrouping patients with and without erosive disease, we found no differences in the frequencies of CD4+ CD28null T cells, neither in the circulation nor in the synovial fluid.

Conclusion: Clearly CD4+ CD28null T cells accumulate in the circulation of arthritis patients, but not in the affected joints. Our results suggest that their proinflammatory capacity are more likely to contribute to extra-articular manifestations (as has been demonstrated previously) rather then having an active role in progressing joint destruction.

166 RITUXIMAB USE IN LUPUS PNEUMONITIS

R. Sharma, A. Hersch, P. Efthimiou.UMDNJ-New Jersey Medical School, Newark, New Jersey, USA

Rationale: Rituximab is a chimeric monoclonal antibody that targets the CD20 B cell surface antigen. We report the first use of rituximab to treat a Systemic Lupus Erythematosus (SLE) and secondary Sjogren’s syndrome (SS) patient who had developed refractory lupus pneumonitis.

Methods: A 29-year-old male with SLE/SS presented with complaint of shortness of breath. His initial chest CT was significant for pneumonitis. The patient was treated with high dose intravenous steroids and was discharged from the hospital on prednisone and hydroxychloroquine. At follow up the patient continued to complain of moderate dyspnea on exertion and pulmonary function tests (PFTs) revealed a restrictive defect with impaired gas exchange (FEV1 49%, FVC 51%, DLCO 42%). He did not respond to the addition of mycophenolate mofetil, and refused treatment with intravenous cyclophosphamide. The patient agreed to rituximab treatment, and was infused with 1000 mg of rituximab at weeks 0 and 2.

Results: Patient returned to clinic one week after second infusion and reported increased activity with resolution of dyspnea. Clinical improvement was confirmed by PFT findings. FVC, FEV1, and DLCO all improved; 74%, 66%, and 59% respectively.

Conclusions: Rituximab may be a valid alternative in the management of autoimmune disorders, especially in cases where the side effect profiles of traditional immunomodulators are not acceptable or well tolerated. We report what we believe to be the first successful use of rituximab for the treatment of refractory pulmonary disease in a patient with SLE/SS.

167 PROTEOMIC ANALYSIS OF HUMAN WHOLE SALIVA IN SJöGREN SYNDROME

C. Baldini1, L. Giusti, L. Bazzichi1, F. Ciriegia, A. Tavoni1, A. Lucacchini, S. Bombardieri1.1Rheumatology Clinic, Department of Internal Medicine, University of Pisa, Pisa, Italy

Background: Sjögren’s syndrome is a chronic autoimmune disease which involves typically the lachrymal and salivary glands with changes in gland structure and function that might be reflected in the composition of salivary fluid.

Aim: To compare the protein patterns of saliva of patients affected by Sjögren’s syndrome with healthy donors, using a proteomic approach.

Materials and methods: Saliva samples were obtained from eleven patients with SS and eleven healthy donors with similar mean age and demographic characteristics. Saliva samples were centrifuged to remove undissolved materials. Aliquots of resulting supernatants were mixed with rehydration solution and subjected to isoelectrofocusing in Immobiline Dry-Strip, pH 3–10. Before the second dimension, dry strips were equilibrated in two steps in equilibration buffer and processed in second dimension using acrylamide gel (12%) applying a continuous buffer system. Gels were stained with silver and images were analysed with Image-Master2DPlatinum. Protein spots from each gel were detected, edited manually and matched automatically. The differentially expressed proteins in patients were identified with peptide mass fingerprinting.

Results: We obtained the resolution of 260 spots approximately. In comparison to healthy donors, the samples of patients with Sjogren’s syndrome showed a reduction of some of the typical salivary proteins. In particular, a remarkable reduction up to the total absence of carbonic anhydrase VI was observed in Sjögren’s syndrome samples with respect to control. Moreover a set of differentially expressed proteins were detected, related both to inflammation and to oxidative stress injury.

Conclusions: This study has shown many qualitative and quantitative differences in the protein patterns of saliva in Sjögren’s syndrome in comparison to normal controls which might be related both to the autoimmune inflammatory involvement of salivary glands and to the tissue damage. Nonetheless, further studies are necessary to confirm our preliminary results and to better understand their potential clinical and pathogenetic implications.

168 ANTIBODIES AGAINST CITRULLINATED VIMENTIN IN RHEUMATOID ARTHRITIS: HIGHER SENSITIVITY AND EXTENDED PROGNOSTIC VALUE CONCERNING FUTURE RADIOLOGICAL PROGRESSION AS COMPARED TO ANTI-CCP

L. Mathsson1, M. Mullazehi1, M. C. Wick2, O. Sjöberg1, R. van Vollenhoven3, L. Klareskog3, J. Rönnelid1.1Unit of Clinical Immunology, Uppsala University, Uppsala, Sweden; 2Dept of Radiology, Innsbruck Med Univ, Innsbruck, Austria; 3Unit of Rheumatology, Karolinska Inst, Stockholm, Sweden

Aims: Citrullinated vimentin can be found in inflamed RA synovium, and at least part of the humoral RA-specific anti-Sa response is directed against citrullinated vimentin. We have evaluated the sensitivity, specificity and prognostic value of determination of antibodies against citrullinated vimentin (anti-MCV) as compared to antibodies against cyclic citrullinated peptides (anti-CCP) in an early RA inception cohort.

Methods: 273 early RA patients were followed with clinical investigations, radiographs and measurement of anti-MCV and anti-CCP antibodies at baseline and after three months, 1, 2, 3 and 5 years. Autoantibodies were also analyzed in 100 healthy controls.

Results: 70.7% (193/273) of the patients were anti-MCV positive and 57.9% (158/273) were anti-CCP positive at the time of diagnosis, with equal specificities (95% and 96%, respectively). 14.7% (40/273) were anti-MCV positive only and 1.8% (5/273) were anti-CCP positive only. Anti-MCV positive and negative patients had similar disease activities at baseline, but presence of anti-MCV was predictive of subsequent high disease activity and continued radiological progression. Changes in anti-MCV showed stronger correlation to changes in clinical parameters than changes in anti-CCP. The anti-MCV-positive and anti-CCP-negative subgroup showed a higher rate of radiological destruction as compared to patients negative for both anti-MCV and anti-CCP.

Conclusions: Analysis of anti-MCV yields a greater sensitivity together with unchanged specificity as compared to anti-CCP in early RA. As compared to anti-CCP, anti-MCV also defines an extended group of early RA patients with poor radiological prognosis.

169 OPSONIZATION OF LATE APOPTOTIC CELLS BY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) AUTOANTIBODIES INHIBITS THEIR UPTAKE VIA AN FCγR-DEPENDENT MECHANISM

E. Reefman1, G. Horst1, M. T. Nijk1, P. C. Limburg2, C. G. M. Kallenberg1, M. Bijl1.1Department of Rheumatology and Clinical Immunology; 2Department of Pathology and Laboratory Medicine, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

Objective: Decreased clearance of apoptotic cells is suggested to be a major pathogenic factor in systemic lupus erythematosus (SLE). We investigated whether binding of SLE-autoantibodies to apoptotic cells influences phagocytosis of these cells by macrophages.

Methods: Apoptosis was induced in human T-cell (Jurkat) and keratinocyte (HaCaT) cell lines by UVB exposure. Binding of purified IgG from 26 SLE patients and 15 controls to apoptotic cells was assessed by flow cytometry and western blotting. Phagocytosis of IgG-opsonized apoptotic cells by monocyte-derived macrophages (MDM) was assessed by light microscopy. Similar experiments were performed with a monoclonal antibody against SSA/Ro and IgG fractions from patients with Sjögren’s syndrome (n = 5) and rheumatoid arthritis patients (n = 5).

Results: IgG fractions from all 26 SLE patients bound to late apoptotic (LA) but not to early apoptotic (EA) cells. IgG fractions isolated from SLE patients with different autoantibody profiles showed comparable levels of binding. Control IgG did not bind. Opsonization of apoptotic cells with IgG fractions from SLE patients resulted in a significant inhibition of phagocytosis as compared to control IgG fractions. A monoclonal antibody directed against SSA/Ro and IgG isolated from 5 ANA-positive patients with Sjögren’s syndrome were also able to elicit these effects, whereas IgG from 5 ANA-negative patients with rheumatoid arthritis did not. The inhibitory effect of patient IgG was abolished by blocking either Fcγ-receptors (FcγR) or the constant region of IgG, using a specific Fc-blocking peptide.

Conclusion: Autoantibodies from SLE patients are able to opsonize apoptotic cells and inhibit their uptake by macrophages via an FcγR-dependent mechanism.

170 PROTECTIVE EFFECT ON RHEUMATOID ARTHRITIS OF NON-INHERITED MATERNAL HLA-DR ANTIGENS

A. L. Feitsma1, J. Worthington2, A. H. M. van der Helm-van Mil1, D. Plant2, I. E. van der Horst-Bruinsma3, T. W. J. Huizinga1, R. E. M. Toes1, R. R. P. de Vries1.1Dept. of Rheumatology and Immunohematology, LUMC, The Netherlands; 2Centre for Integrated Genetic Medical Research, University of Manchester, UK; 3Dept of Rheumatology, VUMC, The Netherlands

Rheumatoid arthritis (RA) is a complex genetic disorder to which HLA confers the largest part of the genetic risk. HLA-DRB1 molecules with the amino acid sequence DERAA at position 70–74 of the DRbeta chain are less often present in patients with RA compared to healthy controls. It is previously proposed that not only inherited but also non-inherited HLA-antigens from the mother (NIMA) as opposed to those from the father (NIPA) can influence the susceptibility to RA. Confrontation of the fetal/newborn immune system with the NIMA is supposed to have a lifelong modulating impact on the immune response of the child and thus on the chance to develop RA. Up to now, no protective NIMAs were described in auto-immunity. Therefore, we were interested to study whether DERAA is associated with a protective NIMA effect. HLA-DRB1 typing was performed in 84 Dutch RA patients and their parents and in 91 English RA patients and their parents, brothers and/or sisters. NIMA and NIPA frequencies were determined. As a control the same analysis was performed for a leukemia patient cohort and their parents, brothers and/or sisters. Within the Dutch RA families, the odds ratio for a DERAA-negative RA patient of having a DERAA-positive mother compared to a DERAA-positive father was 0.36 (p = 0.11). The DERAA frequency of the mothers (16.1%), but not of the fathers (26.2%) was significantly lower (p = 0.02) compared to that of the general Dutch population (29.3%). This difference could not be ascribed to a difference in DERAA-frequency between men and women as this was neither observed in a Dutch healthy control cohort (n = 420, OR 1.10: 95% CI 0.70–1.74; p = 0.65) nor in the leukemia patient control cohort (n = 65 “DERAA-positive” families, OR 0.73: 95% CI 0.35–1.55; p = 0.48). Within the English RA families, the odds of having a DERAA-positive mother was significantly lower compared to having a DERAA-positive father (OR = 0.18: 95% CI 0.04–0.70; p = 0.01). Combined analysis (i.e. Dutch and English families together) also showed a significantly lower DERAA allele frequency in mothers of DERAA-negative RA patients as compared to fathers (OR 0.25: 95% CI 0.09–0.65; p = 0.003). These data indicate that in contrast to a DERAA-positive father, a DERAA-positive mother can transfer the protective effect for RA-development to her DERAA-negative child, presumably by induction of a protective lifelong immune response in the child after being exposed to DERAA-containing antigens of the mother. Further, these data show for the first time that a NIMA may protect the child from an auto-immune disease.

171 UPREGULATED EXPRESSION OF THE NOVEL SUMO SPECIFIC PROTEASE SENP7 IN RA SYNOVIAL FIBROBLASTS

M. A. Peters, T. Pap.Division of Molecular Medicine of Musculoskeletal Tissue, Muenster, Germany

Small ubiquitin-like modifiers (SUMOs) are key post-translational regulators of a large number of proteins that play important roles in diverse cellular processes. It has been shown previously that SUMO-1 is overexpressed in rheumatoid arthritis (RA) synovium and contributes to the resistance of RA synovial fibroblasts (RASF) against apoptosis. The recently described SENP7 protein is a member of the family of SUMO specific proteases (SENPs) that can remove SUMO moieties from its substrates and thereby balance SUMOylation processes. Despite our knowledge about SUMO-1 in RA, the expression of SENP7 has not been analyzed in synovium so far. Synovial tissue samples were obtained from 4 RA and 3 OA patients and used for histological analysis as well as for the isolation of synovial fibroblasts. Using specific antibodies, the expression of SENP7 was analyzed by immunohistochemistry in all synovial tissue specimens. The expression levels of SENP7 in RASF and OASF were compared by PCR and Western-blot analysis. The subcellular localization of SENP7 was studied by immunocytochemical co- staining with cell-compartment specific markers and confocal laser scanning microscopy. By immunohistochemistry, we found high expression of SENP7 in RA synovial tissue. SENP7 was expressed most prominently in the superficial lining layer with strong staining of synovial fibroblasts and around blood vessels. Cultured RASF showed a marked upregulation of SENP7 mRNA as compared to OASF. These data were confirmed by Western-blot and immunocytochemistry, where RASF showed significantly higher SENP7 protein expression levels than OASF. Both RASF and OASF showed nuclear and cytoplasmic staining of SENP7, although the staining was less prominent in OASF. Endogenous nuclear SENP7 protein localized to few prominent nuclear dots. In the cytoplasm SENP7 was localized in the centrosome, which was identified by using the centrosome marker gamma-tubulin and in Golgi-like structures as shown by co-staining with the marker TGN. Our data indicate a disease-specific upregulation of SENP7 in the RA synovium. The data further suggest that SENP7 is upregulated in RASF and is found mainly in the centrosome and in Golgi-like structures. Based on the established role of SUMOs in mediating the resistance of RASF against apoptosis, we hypothesize that SENP7 is a novel player contributing to specific features of activation in RASF and, thus, to the disease process of RA.

