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Extended report
Modulation of established murine collagen-induced arthritis by a single inoculation of short-term lipopolysaccharide-stimulated dendritic cells
  1. L Salazar1,
  2. O Aravena1,
  3. P Abello1,
  4. A Escobar1,
  5. J Contreras-Levicoy1,
  6. N Rojas-Colonelli1,
  7. D Catalán1,
  8. A Aguirre1,
  9. R Zúñiga1,
  10. B Pesce1,
  11. C González2,
  12. R Cepeda2,
  13. M Cuchacovich3,
  14. M C Molina1,
  15. F Salazar-Onfray1,
  16. M Delgado4,
  17. R E Toes5,
  18. J C Aguillón1
  1. 1
    Programa Disciplinario de Inmunología, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile
  2. 2
    Departamento de Patología Animal, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile
  3. 3
    Sección de Reumatología, Departamento de Medicina, Hospital Clínico Universidad de Chile, Santiago, Chile
  4. 4
    Instituto de Parasitologia y Biomedicina, CSIC, Granada, Spain
  5. 5
    Department of Rheumatology, Leiden University Medical Centre, Leiden, The Netherlands
  1. J C Aguillón, Programa Disciplinario de Inmunología, ICBM, Facultad de Medicina, Universidad de Chile. Av. Independencia 1027, Santiago, Chile; jaguillo{at}med.uchile.cl

Abstract

Background: The use of regulatory or immature dendritic cells (DCs) as tools for modulating experimental rheumatoid arthritis is very recent. Tumour necrosis factor (TNF)-stimulated DCs have been shown to restore tolerance in experimental autoimmune encephalomyelitis and collagen-induced arthritis (CIA).

Objective: We investigated the capacity of short-term lipopolysaccharide (LPS)-stimulated DCs pulsed with type II collagen (CII) to induce tolerance against established CIA.

Methods: Bone marrow-derived DCs were generated in the presence of granulocyte monocyte colony-stimulating factor (GM-CSF). After CIA induction, mice were injected at day 35 with a single dose of 4- or 24-h LPS-stimulated DCs that had been loaded with CII (4hLPS/CII/DCs or 24hLPS/CII/DCs). Arthritis progression was monitored by clinical and histological evaluations.

Results: Flow cytometry of 4hLPS/CII/DCs showed intermediate CD40 and CD86 expression, lower than that of 24hLPS/CII/DCs (fully mature) and higher than that of CII/DCs (immature). A functional assay showed that 4hLPS/CII/DCs display increased endocytosis ability with respect to 24hLPS/CII/DCs, indicating a semimature state. The single inoculation of 4hLPS/CII/DCs in mice with established CIA reduced disease severity significantly over time. Histological evaluation of mice treated with 4hLPS/CII/DCs revealed diminished inflammatory synovitis, cartilage damage and fibrosis. Co-cultures of DCs with splenocytes from CIA mice showed that collagen-specific interferon (IFN)γ production was dramatically inhibited by 4hLPS/CII/DCs. 4hLPS/CII/DCs were high IL10 producers, which could explain the inhibition of arthritis progression in mice receiving this treatment because neither antibodies nor regulatory CD4+CD25+Foxp3+ T lymphocytes were demonstrated to be involved.

Conclusion: Short-term LPS-modulated DCs inoculation interferes with CIA progression when loaded with CII.

http://dx.doi.org/10.1136/ard.2007.072199

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    In rheumatoid arthritis (RA) pathogenesis, there is consensus that the autoimmune process involves altered self-antigen presentation by antigen-presenting cells (APCs) or dendritic cells (DCs) leading to the activation of auto-reactive CD4+ T lymphocytes.1 Antigen-activated CD4+ T lymphocytes are the main mediators of inflammation and articular damage through the secretion of interleukin (IL)-17, which activates the release of IL1, IL6 and tumour necrosis factor (TNF) by macrophages.2 3

    Among several arthritogenic antigens, the participation of endogenous proteins such as type II collagen (CII) is well demonstrated.4 Moreover, inoculation of the genetically susceptible DBA/1 mouse strain with bovine CII has allowed the development of the collagen-induced arthritis (CIA) murine model, the most extensively applied experimental model of RA.5

    Current data indicate that DC functions are related to the stage of their maturation. After an antigenic stimulus, immature DCs become fully mature DCs, as shown by upregulation of major histocompatibility complex (MHC) and costimulatory molecules, such as CD80 and CD86.6 Unlike mature DCs, immature DCs are poor APCs, and they have been found to have antigen-specific regulatory properties.7 Thus, DCs play a central role in orchestrating the immune response and inducing tolerisation of immune responses against self and non-self antigens.8

    To promote tolerance, DCs must capture, process and present self antigens in the “steady state”, an immature form of differentiation involved in the induction of peripheral T cell tolerance.9 10 Therefore, the generation of tolerogenic or regulatory DCs in the laboratory has become the focus of new therapies for inhibiting the unwanted immune responses responsible for autoimmunity.11 In order to induce and keep a regulatory phenotype of DCs, several pharmacological strategies have been used, including agents such as IL10, TNF and the vasoactive intestinal peptide (VIP), among others.12–15

    Regulatory DCs have been used to restore tolerance in experimental autoimmune encephalomyelitis (EAE) and CIA.13 14 Hence, repetitive injections of DCs matured with TNF-induced antigen-specific protection of mice from EAE13 and CIA.14

    In this work, we investigate the ability of immature DCs, short-term lipopolysaccharide (LPS)-stimulated DCs (semimature DCs) and long-term LPS-stimulated DCs (fully mature DCs), all of them collagen-pulsed, to induce tolerance in mice with established CIA. Our data confirms that DCs can be differentially modulated by using a wide spectrum of biological agents, but most importantly, it indicates a strong influence of the duration of stimulus.