172 SYNOVIAL B-LYMPHOCYTE REDUCTION PREDICTS CLINICAL RESPONSE IN RHEUMATOID ARTHRITIS PATIENTS TREATED WITH RITUXIMAB

C. H. Burgoyne, S. Dass, E. M. Vital, A. English, L. R. Coulthard, R. J. Reece, A. C. Rawstron, F. Ponchel, P. Emery.Section of Rheumatology, Leeds Institute of Molecular Medicine, Leeds, UK

Aim: B-cell depletion therapy with rituximab (RTX) is increasingly used in rheumatoid arthritis (RA). However, depth and duration of depletion of peripheral blood B-cells does not always explain clinical response. The effect of RTX on B cells in other compartments is not yet clear. High sensitivity flow cytometry was used to follow peripheral B-cell counts in RA patients receiving RTX, in parallel with immunohistochemistry (IHC) analysis of lymphocyte infiltration of synovial tissue.

Methods: 15 RA patients were treated with 2 RTX infusions and their response monitored for 9 to 12 months. EULAR clinical responses were measured 3 monthly. Blood samples were obtained at infusion and 3 monthly thereafter. B-cell counts were measured using MRD analysis. Synovial biopsies were taken by knee arthroscopy pre- and 6 months post-treatment from 10 patients. IHC and semi-quantitative scoring were used to measure B-cells (using anti-CD19), T-cells (anti-CD3) and disease activity (anti-CD68, clone EBM11). Visual analogue score (VAS) for macroscopic inflammation was recorded at arthroscopy.

Results: Results of paired biopsies are available in 10/15 patients. At 6 months, synovial B-cells were reduced in 7 patients (mean score 0.85 at baseline (BL) reduced to 0.13 at 6 months), but were unchanged or increased in 3 patients (mean score 0.44 at BL and 0.76 at 6 months). B-cell reduction was associated with a reduction in T-cell infiltration (mean score 0.58 at BL reduced to 0.40) and in disease activity (mean score 2.23 at BL reduced to 1.32). Disease activity and T-cell infiltration scores directly correlated with VAS (R = 0.67 and R = 0.73 respectively). In addition B-cell infiltration also correlated with VAS (R = 0.63). Of the 7 patients with synovial B-cell reduction, 6 had initial profound peripheral blood B-cell depletion (over 98%). One patient exhibited only partial peripheral blood depletion (60%) after the 2nd RTX infusion and reconstitution by 3 months. Good or moderate EULAR clinical responses, sustained at 9 months, were achieved in all 7 patients. The 3 patients in whom synovial B-cell reduction was not demonstrated had either no clinical improvement or relapse following brief response by 6 months. However, 2 of these patients showed profound blood B-cell depletion lasting 2 to 6 months. At 6 months, in peripheral blood, 13/15 patients had detectable B cells (mean 0.029×109/L), and no correlation was observed with clinical responses that varied between good, moderate and non-response.

Conclusion: These results show that synovial B-cell status is not always reflected in peripheral blood and at 6 months after RTX, reduction in the former seems a more reliable indicator of better clinical outcome. We are currently extending this work to investigate further the mechanism of action of RTX, using additional synovial cell markers and studying the phenotypes of peripheral and synovial B-cells.

173 ACTIVATION OF THE INTERFERON SIGNALLING PATHWAY IN PATIENTS SUFFERING FROM SLE

T. Karonitsch, E. Feierl, G. Steiner, C. W. Steiner, J. S. Smolen, M. Aringer.Dept of Rheumatology, Medical University Vienna, Austria

Purpose: Both type I and type II interferons (IFN) are thought to play an important role in SLE. Serum IFNalpha is increased and associated with SLE disease activity, while murine models suggest a more causative role for IFNgamma. IFNalpha as well as IFNgamma signal via phosphorylating Stat1.

Methods: PBMC of 50 SLE patients and 30 healthy individuals (HC) were isolated over Ficoll-Hypaque gradients. Stat1 immunofluorescence staining was performed immediately after preparation and, in 8 healthy individuals, after 24 hours of incubation in medium with or without IFNalpha, IFNgamma, IL-10, or combinations of these cytokines. Staining for phosphorylated Stat1 (pStat1) was performed immediately or after 15 minutes of incubation in medium with or without IFNalpha or IFNgamma. The expression of both chains of IFNgamma-receptor was quantified by direct immunofluorescence staining, using standard protocols. Cells were analyzed by fluorocytometry, and separate gates were set for monocytes and lymphocytes. SLE disease activity was assessed by SLE Index Score (SIS).

Results: Stat1 mean fluorescence intensity (MFI) was increased in SLE as compared to HC (lymphocytes: 16.2±13.2 vs 5.3±1.9 (mean ± SD), p<0.0001; monocytes: 17.8±12.6 vs 7.4±3.3, p<0.0001), and significantly correlated with disease activity (lymphocytes: r = 0.65, p<0.0001). Within 24 h, IFNalpha increased Stat1 to levels comparable to those of SLE patients. In contrast, the effect of IFNgamma was limited to monocytes, while no significant change in lymphocytic Stat1 was seen. IL-10 reduced Stat1 in both lymphocytes and monocytes, and diminished IFNalpha-induced Stat1 increase. Activated pStat1 (mfi) was likewise increased in SLE lymphocytes (1.64±0.36 vs 1.37±0.2, p = 0.0001) and monocytes (4.53±1.79 vs 3.35±0.92, p = 0.0005). Incubation with IFNalpha for 15 minutes resulted in Stat1 phosphorylation in SLE and HC PBMC. In contrast, IFNgamma induced Stat1 phosphorylation in SLE lymphocytes only (SLE: from 1.56±0.25 to 1.72±0.37, p<0.002 vs HC: from 1.39±0.16 to 1.39±0.17, p = n.s.). Monocytes of both SLE patients and healthy individuals increased pStat1 upon stimulation with IFNgamma, but this effect was much more pronounced in SLE (SLE: from 4.1±1.2 to 6.9±3.3, p<0.0001; HC: from 3.5±0.9 to 4.4±1.5, p<0.005). Nevertheless, both IFNgamma receptor chains were similarly expressed on HC and SLE lymphocytes and monocytes.

Conclusion: Thus, Stat1, the major signalling molecule of both type of IFNs, is upregulated in SLE and significantly correlates with overall disease activity. PBMC of SLE patients are strongly IFNgamma-reactive, but healthy lymphocytes are unresponsive and healthy monocytes are hyporesponsive to IFNgamma. Since there was no difference in the expression of IFNgamma receptor between SLE and HC PBMC, differences in the negative regulation of this pathway seems to be possible. These findings are in line with the concept of an activated IFN system in SLE.

174 QUANTIFERON TB-GOLD TESTING AND RHEUMATOID ARTHRITIS

S. Sood, R. Sharma, P. Efthimiou.UMDNJ, New Jersey Medical School, Newark, New Jersey, USA

Several studies have indicated that tuberculin skin testing (TST) may not be an optimal test for the diagnosis of latent tuberculosis infection (LTBI) in patient with rheumatoid arthritis (RA). We would like to contribute to these findings, based on our clinical experience with a new screening tool called the QuantiFERON TB-Gold (QFT-G) test. While screening policies vary significantly between countries, it is generally recommended that all patients with RA should undergo screening for M tuberculosis latent infection prior to the initiation of treatment with biologic agents, especially the tumor necrosis factor (TNF) alpha inhibitors. Patients with RA may not be able to produce an adequate delayed type hypersensitivity (DTH) reaction to tuberculin because of their deficient cell mediated immunity. QFT-G is a whole blood, antigen-specific, test that utilizes synthetic peptides representing two M tuberculosis proteins, ESAT-6 and CFP-10. After incubation for 16 to 24 hours, the amount of interferon (IFN)-gamma secreted by monocytes in response to these antigens is measured. T-SPOT.TB, not yet available in the US, is another antigen-specific blood test utilizing the enzyme-linked immunospot (ELISPOT) assay technique. These new tests may be applicable to patients with RA, where false negative TST can have adverse consequences in patients treated with TNF inhibitors. We have identified two patients in our rheumatology clinic that were TST negative but were subsequently diagnosed with LTBI by the QFT-G test. Patient 1 is a 73-year-old black female who presented with symmetric bilateral synovitis of the metacarpophalangeal joints, was seropositive for rheumatoid factor (RF) and antinuclear (ANA) antibodies, with radiographic evidence of inflammatory erosive arthropathy. She was diagnosed with RA and had a negative TST. She was an incomplete responder to methotrexate and corticosteroids and the decision was made to initiate anti-TNF treatment. Repeat TST was again negative 2 months after the first testing. However, she tested positive for QTB-G and is currently completing a 9-month isoniazid/vitB6 course for LTBI. A confirmatory chest radiograph showed apical pleural thickening and upper lobe fibrosis, consistent with previous history of primary tuberculosis. Patient 2 is a 61 year-old black male with severe erosive RA, who screened negative by TST prior to immunosuppressive treatment. A pre-operative chest radiograph showed mild interstitial scarring, without any pleural pathology. He partially responded to methotrexate and became a candidate for anti-TNF treatment. Positive QTB-G testing prompted the initiation of a 9-month isoniazid/vitB6 course. In summary, QTB-G may be a more sensitive screening tool for LTBI in RA patients with impaired DTH response to tuberculin. Additional benefits include: it requires a single patient visit to draw the blood sample, results can be available within 24 hours, it is not subject to reader bias, and, most importantly, is not affected by prior BCG (bacille Calmette-Guerin) vaccination.8 However, the test is not widely available yet and careful studies comparing it to the TST are needed to validate our findings in the RA population.

175 INHIBITION OF EGFR BY GW282974 IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS

L. C. Huber, P. Künzler, S. H. Boyce1, B. A. Michel, R. E. Gay, B. S. Ink1, S. Gay.Center of Experimental Rheumatology, University Hospital Zurich and Zurich Center of Integrative Human Physiology (ZIHP), Zurich, Switzerland; 1GlaxoSmithKline, Medicines Research Centre, Stevenage, UK

Objective: To investigate the effects of GW282974, a novel compound against epidermal growth factor receptor (EGFR) ErbB1/2, with respect to inflammation and joint destruction in RA.

Materials and methods: Rheumatoid arthritis synovial fibroblasts (RASFs) were stimulated with EGF (1 ng/ml), IL-1beta (1 ng/ml) and TNF-alpha (10 ng/ml). In addition, GW282974 was added in different concentrations (5–10 uM) to stimulated cells for 24 h. Gene expression was checked by TaqMan PCR, using 18S as housekeeping gene. Protein analysis was quantified by ELISA. Cell growth and proliferation was measured using the ‘ViaLight’ proliferation assay. Cytotoxicity was analysed by FACS using Annexin-V/PI double staining. Expression and phosphorylation of ErbB1/2 receptors were checked by immunostaining with specific antibodies.

Results: EGF had no effect on the gene expression profile of RASFs when used as single stimulatory agent. In combination with pro-inflammatory mediators such as TNF-alpha and IL-1beta, however, EGF showed a synergistic effect. Thus, the expression of matrix metalloproteinases (MMP)-1, -3, -13, as well as cyclooxygenase(COX)-2 on mRNA level was increased by up to 40-, 2000-, 50-, and 1000-fold, respectively. Addition of the inhibitor GW282974 strongly abrogated these effects. In this regard, gene expression of MMP-1, -3, -13, and COX-2 were reduced in a dose-dependent manner. Strongest inhibitory effects were observed when GW282974 was used at a concentration of 10 uM. These data could be confirmed on protein levels analysing the supernatants of RASFs by ELISA. Similarly, cell growth and proliferation of RASFs were inhibited by GW282974 in a dose- and time-dependent manner. On the other hand, no cytotoxic effects were seen within the doses used.

Discussion: GW282974 appears to interfere with both the inflammatory and the destructive pathway in RA and might be used as novel therapeutic approach for the treatment of RA.

176 ACPA AND SHARED EPITOPE: THE BEHAVIOUR OF ANTI-VIRAL CITRULLINATED PEPTIDE ANTIBODIES

C. Anzilotti1, F. Pratesi1, C. Tommasi1, D. Chimenti1, E. Petit-Teixeira, F. Cornelis, P. Migliorini1.1Clinical Immunology Unit, Department of Internal Medicine, University of Pisa, Pisa, Italy

Rheumatoid arthritis (RA) sera contain antibodies specific for peptides in which arginine is substituted by the deiminated form citrulline (anti-citrullinated peptide antibodies, ACPA). A peptide corresponding to the EBNA I 35–58 sequence containing citrulline in place of arginine (Viral Citrullinated Peptide, VCP) was recently proposed as a new probe to detect ACPA. Anti-VCP antibodies for their specificity and clinical associations can be considered bona fide ACPA. Anti-VCP antibodies are detected in 50% RA sera and their level is correlated with anti-CCP antibodies; moreover, purified anti-VCP antibodies bind CCP and deiminated fibrinogen. Anti-CCP and anti-deiminated fibrinogen antibodies are strongly associated with the shared epitope. We analysed the relationship between anti-VCP antibodies and DRB1 alleles in RA patients. 172 RA patients were typed for DRB1 alleles. Among the shared epitope positive (140 pts), 64 were anti-VCP+ and 76 anti-VCP-; of the 32 share epitope negative patients, 12 were anti-VCP + and 20 anti-VCP-. The lack of association with the shared epitope was further confirmed by experiments in which EBNA I-derived deiminated peptides do not associate with shared epitope-positive alleles. These results demonstrate the great heterogeneity of ACPA family and suggest that anti-VCP antibodies represent the first ACPA not associated with shared epitope.

177 SYNOVIAL FIBROBLASTS ARE IMPORTANT MEDIATORS OF SYNOVIAL ANGIOGENESIS IN THE HYPOXIC RHEUMATOID JOINT

H. Larsen, M. Feldmann, E. Paleolog.Kennedy Institute of Rheumatology, Imperial College Faculty of Medicine, London, UK

Introduction: Increased synovial angiogenesis is a hallmark of rheumatoid arthritis (RA), a process thought to be promoted by both local regions of hypoxia and inflammatory cytokines present in the RA synovium. RA fibroblast-like synoviocytes (FLS) are the main cell type at the proliferating and invading edge of the pannus, and are therefore likely to be exposed to the greatest degree of hypoxia in the synovium. RA FLS are capable of secreting a plethora of angiogenic factors including vascular endothelial growth factor (VEGF) in response to hypoxia and inflammatory cytokines and are thus potential inducers of synovial angiogenesis. HIF-1 alpha has been reported to be regulated by both hypoxia and cytokines in other cell systems. To investigate the probable sources of VEGF in the synovium, we compared the effect of hypoxia (1% oxygen) with that of several cytokines (TNF alpha, TGF beta and IL-1 beta) that are associated with arthritis and angiogenesis, on HIF-1 alpha mRNA and protein levels in human RA FLS.

Materials and methods: FLS isolated from human RA joint tissue were exposed to hypoxia and/or 1–10 ng/ml of TNF alpha, TGF beta and IL-1 beta for 6–24 hours and Real-Time PCR, ELISA or a DNA binding assay was performed.

Results: RA FLS showed a marked increase in HIF-1 alpha mRNA levels when stimulated with any of the three cytokines, but interestingly hypoxia had the opposite effect. Hypoxia could also counter-regulate the increase in HIF-1 alpha mRNA levels induced by cytokines. TNF alpha was the most potent cytokine investigated as it induced HIF-1 alpha protein stabilization, HIF-1 alpha DNA binding and VEGF secretion. IL-1 beta and TGF beta induced HIF-1 alpha protein stabilization to a lesser extent and had no ability to induce DNA binding. Interestingly, these two cytokines appeared to have an additive or synergistic effect with hypoxia with regard to HIF-1 alpha protein accumulation, DNA binding and VEGF secretion.

Conclusion: The degree of synovial angiogenesis correlates with the severity of rheumatoid disease. The microenvironment in the RA synovium is characterized by both local regions of hypoxia and the presence of inflammatory cytokines. We have shown that RA FLS stimulated with hypoxia and/or inflammatory cytokines exhibit increased HIF-1 alpha DNA binding activity and subsequent VEGF secretion. The capacity of RA FLS to secrete large amounts of VEGF in response to hypoxia and inflammatory cytokines makes this cell type a potentially important contributor to RA synovial angiogenesis.

178 LOSS OF IMPRINT OF IGF2 CHARACTERIZES LOW INFLAMMATORY TYPE OF FLS IN RHEUMATOID ARTHRITIS

J. G. I. van Rietschoten, A. T. Trunjillo, T. C. G. Timmer, T. C. T. M. van der Pouw Kraan, C. L. Verweij.Molecular Cell Biology & Immunology, VUMC, Amsterdam, The Netherlands

Recently, we have shown by microarray analysis that heterogeneity of gene expression patterns and cellular distribution between rheumatoid arthritis (RA) synovial tissues is reflected in fibroblast-like synoviocytes (FLS) as a stable trait. This analysis identified 2 main groups of FLS with distinctive gene expression profiles. FLS from low inflammatory tissue showed increased expression of IGF2 as compared to FLS from high inflammatory tissue. IGF2 and H19 are a pair of reciprocally imprinted genes. IGF2 is expressed from the paternal allele. The purpose of this study was to analyze whether loss of imprint (LOI), i.e. transcription of the paternal allele and aberrant transcription of the maternal allele, contributes to the increased expression of IGF2 in FLS. In addition we will investigate whether this is accompanied with epigenetic deregulation for the IGF2/H19 imprinted domain, a phenomenon that is often observed in cancer. First, we confirmed the IGF2 expression levels by quantitative RT-PCR. Next, to determine whether increased IGF2 expression is associated with IGF2 LOI, we identified a panel of FLS that were heterozygous for a SNP in exon 9, to discriminate between the allelic transcripts. LOI was observed in 7 out of 9 informative FLS, most of which showed high expression of IGF2. Secondly, we investigated whether the observed LOI is associated with epigenetic changes of the imprinting control regions (ICRs), which are normally differentially methylated. We analyzed the methylation of the ICR, which binds CTCF upstream of H19. We used a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by single nucleotide primer extension (MS-SnuPE). Unexpectedly, this analysis showed the normal imprinted methylation status for this region. Currently, we are investigating a second ICR upstream of IGF2. Taken together, these data show that a subtype of RA-FLS is characterized by high IGF2 expression, which occurs in the presence of LOI, driven by a so far unknown mechanism.

179 VITAMIN D RECEPTOR DEFICIENCY AGGRAVATES TNF-MEDIATED ARTHRITIS

K. Polzer1, J. Zwerina1, K. Redlich2, J. Smolen2, G. Schett1.1Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen, Germany; 2Department of Internal Medicine, Medical University of Vienna, Austria

Introduction: Vitamin D is an important hormone for calcium homeostasis and bone metabolism and may have immunomodulatory effects.

Aim: To investigate the role of the Vitamin D Receptor (VDR) in inflammatory arthritis.

Methods: To determine the role of Vitamin D in TNF-mediated joint disease, we crossed mice deficient for the VDR with arthritic human TNF transgenic (hTNFtg) mice. Hind paws of hTNFtg and VDR-/-hTNFtg mice were analysed for clinical signs of arthritis as well as histological degree of synovitis, cartilage damage and bone erosion. Systemic mineral bone density was evaluated by bone histomorphometry. Furthermore, serum levels of cytokines and other inflammatory mediators were determined by ELISA.

Results: hTNFtg mice with VDR deficiency showed more pronounced weight loss, increased paw swelling and decreased grip strength than hTNFtg mice. In addition, VDR-/-hTNFtg mice exhibited lower bone density compared to hTNFtg mice. In line, serum levels of mTNF were significantly increased in VDR-/-hTNFtg mice compared to hTNFtg mice. Consequently, histological analysis revealed an increase in synovial inflammation in VDR-/-hTNFtg mice as compared to hTNFtg mice. VDR-/-hTNFtg mice showed a high increase in osteoclast number compared to hTNFtg mice, accompanied by strengthened bone erosions in the joints. These data were verified in osteoclast assays in vitro. In addition, cartilage damage as measured by proteoglycan loss was also enhanced in VDR-/-hTNFtg mice compared to hTNFtg mice.

Conclusion: These findings suggest that deficiency of the VDR leads to an increase in inflammation and bone erosion in an animal model of inflammatory arthritis. This leads to the assumption that treatment with an active metabolite of vitamin D may have potentially positive immunomodulatory effects in arthritis.

180 PATHOMECHANISMS OF TNF-DEPENDENT INFLAMMATORY CARTILAGE DESTRUCTION

A. Korb1, B. Tuerk1, F. Echtermeyer2, T. Pap2, J. S. Smolen1, K. Redlich1.1Dept Rheumatology, Medical University of Vienna, Austria; 2Div Mol Med Musculoskeletal System, University of Muenster, Germany

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that primarily affects the joints and results in the destruction of cartilage and subchondral bone by the inflamed synovium. To study the time course and mechanisms of inflammatory cartilage destruction, we used the human TNF-alpha transgenic (hTNFtg) mouse model of RA. Mice were sacrificed from week 2 to 14 in two-week intervals, and sections of the hind paws were analyzed for histopathological changes. Proteoglycan loss was assessed by toluidin-blue staining. Inflammation was quantified by measuring the area of hyperplastic synovial tissue. To study the effects of proteoglycan loss on the attachment of synoviocytes in vitro, synovial fibroblasts were seeded onto isolated and IL-1 treated (2 ng/ml and 10 ng/ml, 24 hours) murine femoral heads, and the attachment was quantified by light microscopy. Synovial inflammation increased over time (0.025 mm2 at week 2 vs. 0.65 mm2 at week 14; p<0.05), most prominently between weeks 4 and 6 (0.029 mm2 and 0.78 mm2, respectively, p<0.05), however. Loss of cartilage was seen only between weeks 2 and 4. After week 4, cartilage area and thickness remained stable indicating a dissociation between cartilage degradation and pannus formation. As seen in toluidin-blue staining, loss of proteoglycans occurred over time, starting at week 4 with a peak at week 8. There was a prominent attachment of pannus tissue to articular cartilage. Attachment started after week 4, peaked at week 10 and showed a decrease thereafter. This observation was supported by in vitro attachment data showing that early moderate loss of proteoglycans (induced by 2 ng/ml IL-1) strongly enhanced attachment of synovial fibroblasts while an almost complete loss of proteoglycans (induced by 10 ng/ml IL-1) did not facilitate fibroblast attachment. Our data indicate that in hTNFtg mice, inflammatory pannus formation does not result in a significant reduction of cartilage area and thickness. However, loss of proteoglycans in early phases of disease is closely linked to the attachment of the inflammatory tissue to the cartilage matrix. We propose that the loss of proteoglycans regulates the attachment of synovial cells to the cartilage surface.

181 VON WILLEBRAND FACTOR AS A MARKER FOR ENDOTHELIAL DAMAGE IN PATIENTS WITH RHEUMATOID ARTHRITIS AND LOW INCIDENCE OF TRADITIONAL RISK FACTORS FOR ATHEROSCLEROSIS

G. G. Ristic1, V. Subota2, T. Lepic3, B. Glisic1, M. Petronijevic1, M. Cirkovic1, D. Z. Stefanovic1.1Department of Rheumatology and Clinical Immunology; 2Institute for Medical Biochemistry; 3Department of Neurology, Military Medical Academy, Belgrade, Serbia

Background: High plasma level of von Willebrand factor (vWF) is a marker of the extent of endothelial damage predicting the subsequent occurrence of cardiovascular disease. Furthermore, ultrasound assessment of carotid intima-media thickness (IMT) has been confirmed as a reliable marker of generalized atherosclerosis during its subclinical stage.

Objectives: To evaluate the incidence of subclinical atherosclerosis in otherwise low-risk patients with rheumatoid arthritis (RA) measuring the IMT of the carotid arteries and to determine its association with vWF serum levels and other laboratory and clinical parameters.

Methods: IMT was measured by ultrasonography in the carotid arteries of 42 non-diabetic, normotensive RA female patients with otherwise low cardiovascular risk (mean age 45.3±10.0 years) and 32 healthy female control subjects (mean age 45.2±9.7 years) matched regarding age, body mass index (BMI), lipid status, and smoking habits. The IMT was evaluated on both left and right carotid arteries in the common carotid (CC), bifurcation (BI), and internal carotid (IC) arteries. Three measurements in each site were performed, and mean and maximal (max) IMT were calculated. The presence of atherosclerotic plaque was defined as IMT>1.5 mm. Clinical work-up included determination of the disease activity score (DAS 28), physical disability score (mHAQ), patient’s global assessment, as well as the estimation of the functional status. Two-dimensional and color Doppler echocardiography was performed in all patients. Laboratory assessment included measurements of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor, lipid status, and vWF. Clinical and laboratory findings were compared in RA patients with and without subclinical atherosclerosis.

Results: RA patients had significantly higher maximal and mean IMT (mm) than healthy controls at all measuring points (Max-CC: 0.764±0.148 vs. 0.703±0.100; P<0.05, Mean-CC: 0.671±0.119 vs. 0.621±0.085; P<0.05, Max-BI: 1.055±0.184 vs. 0.941±0.161, P<0.01, Mean-BI: 0.889±0.168 vs. 0.804±0.124; P<0.05; Max IC 0.683±0.108 vs. 0.613±0.093; P<0.01, Mean IC 0.577±0.101 vs. 0.535±0.076; P<0.05). Fifteen out of 42 RA patients (35.7%) and 1/32 controls (3.1%) had subclinical atherosclerosis defined as mean IMT above mean+2 SD of the control group at any measurement point (>0.791 at CC, >1.042 at BI, and >0.684 at IC). Atherosclerotic plaques (IMT>1.5 mm) were revealed in carotid bifurcation in 12/42 RA patients (28.6%) in contrast to 3/32 controls (9.4%), P<0.05.

Conclusion: Despite investigated RA population had a low incidence of traditional risk factors for atherosclerosis, both maximal and mean IMT were significantly higher than in matched healthy controls. von Willebrand factor serum levels were significantly increased in RA patients with subclinical atherosclerosis in contrast to patients with normal IMT revealing their potential role as an early marker of endothelial dysfunction and atherosclerosis in RA.

182 INTRAARTICULAR ADMINISTRATION OF ANTI-TNF ALPHA DRUGS UNDER ECHOGRAPHIC CONTRAST TECHNIQUE

L. S. Martin1, V. Anelli2, A. Ragno1, M. Russini1, A. Pierangeli1, A. Pagano1, A. Latini1, A. Migliore3, S. Tormenta3, C. M. Pacella2.1Department of Internal Medicine, “Regina Apostolorum” Hospital; 2Department of Diagnostic Imaging and Interventional Radiology, Regina Apostolorum Hospital, Albano Laziale (Rome); 3Unit of Rheumatology

Introduction: AntiTNF-alpha biological drug therapy has shown a great efficacy in the treatment of inflammatory arthropathies (rheumatoid arthritis, psoriasic arthritis and ankylosing spondylitis), if administered systemically and in accord with the specific therapeutic regimens for each drug. In the last few months sporadic cases of local therapy with antiTNF-alpha on single joints have been published; in some cases the efficacy of therapy has been evaluated through Infliximab marked scintigraphy. Here the case of a female patient treated with intraarticular Infliximab is presented; drug efficacy has been evaluated through articular echography with echographic contrast agent.

Patient and method: The patient, C.T., aged 62, has been suffering from psoriasic arthritis for several years now. She was visited two years ago for a worsening of articular pain, while taking Cyclosporin 250 mg and Diclofenac 150 mg. She presented with swollen and sore joints, severe morning stiffness and a serious reduction in self-sufficiency, with DAS28 = 5.0. She has been on therapy for 12 months with Infliximab 5 mg/kg according to the planned therapeutic regime, then changed to methotrexate 15 mg/w and Celecoxib 200 mg twice a day. Patient response to therapy has been excellent, achieving a reduction in the number of swollen and sore joints and reaching a DAS28 = 2.7. Six months ago a swollen and tender joint was observed in the patient’s right knee but no other joint was involved. Serological tests have shown an ESR = 30 (nv 18) and a CRP = 1.7 (nv 0.5), with DAS28 = 2.2. Considering the excellent tolerability of Infliximab, as already shown, it was decided to inject the drug in the inflamed right knee during echography.

Results: Before therapy the echography showed a serious synovial thickening; with Power-Doppler technique and echographic contrast agent a high intraarticular vascularization was observed, indicating an inflammatory process. Intraarticular Infliximab 100 mg has been injected during echography. During the administration there were no problems nor complaints on part of the patient. A progressive reduction of pain and swelling has been observed in the patient’s right knee. After 10 days a control echography with Power-Doppler technique and echographic contrast agent has shown a persistent synovial thickening but no signs of inflammation. At the moment no joint swelling is present in the patient and she feels a mild pain in her right knee, which is easily controlled with Celebrex 200 mg/day.

Discussion: Our case report suggests the possible intraarticular administration of antiTNF-alfa biological drugs in patients with early monoarthritis or in patients with a good response to systemic therapy but poor or no response in the individual joints. In these patients the intraarticular treatment could represent the best cost/benefit balance, considering the costs of biological drug therapy.

183 RELATIONSHIP BETWEEN T CELL AREA LYMPHOID CHEMOKINE (CCL21, CCL19) INDUCTION AND CD4+ NAïVE T CELL LOCALIZATION IN SYNOVIAL ECTOPIC LYMPHOID TISSUE

C. Alberto Scirè1, A. Manzo2, B. Vitolo1, V. Codullo1, S. Bugatti1, C. Pitzalis2, C. Montecucco1.1Chair and Division of Rheumatology, University of Pavia, IRCCS Fondazione San Matteo, Pavia, Italy; 2Rheumatology Unit, Guy’s, King’s and St Thomas’ School of Medicine, Guy’s Campus, London, UK

Aim: Naïve T-cell recirculation through secondary lymphoid organs (SLO) is a critical step for the genesis of adaptive immune responses, and is physiologically driven by lymphoid chemokines (CK) constitutively expressed in the T-cell area, such as CCL21 and CCL19. In chronic inflammatory states associated with persistent antigenic stimulation, such as rheumatoid arthritis (RA), the inflammatory infiltrate can acquire some morphological and functional features of SLO, including the expression of lymphoid CK. An unresolved issue remains whether the ectopic expression of lymphoid CK can mediate the misdirection of naïve T cells from their homeostatic route to SLO to inflammatory ectopic lymphoid tissues (ELT) in humans. In this study we analyzed, by using specific combinations of cell surface markers, the in situ relationships between T-cell area lymphoid CK ectopic expression and naïve T-cell localization in rheumatoid synovial ELT.

Methods: Twelve synovial tissue (ST) samples from RA patients were selected for the study. Paired ST, synovial fluid (SF) samples were also collected in 5 cases. Expression of CCL21 and CCL19 was examined by immunohistochemistry and quantified by digital image analysis. Naïve T CD4 cells (CD3/CD4/CD45RA) were detected by triple immunofluorescence and confocal microscopy in the ST and by FACS in the SF.

Results: Comparative analysis in the inflamed sublining demonstrated a significant association between CCL21 and CCL19 expression with the formation of synovial ELT (Mann-Whitney-test p<0.001). Both these CK showed also a strong association with the dimensional enlargement of the lymphoid aggregates, and a significant reciprocal association within the same aggregates both in terms of global prevalence (Fisher exact test p<0.001) and quantitative expression (Spearman Rho = 0.78, p<0.001). Microanatomical studies revealed the possible cognate perivascular expression of CCL21/CCL19 around the CXCL12/PNAd positive HEVs, as physiologically required in SLO for naïve T-cell recruitment. Notably, phenotypic tissue analysis did not reveal the expected co-localization of CD3/CD4/CD45RA cells inside synovial ELT both in small perivascular aggregates and in large organized lymphoid clusters. FACS analysis of SF samples from RA patients confirmed the rare presence of CD3/CD4/CD45RA cells. Similar findings were obtained when paired SF and ST were collected and analysed in parallel for CK expression and T cell subset distribution. As expected, CD3/CD4/CD45RA cells were found both in peripheral blood and lymph nodes used as positive control.

Conclusions: Our data support the concept that a T-cell area lymphoid CK system can associate with lymphoid neogenesis in chronic inflamed synovium, leading to highly organized structures with qualitative features of SLO. Nevertheless, this expression within the synovial lymphoid tissue does not appear sufficient to allow constitutive naïve T cell misdirection in ELT, at least in specific disease stages. Altogether, our findings open new perspectives for future studies aimed to address the molecular rationale and physio-pathological consequences of naïve-cell exclusion from RA ELTs.

184 MESENCHYMAL CELLS ARE SUFFICIENT TARGETS FOR TNF IN MODELLED JOINT AND GUT PATHOLOGIES

M. Armaka, D. Kontoyiannis, G. Kollias.Institute of Immunology, BSRC “Al. Fleming”, Vari/Athens, Greece

TNF plays an essential role in the pathogenesis of rheumatoid arthritis and Crohn’s inflammatory bowel disease. Anti-TNF therapies have proved successful in their clinical treatment. The cellular mechanisms of TNF/TNF-R function in these diseases have remained poorly characterized. In general it is thought that TNF delivers mostly innate activation and pro-inflammatory signals through its action on myeloid/monocytic cells or other haemopoietic cell types. Using reciprocal bone marrow grafting experiments in previously established TNF transgenic animal models of arthritis and Crohn’s-like IBD (Tg197 and TNFΔARE mice), we show that development of arthritis requires the expression of TNFRI in cells of the radiation-resistant compartment, which are also sufficient targets for TNF in the development of the Crohn’s-like IBD as previously demonstrated. Moreover, we have generated a Cre-expressing mouse line (Cre expression driven by Collagen VI (a1) promoter). As expected, Cre activity could be detected in mesenchymal cells such as synovial fibroblasts, articular chondrocytes, skeletal myocytes, keratinocytes, dermal fibroblasts and intestinal myofibroblasts. Mesenchymal cell-specific reactivation of a mutant floxed TNFRI allele in Col(VI)-Cre/TNFΔARE/TNFcneo mice led to a full blown arthritic and intestinal phenotype indicating that cells of mesenchymal origin are sufficient targets of pathogenic TNF function. Our results offer a novel mechanistic perspective for TNF function in gut and joint pathologies and indicate a common TNF target that may explain the often observed synovial/gut axis in human disease.

185 PHOSPHATIDYLINOSITOL 3- KINASE GAMMA REGULATES MATRIX METALLOPROTEINASES PRODUCTION IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS

I. Kühnel1,2, N. Pundt2, A. Baier1, T. Bondeva3, I. Meinecke1,2, S. Gay4, T. Rückle5, M. Camps5, M. K. Schwarz5, C. Rommel5, R. Wetzker3, T. Pap.1Dept Orthopedic Surg, Univ Magdeburg, Germany; 2Div Mol Med Musculoskeletal Tissue, Univ Munster, Germany; 3Unit Mol Cell Biol, Univ Jena, Germany; 4Ctr Exp Rheum, Univ Hosp Zürich, Switzerland

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are secreted by rheumatoid arthritis fibroblasts (RASF) and play an important role in cartilage destruction in patients with RA. Different studies have shown that the MMP expression is linked to signalling pathways in which phosphatidylinositol 3- kinase (PI3K) is also implicated. However there is only sparse knowledge about the relevance of the PI3K isoforms for the activation of RASF. Therefore, we analyzed the expression of the gamma isoform of PI3K (PI3Kgamma) in RASF and osteoarthritis SF (OASF) and investigated its effects on epidermal growth factor (EGF) induced phosphorylation of AKT and on the EGF-mediated expression of MMP-1 and -3. Synovial fibroblasts from RA- and OA- patients were analyzed for the expression of mRNA for the catalytic p110 and regulatory p101 subunit of PI3Kgamma by PCR. Expression of PI3Kgamma was confirmed by Western blot. The involvement of PI3Kgamma in Akt-phosphorylation was studied in EGF- stimulated cells (10–100 ng/ml, 5 min) using the pan-PI3K-inhibitor LY294002 (200–500 nM) and a PI3Kgamma specific inhibitor 1 (50–200 nM) together with phospho-specific antibodies. The expression of MMP-1 and MMP-3 was studied by ELISA following stimulation of synovial fibroblasts with EGF (10 ng/ml) over 24 hours. PCR and Western blot revealed the expression of the p110 catalytic subunit of PI3Kgamma but not of the regulatory p101gamma subunit in RASF. OASF showed only negligible levels of the p110 subunit. Stimulation of RASF and OASF resulted in the phosphorylation of Akt with LY294002 inhibiting completely the EGF- mediated activation. The PI3Kgamma specific inhibitor 1 reduced the EGF-stimulated phosphorylation of Akt in RASF but had no effect in OASF. Interestingly, treatment of RASF with both LY294002 and compound 1 significantly reduced the EGF-mediated induction of MMP-1 (63% and 42%, respectively). Similar effects were seen with MMP-3 with a reduction of 20% and 40%, respectively. These data suggest a disease-specific expression of PI3Kgamma in RASF that contributes to EGF-mediated phosphorylation of Akt and subsequent induction of MMP-1 and MMP-3. Therefore specific inhibitors of PI3Kgamma may be a possibility to interfere with the activation of RASF and to reduce cartilage destruction in RA.

186 ANALYSIS OF PATHOGENIC AND PROTECTIVE T CELL SUBPOPULATIONS IN A MURINE ARTHRITIS MODEL

M. Janke1, F. Hardung1, S. Rutz1, L. Morawietz2, A. Scheffold1.1German Arthritis Research Center, Berlin; 2Institute for Pathology, Charité, Berlin, Germany

Introduction: Rheumatoid arthritis is a chronic disease with an autoimmune character which affects about 1 percent of the world population. Patients mainly suffer from severe destruction of bone and cartilage. Although there is indirect evidence that CD4+ T cells are key players in rheumatoid arthritis, their exact role in disease pathogenesis is still not clear. Therefore, our aim was to define protective or pathogenic effector functions of T cells recognising joint-specific antigens using the ovalbumin-induced arthritis model. In this model, arthritis is induced by intra-articular (i.a.) injection of cationic ovalbumin into mice which have been previously sensitised to the same antigen either by immunisation or by adoptive transfer of Ova-specific CD4+ T cells.

Results: We show that i.a. injection of ovalbumin into immunised mice leads to a strong arthritis characterised by massive cell infiltration, hyperplasia and severe bone and cartilage destruction. Adoptive transfer of Ova-specific Th1 cells alone into naive mice which received i.a. Ova also induced arthritis. Th1-induced disease is less pronounced than after Ova-immunisation and completely lacks bone or cartilage destruction. Th2 cells, which were thought to counterbalance the pathogenic effect of Th1 cells, were only partially able to abolish Th1-induced arthritis after co-transfer and even induced mild inflammatory symptoms when transferred alone. In contrast, Ova-specific CD4+CD25+ regulatory T cells (Treg) completely suppressed Th1-induced arthritis. However, if mice were sensitised by Ova-immunisation, CD4+CD25+ antigen-specific regulatory T cells (Treg) were only partially protective. Non-specific Treg had little to no protective influence on arthritis severity.

Conclusion: Induction of full blown arthritis depends on participation of both joint-specific B cells and T cells. Th1 cells alone induce joint inflammation without cartilage destruction. Antigen-specific Foxp3+ Treg completely suppress Th1-mediated disease but only ameliorate arthritis induced by B/T cooperation. Th2 cells and non-specific Treg have little protective effect. Thus, Foxp3+Treg specific for joint antigens have the maximal protective effect in vivo. However, we identified several layers of arthritis pathogenesis and only some of them seem to be under the control of Treg.

187 QUANTIFICATION OF THE INVASIVE POTENTIAL OF RA SYNOVIAL FIBROBLASTS BY A NOVEL INVASION ASSAY USING TRANSEPITHELIAL ELECTRICAL RESISTANCE MEASUREMENT (TEER)

C. Wunrau1, E-M. Schnaecker2, B. Dankbar1, N. Pundt1, T. Pap1.1Division of Mol Med of Musculoskeletal Tissue and 2Division of Physiology-Nanolab, Univ Hosp Muenster, Germany

RA synovial fibroblasts (RASFs) are key effector cells in the rheumatoid synovium and have been associated strongly with the destruction of articular structures. RASFs exhibit features of stable cellular activation that result in their attachment to the articular cartilage and deep invasion of extracellular matrix. However, quantification of the invasive potential of RASF has been a major challenge, and the question of whether these cells can transmigrate actively from one joint to another has been a matter of debate. Here, we established a novel in vitro assay to exactly determine the invasive potential of SFs from patients with RA and study their transmigration through epithelial cell layers. Synovial tissue samples were obtained from three RA patients and RASFs were isolated and cultured under standard conditions. For the measurement of their invasiveness, we used a highly sensitive electrophysiological technique that is based on the measurement of the electrical resistance of a monolayer of the C7 subclone of Madin-Darby canine kidney cells (MDCK-C7). This high resistance clone was grown to confluency in a filter cup and the transepithelial electrical resistance across the monolayer was measured with an electrode. Following full establishment of the MDCK-C7 monolayer, RASFs were added on top of the MDCK-C7 cells, and their invasion was assessed four times of every patient through measurement of electrical resistance breakdown in the assay. Using this assay, RASF exited a strong invasiveness through the monolayer. Resistance decreased to 60% of baseline value after two days and total breakdown was achieved after four days. The invasive potential of RASF was comparable to that of the chondrosarcoma cell line CAL-78, which exhibited a similar decrease of resistance after two days. Collectively, our data demonstrate that the TEER assay constitutes a highly sensitive electrophysiological method to analyse the invasive potential of RASF as well as their transmigration capacity. It allows us to measure a decrease in the resistance of an epithelial monolayer before morphological destruction of the barrier or penetration is evidently visible. Specifically, our data indicate a high invasiveness of RASFs comparable to that of tumor cells. In addition, differences in the invasive potential of different RA patients and under different conditions can be measured in a highly precise manner.

188 SYNDECAN-4 DEFICIENCY RESULTS IN DELAYED ENCHONDRAL OSSIFICATION AND REDUCED AGGRECAN CLEAVAGE IN OSTEOARTHRITIC CARTILAGE

F. Echtermeyer1, I. Meinecke1,2, K. Neugebauer1, C. Herzog3, R. Dreier4, T. Pap1.1Div Mol Med of Musculoskeletal Tissue; 2Dept Traumatol; 3Dept Anatomy and Anaesthesiol; 4Dept Phys Chem and Pathobiochem, Univ Hosp Munster, Germany

During limb development chondrocyte differentiation into hypertrophic chondrocytes is essential for enchondral ossification of long bones. In pathologic situations such as osteoarthritis chondrocytes differentiate again into hypertrophic-like chondrocytes in affected cartilage areas. However, the mechanisms that link chondrocyte hypertrophy to cartilage remodelling are poorly understood. Based on recent data that have implicated transmembrane heparan sulfate proteoglycans in matrix turnover and cell differentiation, we analyzed the distribution and functional role of syndecan-4 during limb development in mice and studied its expression and function in osteoarthritis (OA). Syndecan-4 promoter activity was detected in whole embryos by staining for beta-Galactosidase in syndecan-4-/- LacZ knock-in mice. For cellular localization of syndecan-4 expression within cartilage immunohistochemistry with antibodies against syndecan-4 and type X collagen was performed. Alizarin red S staining was carried out to analyze the mineralization of bones in wild type and syndecan-4-/- littermates at days E13.5 and E14.5. To study syndecan-4 in OA, we compared its expression in normal and OA articular cartilage by Northern-blot analysis and immunohistochemistry. For functional analysis, osteoarthritic changes were induced in syndecan-4-/- mice and their wild-type controls by surgically achieved joint instability, and safranin-orange staining assessed the loss of proteoglycans. Staining for syndecan-4 and ADAMTS generated aggrecan neo-epitopes was performed in the knees of these mice. Beta-Gal-staining of syndecan-4-/- mice at E12.0 showed a strong activity of the syndecan-4 promoter at sites of cartilage condensations. In later stages syndecan-4 was detected in the growth plates of long bones. In wild-type embryos, syndecan-4 protein was also found mainly in chondrocytes of the hypertrophic zone, where it co-localized with type X collagen. The loss of syndecan-4 was associated with a significant retardation in the mineralization of axial and appendicular bones. There was an upregulation of syndecan-4 in human OA cartilage both at the mRNA and the protein level. Analysis of osteoarthritic changes in mice revealed a strong and early induction of syndecan-4, and there was a significant reduction of proteoglycan loss in the syndecan-4-/- mice compared to their wild-type controls. This was accompanied by a significantly reduced staining for ADAMTS generated aggrecan neo-epitopes in syndecan-4-/- mice. Our data demonstrate that syndecan-4 is induced in hypertrophic chondrocytes both during embryogenesis and in OA cartilage. By promoting ADAMTS mediated cleavage of aggrecans, syndecan-4 facilitates enchondral ossification but is involved also in cartilage degradation by hypertrophic chondrocytes in OA. Inhibition of syndecan-4 may, therefore, constitute a promising strategy to interfere with osteoarthritic cartilage damage.

189 REGULATION OF CYTOKINE-INDUCED HIF-1ALPHA EXPRESSION IN RHEUMATOID SYNOVIAL FIBROBLASTS

J. Westra, E. Brouwer, M. D. Posthumus, B. Doornbos-van der Meer, C. G. M. Kallenberg, P. C. Limburg.Rheumatology and Clinical Immunology, University Medical Center Groningen, The Netherlands

Aim: The transcription factor hypoxia-inducible factor (HIF)-1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilisation of the subunit HIF-1alpha. Activation can also occur by pro-inflammatory cytokines during inflammation. Hypoxia as well as proinflammatory cytokines play an important role in the synovial inflammation in rheumatoid arthritis (RA) patients. Expression of HIF-1alpha has been demonstrated in RA synovial lining layer. The aim of the study was to investigate the regulation of the intracellular signal transduction pathways, involved in the expression of HIF-1alpha, and the expression of genes regulated by HIF-1alpha in rheumatoid synovial fibroblasts.

Materials and methods: Rheumatoid synovial fibroblasts (RSF) were cultured under proinflammatory conditions (IL-1beta and TNF-alpha stimulation) and under chemical hypoxia (CoCl2 treatment). Expression of HIF-1alpha was analyzed in nuclear extracts by Western blotting. The effects of inhibitors of the phospho-inositide 3 kinase (PI3K) pathway, the extracellular signal-regulated kinase (ERK) pathway, and the Ca2+/Calmodulin kinase II (CAMKII) pathway on HIF-1alpha expression was measured. mRNA expression of HIF-1apha, COX-2, vascular endothelial growth factor (VEGF) and stromal cell-derived factor (SDF)-1 was determined by real-time RT-PCR and protein production of VEGF and SDF-1 was determined by ELISA.

Results: Treatment of the synovial fibroblasts with 150 mM CoCl2 as well as stimulation with 10 ng/ml IL-1beta or TNF-alpha resulted in strong protein expression of HIF-1alpha, measured with Western blotting. Pre-treatment with the MEK1/2 inhibitor PD98059, the PI3K inhibitor LY294002 and the CAMKII inhibitor KN93 resulted in inhibition of the cytokine-induced HIF-1alpha. Furthermore it was shown that cytokine-induced mRNA expression of HIF-1alpha was inhibited by LY294002. IL-1beta and TNF-alpha stimulation was able to induce mRNA expression of VEGF and COX-2, which was inhibited by the MEK1/2 inhibitor. VEGF but not SDF-1 protein production was induced after cytokine stimulation, but no significant effect of kinase inhibitors was found.

Conclusions: Expression of cytokine-induced HIF-1alpha at the mRNA level in rheumatoid synovial fibroblasts is regulated by the PI3K pathway, whereas HIF-1alpha protein expression is also influenced by other pathways. We found that cytokine stimulation induced VEGF mRNA and protein production, but no significant effect of kinase inhibition was found on VEGF production in cytokine-stimulated rheumatoid synovial fibroblasts.

190 ROLE OF CAVEOLIN-1 IN THE PATHOGENESIS OF TISSUE FIBROSIS IN SYSTEMIC SCLEROSIS

F. Del Galdo, C. de Almeida, J. F. Jasmin, M. P. Lisanti, S. Jimenez.Thomas Jefferson University, Philadelphia, USA

Systemic sclerosis (SSc) is a multi-systemic disease associated with autoimmune activation and widespread fibroproliferative vascular damage, causing progressive tissue fibrosis and subsequent multi-organ dysfunction. The fibroproliferative process occurs essentially in all organs, but is often particularly evident and aggressive in the kidneys and lungs, causing scleroderma renal crisis or pulmonary arterial hypertension (PAH) respectively, which combined represent the major cause of mortality in SSc patients. Recent studies on idiopathic pulmonary hypertension and idiopathic pulmonary fibrosis independently identified caveolins as important inhibitors of progression of both diseases. Caveolins comprise a family of membrane proteins involved in the regulation of TGF-β, VEGF, and endothelin pathways, three pathways universally accepted to play a crucial role in the SSc progression. Similarly, recent data indicate that caveolin-1 (Cav-1) is down regulated in SSc lung and dermal fibroblasts in vitro. In this study, we analyzed the expression of Cav-1 in vivo in SSc patients and the role of Cav-1 protein in the regulation of collagen production by human normal and scleroderma fibroblasts in vitro.

Materials and methods: Lung biopsies from SSc patients affected by pulmonary hypertension and pulmonary fibrosis were analyzed by confocal laser microscopy for Cav-1 expression. Cav-1 knock out mice lungs were analyzed by histology/histochemistry studies and by hydroxyproline content to assess pulmonary fibrosis. Normal and scleroderma derived fibroblasts were stimulated with TGF-β and treated with recombinant caveolin. Cellular mRNA, intracellular proteins and tissue supernatants were analyzed by real time PCR and immunoblotting. Cell viability was assessed by WST-1 assay.

Results: We observed that Cav-1 expression was markedly reduced in affected SSc tissues in vivo, as demonstrated by quantitative confocal microscopy analysis of lung biopsies of SSc patients affected by pulmonary hypertension and/or pulmonary fibrosis. Consistent with these findings, lungs from Cav-1 knockout mice showed pulmonary fibrosis remarkably similar to that seen in SSc patients, as evidenced by histological analysis and hydroxyproline content of the lungs. Additionally, TGF-β, the main profibrotic cytokine in SSc pathogenesis, down-regulated Cav-1 expression in normal human fibroblasts in vitro. Moreover, Cav-1 markedly reduced collagen production of SSc fibroblasts in vitro and suppressed TGF-β induced ECM gene upregulation without affecting cell viability.

Conclusions: This study indicates that Cav-1 expression is reduced in SSc in vivo and that Cav-1 plays a key role in TGF-β induced tissue fibrosis and in the development of the scleroderma fibrotic phenotype of fibroblasts. Furthermore, Cav-1 can suppress collagen production of SSc fibroblasts and of TGF-β stimulated fibroblasts in vitro without displaying evidence of cell toxicity. These results suggest that Cav-1 is a novel therapeutic target for the treatment of SSc patients.

191 SYSTEMIC SCLEROSIS (SSC) LYMPHOCYTES INDUCE ANGIOGENESIS IN HEALTHY MICROVASCULAR ENDOTHELIAL CELLS

M. Cinelli, S. Guiducci, V. Rogai, I. Miniati, G. Fiori, M. L. Conforti, F. Nacci, M. Matucci-Cerinic.Department Medicine, Division of Rheumatology, University of Florence, Florence, Italy

Background: SSc pathogenesis and progression may involve three cellular types: endothelial cells (leading to vascular damage), fibroblasts (inducing fibrosis) and lymphocytes (producing autoimmune reaction). Endothelial damage and defective neoangiogenesis are hallmarks of SSc and often precede the other disease features. We previously showed that the conditioned medium from SSc-fibroblasts and SSc-microvascular endothelial cells (SSc-MVEC) inhibits angiogenesis in healthy MVEC (H-MVEC).

Aim: To evaluate the effects of SSc peripheral blood lymphocytes on H-MVEC angiogenic potential.

Methods: H-MVEC were incubated with the conditioned medium (CM) from healthy lymphocytes (H-CM), from limited (lSSc-CM) and diffuse SSc (dSSc-CM) lymphocytes. 5 CM for each subset were evaluated. Angiogenesis was assayed by chemoinvasion (Boyden chamber invasion assay, using Matrigel as invasion matrix) and in vitro angiogenesis (capillary morphogenesis assay). Induction by vascular endothelial growth factor (VEGF) 50 ng/ml was considered as positive control. CM from SSc-MVEC (SSc-MVEC-CM) or from SSc-fibroblasts (SSc-Fb-MC) was used to inhibit angiogenesis. All CM were obtained by incubating cells in MVEC basal medium (MCDB131 plus 0.2% FCS, without other growth factors) for 48 hours. Basal chemoinvasion and capillary morphogenesis (0.2% FCS) were considered as 100%.

Results: H-CM had slight but significant stimulatory effects on basal chemoinvasion (138±8.7%, p<0.05) and capillary morphogenesis (123±4.8%, p<0.05). Furthermore, both lSSc-CM and dSSc-CM significantly increased basal migration (303±14.2%, p<0.001 and 247±19.8% p<0.01, respectively) and tubular-like structure formation (141.7±5.2 p<0.005 and 129.0±4.6 p<0.05, respectively). Chemoinvasion was significantly reduced by SSc-MVEC-CM or SSc-Fb-MC (55±8.1% and 64±9.2% respectively, p<0.05 vs basal), and also capillary morphogenesis was inhibited (27±5.1% and 38±6.2% respectively, p<0.001 vs basal). Co-incubation with lSSc-CM or dSSc-CM was able to contrast the anti-angiogenic potential of SSc-MVEC-CM and SSc-Fb-CM, maintaining the capacity of H-MVEC to form capillaries (64±3.3% and 85±4.8%, p<0.001) and to migrate (72±8.8% and 88±5.0%, p<0.05), even if reduced (p<0.05) in comparison to basal unstimulated cells.

Conclusion: These data show that SSc lymphocytes may influence the H-MVEC microenvironment, probably secreting pro-angiogenic molecules that stimulate their angiogenic properties and partially counteracting the response to anti-angiogenic factors. For this reason, the role of lymphocytes in SSc breakdown of capillary formation might be reconsidered. This may suggest that the early impairment of MVEC may depend by factors secreted by fibroblasts.

192 EXPRESSION AND REGULATION OF ST2 ISOFORMS IN SYNOVIAL MEMBRANE CELLS FROM PATIENTS WITH ARTHRITIS

A. R. Fraser, M. Moore, A. Crilly, D. Xu, I. B. McInnes.University of Glasgow, Glasgow, UK

ST2 is a member of the IL-1 receptor family widely reported to have a negative regulatory function on IL-1R and TLR signalling. However, recent reports have identified a novel cytokine (IL-33) that binds ST2L (the membrane bound isoform) and induces Th2-type cytokine release from Th2 T cells. We previously reported that ST2 is significantly elevated in synovial fluids from patients with inflammatory synovitis and have now investigated the presence of both ST2L and the soluble isoform sST2 in synovial tissue from OA and RA patients. Our aim was to determine the source of ST2 present in inflammatory synovitis and factors regulating ST2 expression. Synovial membranes were obtained from consenting OA and RA patients and prepared as whole tissue digests or passaged fibroblast cultures. The isolates were stimulated as described and probed for isoform expression by RT-PCR and Western blot. Membrane bound ST2L was detected as mRNA and protein in synovial tissue extracts and in fibroblast cultures. The cognate ST2 co-receptor (IL-1RAcP) was also identified in tissue and fibroblast isolates by Western blot. Functionality of the ST2 receptor was confirmed by detection of p38 MAPK phosphorylation in response to IL-33 stimulation of fibroblasts. Expression of ST2L was regulated by pro-inflammatory factors such as TNF-alpha and IL-1 beta which markedly increased ST2L mRNA expression. The soluble variant (sST2) was also detected by RT-PCR, and could be upregulated in isolates by stimulation with IL-33. Th2 T cells are known to produce sST2 upon activation, but the low level of Th2 cells associated with RA suggests that the stromal environment contributes the majority of ST2 detected in RA synovial fluid. Increased sST2 expression provides a mechanism whereby stromal cells could regulate synovial T cell maturation.

193 EVALUATION OF ANGIOGENESIS AND FIBROSIS IN PLACENTA FROM SYSTEMIC SCLEROSIS

L. Ibba-Manneschi1, M. Manetti1, A. F. Milia2, I. Miniati2, S. Guiducci2, L. Polidori1, M. Cinelli2, G. Mello3, M. Matucci-Cerinic2.1Dept. Anatomy, Histology and Forensic Medicine; 2Dept. Internal Medicine, Division of Rheumatology; 3Dept. Gynecology, Perinatology and Human Reproduction, University of Florence, Florence, Italy

Aim: Systemic sclerosis (SSc) is an autoimmune disease affecting the skin and internal organs, characterized by impaired angiogenesis and abnormal fibrosis. Several angiogenic and connective tissue remodelling mediators are known to be involved in SSc pathogenesis. Angiogenesis and vascular transformation are important processes in the normal development of the placenta during pregnancy. Therefore, a tight regulation of angiogenic factors is essential for reproductive success. The aim of this study was to evaluate the expression of angiogenesis and fibrosis markers in placenta from a 29-year-old woman affected by diffuse SSc.

Materials and methods: Placenta biopsies were obtained from one SSc patient after caesarean delivery at 34 weeks and from three normal uncomplicated pregnancies (34–35 weeks), as controls. The samples were routinely processed and embedded in paraffin. Immunohistochemistry was performed on serial sections (5 μm). We analysed the expression of VEGF, PLGF, VEGFR-1 and VEGFR-2, as angiogenic factors. Moreover, Masson’s stain and immunohistochemistry for CTGF, collagen type I and α-smooth muscle actin (α-SMA) were performed to analyse tissue fibrosis and stromal fibroblast transdifferentiation to myofibroblasts.

Results: The trophoblast, decidual cells, endothelial cells (ECs), fibroblasts and Hofbauer cells showed immunoreactivity for VEGF. In SSc placenta, ECs displayed a stronger immunopositivity for VEGF, whereas the trophoblast showed a disomogeneous and weaker immunostaining than controls. VEGFR-1 was detected in SSc trophoblastic layer and ECs similar to control placentas. VEGFR-2 expression was evident in ECs of the villi, but the staining intensity and the number of positive vessels were higher in SSc compared with controls. PLGF immunopositivity was weaker in decidual cells, ECs and fibroblasts in SSc than in healthy placentas, while it was similar to controls in trophoblastic cells. Masson’s stain revealed a diffuse fibrosis in SSc placenta, mostly around the vessels. Similarly, the perivascular immunoreactivity for collagen type I was stronger in SSc placenta compared with controls. In SSc, a very strong immunopositivity for CTGF was detected in decidual and Hofbauer cells and, in particular, in the larger vessel wall, ECs and fibroblasts. α-SMA was evident around the larger vessels in both healthy and SSc placentas, whereas it was disomogeneously distributed around SSc microvessels. Stromal cells showed a strong immunopositivity for α-SMA only in SSc placenta.

Conclusion: This is the first morphological study on the expression of angiogenesis and fibrosis markers in placental tissue from a SSc patient. Our preliminary results suggest that the dysregulation of angiogenic factors, as well as the tissue fibrosis and the transdifferentiation of stromal fibroblast to myofibroblasts could lead to functional abnormalities in SSc placenta.

194 FUNCTIONAL CHANGES IN SCLERODERMA FIBROBLASTS CO-CULTURED WITH AUTOLOGOUS PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS)

S. Vettori1, R. De Palma2, D. Malanga2, E. D’Aiuto2, M. Zekusic2, G. Abbate2, G. Valentini1.Section of 2Internal Medicine and 1Rheumatology, Department of Clinical and Experimental Medicine Second University of Naples, Naples, Italy

Introduction: Increasing evidence suggest that an interplay between T cells and fibroblasts plays a pivotal role in promoting matrix accumulation in systemic sclerosis (SSc).

Aim: We investigated if the co-culturing of fibroblasts derived from SSc patients with autologous PBMCs could induce peculiar modifications in any cell types.

Materials and methods: Fibroblasts were obtained from the skin of patients undergoing biopsy for diagnostic purposes. PBMCs were obtained from the same patients. All the patients were fully informed of the meaning of the study and accepted to donate blood. Cells were taken in culture at 37°C 5% CO2 for ten days, then cells were stained with monoclonal antibodies for HLA-DR, CD3, CD4, CD56, CD95, TCRαβ, TCRγδ, After the staining, cells were analyzed by flow-cytometry using a FACScalibur.

Results: We found that T cells bearing an αβ receptor were expanded in the co-cultures. Moreover, these cells were positive for the expression of HLA-DR, suggesting an activation of T cells induced by co-culturing with autologous fibroblasts. No expansion of γδ T cells nor CD56 positive T cells was found. SSc fibroblasts, which have already been reported to have a basal high expression of CD95 (FAS), were found to up-regulate FAS in these experiments.

Conclusions: We recently showed that peripheral blood T cells from SSc patients expand when co-cultured with autologous fibroblasts and acquire the same T cell clonotypes that are increased in the affected skin. The results presented here further support that such expansion may be due to a specific antigenic activity of fibroblasts on T cells. In addition, the up-regulation of FAS expression on SSc fibroblasts may be related to phenotypic changes that indicate an increased ability of these cells to escape normal pathways leading to cell death. The meaning of these findings in the pathogenesis of SSc remains to be further investigated.

195 EVIDENCE FOR REDUCED ANGIOGENESIS IN BONE MARROW (BM) IN SYSTEMIC SCLEROSIS (SSC)

I. Miniati, V. Carrai1, L. Nassi1, G. Capaccioli1, S. Guiducci, M. Cinelli, study on G. Salvadorini, V. Rogai, R. Saccardi1, L. Rigacci1, R. Giacomelli2, study on A. Tyndall3, R. Alterini1, M. Matucci-Cerinic.Dept of Medicine & Surgery Div Medicine I & Rheumatology; 1Division of haematology, Univ of Florence, Italy; 2Dept. Internal Medicine and Public Health, University of L’Aquila, 3Dept of Rheumatology, Univ Basle, Switzerland

Background: Dysfunctional angiogenesis is a pathogenetic marker of SSc. Microvascular endothelial cells have a reduced capacity to form capillaries,1 reduced circulating levels of endothelial progenitor cells have been found2 and mesenchymal stromal cell differentiation into endothelial cells has shown a defective capacity to form capillaries.3 This suggests that pathophysiologically relevant changes may already exist in SSc BM stromal cells.

Aim: To study in SSc BM angiogenesis, the cellular immune system and fibrosis.

Methods: 8 SSc patients affected by a severe diffuse cutaneous SSc and screened for autologous hematopoietic stem cells transplantation, underwent a BM biopsy to assess cellularity and morphology. BM biopsies were compared with 5 healthy controls.

To evaluate angiogenesis, cellular immunity and fibrosis the following antibodies were used: VEGF, KDR/flk-1, MMP-9, CD34/QBEND10, vWF, CD20, CD3, CD4, CD8, CD38, K, λ, CD68/PGM-1, CD61. To evaluate fibrosis silver impregnation for reticulum was used. The number of vessels, the mean area of vascularisation (μm2, %), the perimeter and microvessel density (MVD) were measured with a multiparametric computerized imagine analysis.

Results: The morphology of BM was similar in SSc and controls. Also the B cell population was similar but the T cells showed a reduction of CD4/CD8 ratio in SSc. A significant reduction in BM vascularity was found: both microvessel density and number of vessels were lower, while VEGF expression (observed in myeloid cells and in megakaryocytes and histiocyte-macrophage system) was much higher in SSc BM samples than in controls. In seven patients, a weak expression of KDR/flk-1 was observed and MMP-9 expression was low in all cases. Out of 8 patients 2 had a maximal while another 2 had a moderate degree of BM fibrosis.

Conclusion: In SSc, BM is characterised by a reduction of angiogenesis that may induce the increase of VEGF.

1

2

3

196 EVALUATION FOR MARKERS OF FIBROSIS IN THE UPPER GASTROINTESTINAL TRACT OF PATIENTS WITH SYSTEMIC SCLEROSIS

M. Manetti1, I. H. Tarner2, P. Saar2, A. F. Milia3, L. Polidori1, A. Knedla2, study on S. Lefevre2, M. Matucci-Cerinic3, U. Müller-Ladner2, L. Ibba-Manneschi1, study on E. Neumann2.1Dept. Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy; 2Dept. Internal Medicine and Rheumatology, Justus-Liebig-University Giessen, Kerckhoff-Klinik, Bad Nauheim, Germany; 3Dept. Internal Medicine, Division of Rheumatology, University of Florence, Florence, Italy

Aim: Systemic sclerosis (SSc) is a chronic connective tissue disease characterized by fibrosis and destruction of the microvasculature. Increased deposition of collagen and other extracellular matrix components affect not only the skin but most of the internal organs including the gastrointestinal (GI) tract. Few reports assessed the morphology of the stomach in SSc. We studied a case of a 52-year-old female with SSc, showing an important involvement of upper digestive system. She underwent distal esophageal myotomy and subsequently total gastrectomy. Moreover, we analysed gastric biopsies obtained during gastroscopical examination from SSc patients to evaluate the expression of markers for fibrosis in the gastric wall of SSc patients.

Materials and methods: Full-thickness gastric wall samples were obtained from one SSc patient and biopsies were taken from 10 SSc patients, which underwent esophagogastroscopy. The full-thickness gastric samples were fixed in formalin and embedded in paraffin. The endoscopic samples were embedded in OCT, snap frozen in liquid nitrogen and stored at −80°C. In order to investigate tissue fibrosis, the sections were stained by Masson’s thrichromic method and analysed by immunohistochemistry for the expression of connective tissue growth factor (CTGF), collagen type I, collagen type IV, α-smooth muscle actin (α-SMA) and endothelin-1 (ET-1). Full-thickness gastric wall biopsies, free from inflammatory responses and neoplastic infiltration, from 3 patients who underwent total gastrectomy due to other disease entities, and endoscopic biopsies from 5 controls were also examined.

Results: In SSc gastric wall, Masson’s stain revealed a mild fibrosis in the lamina propria that became more severe in the muscularis mucosae. In the muscle layers, wide areas of focal fibrosis surrounded smooth muscle cells (SMCs) increasing intercellular spaces. A marked fibrosis was evident also around the vessels. Collagen type I expression mirrored the fibrosis areas revealed by Masson’s stain. The immunopositivity for collagen type IV was well detectable in the basal lamina around the glands and the vessels in SSc compared with the control gastric wall. In SSc, a strong CTGF expression was evident in the muscularis mucosae, and it was focally distributed in the muscle layers when compared with the control. Moreover, most of the vessels showed a strong immunopositivity for CTGF in SSc gastric wall, while they were weakly positive in the controls. In SSc the α-SMA reactivity was stronger in the lamina propria, muscularis mucosae, muscle layers and in the vessel wall than in controls, whereas ET-1 positive cells were evident mostly in SSc muscle layers.

Conclusion: Our results show that the expression of markers for fibrosis is relevant in SSc stomach. The involvement of gastric wall components in fibrosis process likely accounts for the decreased mobility and atrophy of the stomach in SSc patients.

The study was supported by grant of the German Ministry of Education and Research/German Network for Systemic Sclerosis and EUSTAR.

197 COMPLEX METABOLIC COMPOSITION OF SYNOVIAL FLUID IN SCLERODERMA DETECTED BY PROTON MAGNETIC RESONANCE SPECTROSCOPY

C. Ciurtin, M. Cojocaru.Carol Davila University of Medicine and Pharmacy, Bucharest, Romania

Objectives: Because of the difficulties of synovial fluid elevation in scleroderma, previous studies have given little information about possible correlation between the disease’s pathogenesis and the complex composition of joint effusions.

Study methods: We tried to characterize the complex metabolic environment of the joints affected by diffuse scleroderma in a 35 year old patient. Our previous study used proton magnetic resonance spectroscopy (MRS) for the simultaneous detection and measurement of the metabolic components of synovial fluid in different pathologies. In the present study, we focused on simultaneous detection of synovial potential markers for scleroderma. Our patient met criteria for scleroderma diagnosis and underwent determination of synovial fluid composition after 3 h, 10 h and 24 h refrigeration. The patient was treated with vasodilators, non-steroid anti-inflammatory drugs and antiaggregant medication, having Raynaud, esophageal symptoms and arthritis. Rodnan score was 15. Spectra were recorded on a Bruker apparatus operating at 400 MHz.

Results: This method led to the possibility to attribute the signals for glutamine, threonine, lactate, hydroxybutyrate, glycine, dimethylamine and lipoprotein-associated fatty acids, ceramide and citrulline. We didn’t detect a highly intense signal for ceramide as in RA samples, showing that apoptosis is not an important process in sclerodermic joints. We have shown extremely weak signal intensity for citrulline at 3.15 ppm (highly specific for RA synovial fluid—sensitivity 80%, specificity 100% in our previous study). Increased synovial fluid levels for chylomicron- and VLDL- associated triacylglycerols was found, the same observation being valid for lactate levels as well.

Conclusion: MRS investigation of synovial fluid provides valuable information consisting of simultaneous detection of different metabolites in sclerodermic synovial fluid. We can correlate the presence of lipid components with the endothelial dysfunction and possible early progression of atherosclerosis in scleroderma patients.

198 CIRCULATING ENDOTHELIAL PROGENITOR CELLS IN SYSTEMIC SCLEROSIS ARE RELATED TO IMPAIRED ANGIOGENESIS AND VASCULAR DISEASE MANIFESTATIONS

T. Nevskaya, S. Bikovskaya, E. Lyssuk, M. Zaprjagaeva, E. Much, N. Guseva, study on L. Ananieva, E. Nassonov.State Institute of Rheumatology of RAMS, Moscow, Russia

Background: Endothelial damage is one of the earliest events in the pathogenesis of vascular injury in systemic sclerosis (SSc). Given the essential role of endothelial progenitor cells (EPCs) in ongoing endothelial repair and neovascularization, it is likely that insufficient angiogenesis seen in SSc is related to EPC alterations.

Material and methods: We measured EPC numbers in blood of 40 SSc patients (all female, mean age 44±10 yr, 15 dSSc and 25 lSSc) and 24 controls by flow cytometry analysis and studied their relation to disease activity, severity of internal organ damage and vascular manifestations. Endothelium dependent and endothelium-independent vasodilatation was assessed by high-resolution ultrasonography of the brachial artery. Structural capillary changes were studied quantitatively using nailfold video capillaroscopy.

Results: Compared to control subjects, SSc patients showed higher levels of both early CD133+/VEGF-R2+ (0.0159±0.0026%, p<0.05) and more mature CD34/VEGF-R2-positive (0.0041±0.0027%, p<0.01) EPC populations in association with increased membrane expression of Fas (CD95) on CD34+VEGF-R2+ progenitor cells (0.0076±0.0025, p<0.005). Early stage of SSc and high disease activity were accompanied by a rise in EPC numbers in blood that correlated positively with severity of peripheral vascular manifestations (r = 0.34, p<0.05). EPC reduction was attributed to the late stage of disease and had a strong relation to the development of severe internal organ (predominantly cardiac) involvement and pulmonary hypertension. There was a lack of EPC mobilization from BM in response to ischemia and tissue damage late in disease course. A decrease in circulating EPC numbers was closely associated with endothelial dysfunction and morphological signs of destructive microangiopathy.

Conclusions: At an early stage of systemic sclerosis EPC mobilization in response to tissue ischemia was preserved, but the levels of these cells dramatically dropped along with disease progression. EPC reduction strongly contributed to endothelial dysfunction and impaired angiogenesis leading to the development of severe cardiac disease and pulmonary hypertension—life-threatening complications of SSc.

199 CULTURAL AND HIGHLY SENSITIVE MOLECULAR TECHNIQUES FOR DETECTION OF Chlamydia pneumoniae in synovial fluid and blood specimens from patients with chronic synovitis

C. Contini, S. Seraceni, R. Cultrera, A. Grilli, F. Trotta1, A. Dautry-Varsat2.Institute of Infectious Diseases and 1Institute of Rheumatology, Ferrara, Italy; 2Institut Pasteur, Paris, France

Background and aim:Chlamydia pneumoniae (C pneumoniae) is a ubiquitous pathogen associated with upper respiratory tract entities and, although controversial, with a number of illness such as atherosclerosis, multiple sclerosis and joint diseases. While Chlamydia trachomatis-triggered reactive arthritis (ReA) is a well-documented entity extensively described, the role of C pneumoniae in the inflammatory oligoarthritis is less known. C pneumoniae is difficult to grow from synovial fluid (SF), and serology is not always effective for final diagnosis. We report a case of ankylosing spondylitis in whom C pneumoniae was repeatedly isolated from the SF and blood specimens by cultural and molecular methods.

Methods: SF and PBMC specimens were collected from 6 patients with chronic synovitis, one of them with relapsing joint swelling of the knee affected by ankylosing spondylitis. Serum anti-Chlamydia immunoglobulins were searched by ELISA (Cp Quant®, Eurospital, Italy). Specimens were inoculated on Hep-2 cells in duplicate wells, and incubated in CO2 for 3 h at 35°C. Cells grown in the half wells were harvested and fixed onto immunofluorescence (IF) slides using monoclonal antibodies FITC-conjugate specific for C pneumoniae (Institute Pasteur, Paris, Unit of Biological Cellular Interactions). To increase the number of bacterial inclusions, the resting wells were centrifuged and incubated for other 72 h with additional centrifugations on 3rd and then on 4th and 5th culture day, with medium refreshment on 72 h only. Culture supernatants underwent DNA extraction. PCR, Reverse Transcriptase (RT) PCR targeting the 16S rRNA, the Momp gene and the Heat shock Protein 60 of C pneumoniae were employed. Real-time PCR (Light-Cycler) was also performed.

Results: Of the 6 patients, 5 had a positive PCR result which was confirmed by RT-PCR. When these samples were put into culture, only three did grow Chlamydia after culture. For the patient with ankylosing spondylitis, the results are shown in table 1. Serology did detect IgA and IgG antibodies in absence of IgM.

Conclusions: Our culture method proved efficacious; the sensitivity was improved by additional centrifugation associated with extension of culture time. C pneumoniae reaches the articular cavity within monocytes surviving in a vegetative state, and triggers joint inflammation by mechanisms which are still unknown. The PBMC specimens, negative for DNA when directly extracted, became PCR positive after 144 h. This indicates that C pneumoniae is able to survive in PBMC in an infective stage, as demonstrated by their passage on Hep-2 cells. The high expression of C pneumoniae HsP-60 in the PBMC and synovial fluid confirms the ability of C pneumoniae to survive inside these compartments in vital and metabolically active forms. With concern with real-time PCR, the number of chlamydial DNA copies found with HsP 60 gene was higher before than after culture. By contrast, the selective decrease of 16sRNA before culture in SF and PBMCs leads to hypothesize, as shown by RT-PCR, a different expression of Chlamydophila genes during the different phases of infection.

Work in part supported by Fondazione CARIFE (Ferrara) and CARICE (Cento), and PRIN (MIUR) 2005–2007, Italy.

200 ENDOTHELIAL PROGENITOR CELLS IN SYSTEMIC SCLEROSIS: QUANTIFICATION, COLONY FORMING AND PROLIFERATION ASSAYS

J. Avouac1,2, G. Uzan3, C. Boileau2, A. Kahan Y et Allanore11.1Rheumatology A department, René Descartes University, Cochin hospital, Paris; 2INSERM U781, Necker hospital, Paris; 3INSERM U602, Paul Brousse hospital, Villejuif, France

Background: Contradictory results have been reported regarding the number of circulating endothelial progenitor cells (EPCs) participating in vasculogenesis in systemic sclerosis (SSc).

Objective: To determine the number of circulating EPCs, assessed by flow cytometry and count of EPCs colony forming unit (CFU) in SSc patients and healthy controls.

Patients and methods: 30 SSc patients and 18 controls matched for age and sex were included. SSc patients had a mean disease duration of 9±10 years, 66% had the limited cutaneous subtype, 12% pulmonary arterial hypertension and 20% digital ulcers. Quantification by flow cytometry: the negative lineage mononuclear cells were enriched from peripheral-blood mononuclear cells using a human progenitor cell enrichment cocktail and collected on a Ficoll density gradient centrifugation. This negative lineage population was then subjected to a triple labelling with VEGFR2-APC, CD133-PE and CD34-FITC antibodies associated with 7AAD labelling (identification of dead cells). EPC population was defined as the Lin-CD34+/CD133+/KDR+/7AAD-. Evaluation of the number of EPCs colonies: blood mononuclear cell fraction was collected on a Ficoll density gradient centrifugation and cultured on collagen-coated chamber slides in EGM2 medium for at least four weeks. The number of CFU-EPCs and their delay of appearance in patients and controls were compared. Population doublings: The proliferation curves of patients and controls CFU-EPCs were compared to those of human umbilical vein endothelial cells (HUVECs).

Results: SSc patients had significantly higher circulating EPC levels than controls (median 92 [11-248] vs 54 [7-275] EPCs for 1 million lin- mononuclear cells; p = 0.002). There was no association between the number of EPCs and patients’ phenotype.

The colonies’ formation and delay of appearance did not differ between SSc patients and controls, but the proportion of subjects having more than 5 EPC-CFU was significantly higher in the SSc group than in controls (8/15 versus 1/7; p<0.05). In SSc patients, the formation of colonies was associated with a high number of EPC detected by flow cytometry (p = 0.002). EPCs isolated from SSc patients and controls had a significantly higher proliferation capacity than HUVECs.

Conclusion: The number of circulating EPC is increased in SSc. This high level may have a multifactorial origin as we found no association between the number of these cells and disease phenotype. These cells have the ability to form colonies in vitro and have high proliferation capacity. Further analyses are now warranted to study the differentiation ability toward mature endothelial cells and the functional characteristics of EPCs in SSc.

201 SYNDECAN-4 MODULATES TNF-ALPHA INDUCED MMP PRODUCTION IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS

K. Neugebauer1, C. Herzog2, N. Pundt1, L. H. Meyer1, G. Kollias3, P. J. Blackshear4, G. Schett5, T. Pap1, F. Echtermeyer1.1Div Mol Med Musculoskel Tissue; 2Dept Anatomy and Anaesthesiol, Munster, Germany; 3Alexander Fleming Biomedical Sciences Research Center, Vari, Greece; 4NIEHS Res Triangle Park, NC, USA; 5Dept Rheumatol, Univ Hosp Erlangen, Germany

Recently syndecans, a new family of transmembrane heparansulfate proteoglycans, have been implicated in matrix turnover and cell differentiation. We analysed the expression of syndecan-4 in the synovial membranes of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and studied its regulation and its influence on the expression of matrix metalloproteinases (MMPs) in synovial fibroblasts (SF) following short- and long-term exposure to TNFalpha. Synovial tissue samples were obtained from 6 patients with RA and 3 patients with OA, and syndecan-4 expression was analysed by immunohistochemistry. In addition, SF were isolated from these tissues and mRNA and surface expression of syndecan-4 was determined by quantitative PCR and immunocytochemistry. The regulation of syndecan-4 expression by TNFalpha (10 ng/ml) was determined by quantitative PCR in RASF and OASF. Analysis of SF from arthritic human TNFalpha transgenic (hTNFtg) mice that constitutively overexpress TNFalpha and from arthritic tristetraprolin (TTP)-/- mice that are exposed to high TNFalpha levels in vivo but do not express this cytokine were performed to determine the expression of syndecan-4 mRNA following long-term exposure to TNFalpha. The role of syndecan-4 in the synthesis of MMPs was analysed by measuring MMP-1 and -3 levels by ELISA in untransfected, syndecan-4 siRNA transfected and mock-transfected RASF. High expression of syndecan-4 was found in all RA synovial tissues, while only negligible staining was seen in OA tissues. As determined by quantitative PCR, RASF exhibited a 4-fold higher expression of syndecan-4 than OASF, and this was seen also in immunocytochemistry. Stimulation of RASF with TNFalpha resulted in a 17-fold up regulation of syndecan-4 mRNA while induction of syndecan-4 was only 9-fold in OASF (p<0.05). Compared to their wild-type controls, hTNFtg SF showed a 36-fold upregulation of syndecan-4, while syndecan-4 was upregulated 23- fold in TTP-/- fibroblasts. Knockdown of syndecan-4 by siRNA in RASF results in a significant downregulation of the production of MMP-1 (27%, p<0.05) and -3 (39%, p<0.05). Our data suggest a disease-specific and stable upregulation of syndecan-4 in human RASF even without continuous high levels of TNF-alpha. Syndecan-4 seems to be involved in the regulation of MMP-1 and -3 levels and knock-down of syndecan-4 causes a decrease in MMP synthesis. Therefore, syndecan-4 may constitute an interesting target for interfering with fibroblast activation and matrix degradation in RA.

202 LEVEL AND CHARACTERISTICS OF NATURAL T REGULATORY CELLS IN VASCULAR-CONNECTIVE AUTOIMMUNE DISEASES

L. Banica1, A. Besliu1, G. Pistol1, C. Stavaru1, R. Ionescu2, C. Tanaseanu3, I. Tamsula3, M. Stefanescu1, C. Matache1.1Cellular Receptor Laboratory, Center for Advanced Studies, Cantacuzino NIRDMI; 2Carol Davila UMF; 3Sf. Pantelimon Emergency Hospital, Bucharest

The contribution of natural T regulatory (Tr) cells to the development of autoimmunity became most evident in the last years. However, there is not enough information regarding the role of natural Tr cells in the development of vascular-connective autoimmune diseases. Natural Tr cells constitute a homogenous population, derived from thymus, phenotypically characterized by CD25high CTLA-4+ GITRhigh CD45RO+, and molecular marker FOXP3. Cells with this phenotype are regulatory/suppression cells and play a major role in the maintaining of immune tolerance. Based on this evidence, we initiated a study on some vascular-connective autoimmune diseases [Systemic Lupus Erythematosus (SLE), Systemic sclerosis (Sc) and Sjögren’s Syndrome (SS)] in order to evaluate the level of natural Tr cells in peripheral blood mononuclear cells. For this purpose, FACS analysis was used. Preliminary results, in comparison with healthy subjects, suggested that in all of these pathologies there is a reduced level of natural Tr cells, more significant in SLE and SS patients. Important alterations in the expression of GITR and intracellular CTLA-4 receptors on Tr cells of SLE and Sc patients were found.

203 INCREASED NITRIC OXIDE PRODUCTION REGULATES T CELL FUNCTION IN RHEUMATOID ARTHRITIS

G. Nagy1, J. M. Clark2, E. Buzas1, C. Gorman2, A. Falus1, A. P. Cope2.1Department of Rheumatology, and Department of Genetics, Cell and Immunbiology, Semmelweis University, Medical School, Budapest, Hungary; 2Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, London, UK

Experimental and clinical evidence for T cell involvement in the pathology of rheumatoid arthritis (RA) is compelling, and points to a local dysregulation of T cell function in the inflamed joint. Nitric oxide (NO) has been shown to regulate T cell function under physiological conditions, but overproduction of NO may contribute to lymphocyte dysfunction in RA. Impaired responses to stimulation with antibodies to the CD3 complex of the T cell receptor (TCR), as well as decreased production of interleukin-2 (IL-2) and interferon-γ (IFN-γ) following activation, have been described in RA patients. It has been observed that the expression of the signaling chain subunit of the TCR/CD3 complex, the TCR ζ chain, is downregulated in T lymphocytes of RA patients. Chronic TNF-α treatment appears to reproduce many of the TCR signaling defects observed in T cells from RA patients, suppress a broad range of T cell responses and attenuates intracellular Ca2+ mobilization. Several studies carried out on patients with rheumatoid arthritis have documented increased endogenous NO synthesis, but its contribution to T cell dysregulation is not known. We investigated the possible role of NO in T cell dysfunction in RA.

Our present data indicate that T cells from RA patients produce >2.5 times more NO than control healthy donor T cells (p<0.001). Although NO is an important physiological mediator of mitochondrial biogenesis, mitochondrial mass is similar in RA and control T cells (p = 0.65), whilst increased NO production is associated with increased cytoplasmic Ca2+ concentrations in RA T cells (p<0.001). We observed that T cell NO production decreased in most RA patients following anti-TNF treatment. TNF treatment of Jurkat cells (10–50 ng/ml) induces dose dependent NO production (p<0.001). Furthermore, chronic NO treatment, like TNF, downregulates TCRζ expression. Experiments also indicate that NO seems to regulate TNF induced apoptosis. These data suggest that TNF induced NO production in T lymphocytes is a key modulator of T cell responses, and its overproduction may contribute to perturbations of immune homeostasis in RA.

204 THERAPEUTIC INTRAVENOUS IMMUNOGLOBULIN INCREASES THE EXPRESSION OF FCγIIB RECEPTORS ON B CELLS FROM LUPUS PRONE MRL/LPR MICE

K. Nikolova, A. Tchorbanov, I. Djoumerska, T. Vassilev.Department of Immunology, Institute of Microbiology, The Bulgarian Academy of Sciences, Sofia, Bulgaria

Infusion with normal pooled IgG (intravenous immunoglobulin, IVIg) has been shown to be beneficial to patients with various autoimmune diseases. Its mechanism of action is complex. It has been previously reported that exposure to IVIg increases the expression of the inhibitory FcγIIB receptor on macrophages in a mouse model of ITP (Science, 2001, 291, 484). We find that IVIg enhances in a dose dependent manner the expression of FcγIIB on B cells from lupus prone mice. We show further that the administration of Fc fragments of IVIg and of pooled human IVIgM has no effect. However, the (Fab)2 fragments of IVIg have the same effect as the intact IVIg. We have shown previously that chimeric antibodies able to cross-link FcγIIB receptors with DNA-specific BCRs on disease associated B lymphocytes, inhibited specifically their ability to produce antibodies against dsDNA. We hypothesize that the effect of these chimeras would be stronger if they are administrated to animals pretreated with IVIg. To test this, we preincubated in vitro splenocytes from sick MRL/lpr mice with increasing concentrations of IVIg and incubated them with an antibody chimera. Our results show that the preincubation of the spleen cells with IVIg increases the inhibitory effect of the chimeric molecule shown in an ELISPOT assays, measuring the number of DNA – binding IgG antibody producing plasma cells. Disease activity has been shown to depend on the balance from stimulatory and inhibitory Fc receptors (J Exp Med, 2006, 203, 789). We thus conclude that the modulation of the FcγIIB expression on lupus B cells could be clinically relevant.

Abstract 074 Table 1

Genotypes and modified Larsen score

Abstract 074 Table 2

Radiographic damage and genotypes stratified by CCP and RF status

Abstract 076 Table 1
Abstract 181 Table 1
Abstract 199 Table 1
Abstract 057 Figure 1

Pre- and postvaccination serum geometric mean titres (GMT with standard deviation) against influenza A/H3N2, A/H1N1 and Influenza B for a group of RA patients treated with rituximab (RA-RTX), compared to RA patients treated with anti-TNF (RA-TNF) and healthy controls (HC). *p = 0.02; **p<0.001.

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