    METHODS

    Mice

    We used 7–8-week-old DBA1/lacJ (H2q) mice, obtained from Jackson Laboratories (Bar Harbor, ME, USA) and maintained in accordance with international guidelines for animal care; protocols were approved by the University of Chile Bioethics Committee.

    Bone marrow (BM)-derived DCs

    BM cells were cultured for 7 days in medium containing granulocyte monocyte colony-stimulating factor (GM-CSF).16 17 After 6 days of culture, DCs were loaded for 24 h with bovine CII protein (Chondrex, Redmond, Washington, USA) (CII/DCs) or were left unloaded. Then, DCs were removed and used as immature DCs (untreated DCs) or they were stimulated with 1 μg/ml of LPS (Escherichia coli, serotype 026:B6, Sigma, St Louis, Missouri, USA) for 4 h (CII loaded: 4hLPS/CII/DCs or unloaded: 4hLPS/DCs) or for 24 h (CII loaded: 24hLPS/CII/DCs or unloaded: 24hLPS/DCs), respectively.

    Antibodies and fluorescence-activated cell-sorting (FACS) analysis

    Antibodies were purchased from eBiosciences (San Diego, California, USA): fluorescein isothiocyanate (FITC)-labelled anti-CD11c, anti-CD86, anti-CD80, anti-MHC class II (AI/EI) and anti-CD40. Differentially treated DCs were incubated with the appropriate antibodies, fixed in paraformaldehyde and analysed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, California, USA).

    Endocytosis assays

    DCs, 2×105, were incubated with 1 mg/ml of the endocytosis marker FITC-conjugated dextran (Molecular Probes, Leiden, The Netherlands) for 30 min at 37°C or at 4°C (control) and then analysed by FACS. Mean fluorescence intensity values within the gate for the different endocytically active stages were plotted.18

    Cytokine determination in non-purified DCs

    Cytokines were captured by specific anti-IL10 or anti-IL12p70 monoclonal antibodies (eBioscience), detected by biotinylated anti-cytokine antibodies and revealed by luminescence (Luminoskan Ascent; Thermo, Vantaa Finland).

    Cytokine determination in purified DCs

    DCs were washed in PBS/0.1% BSA and stained with FITC-labelled anti-mouse CD11c antibody (Becton Dickinson). CD11c+ cells were sorted in a FACSCalibur sorter (Becton Dickinson). Sorted CD11c+ cells (>98%) were differentiated from untreated DCs, 24hLPS/DCs and 4hLPS/DCs, and cytokine contents in supernatants were assayed by ELISA (eBioscience).

    CIA induction and clinical evaluation of arthritis

    Bovine CII protein was emulsified with complete Freund adjuvant, and then injected subcutaneously into the base of the tail (100 μg CII/mouse). Immunisation was boosted 3 weeks later by a subcutaneous injection of 100 μg of CII. Beginning 3 weeks after immunisation, mice were clinically examined 3 times each week. Arthritis severity was graded as follows: 0 = normal joints, 1 = 1 or 2 swollen joints, 2 = 2–4 swollen joints, 3 = >4 swollen joints and 4 = extreme swelling of the entire paw and/or ankylosis. An arthritis score was calculated by adding the scores for each paw/mouse.

    DCs inoculation

    Differentially treated DCs (5×105) were intraperitoneally administered to 4–6 mice per group by a single inoculation at day 35 after the first inoculation of CII.

    DC and splenocyte co-cultures

    Splenocytes from CIA mice were co-cultured with 4hLPS/CII/DCs, 24hLPS/CII/DCs, CII/DCs or untreated DCs for 7 days.19 Then, 100 000 cells, isolated from the co-cultures, were co-cultured with 20 000 differentially treated DCs in a 96-well enzyme-linked immunosorbent spot (ELISPOT) assay plate (Millipore, Billerica, Massachusetts, USA), coated with an anti-mouse IFNγ monoclonal antibody (eBiosciences). After adding a secondary anti-IFNγ antibody, the reaction was revealed with streptavidin-alkaline phosphatase (Mabtech, Cincinatti, Ohio, USA). Spots representing IFNγ secreted by a single cell were counted.

    Histological analysis

    Tissue sections were cut and stained with haematoxylin/eosin. A morphometric analysis was performed evaluating all joints in the paws with the most evident clinical damage. Synovial membrane leukocyte infiltration was determined as an index (%), calculating the proportion of the area corresponding to cellular nuclei.20 The loss of articular cartilage by destruction was graded on a scale of 0–5 (0 = normal cartilage, 1 = minimum damage in chondrocytes, 2 = moderate chondrocyte death, 3 = moderate chondrocyte death and loss of articular surface, 4 = active chondrocyte death and articular surface damage and 5 = maximum chondrocyte death and cartilage erosion). Fibrosis was evaluated by a score ranging from 0–3 (0 = normal tissue, 1 = 1–3 fibrous layers, 2 = 4–10 fibrous layers or 1–4 fibrous foci and 3 = >5 fibrous foci).

    Antigen-specific antibodies in serum by ELISA

    Microtitration plates, sensitised with bovine CII protein (Chondrex), were incubated with mice serum and then treated with anti-mouse-specific subclass antibodies.

    Statistical analysis

    Differences in disease severity were analysed by the Mann–Whitney U test. A one-way analysis of variance (ANOVA) was used for other comparisons. A p value <0.05 was considered statistically significant with a 95% CI.

    RESULTS

    Influence of LPS on DC maturation

    DCs were obtained from BM cells after 7 days of culture in medium containing GM-CSF, with a yield of approximately 60%. Figure 1A shows that these cells expressed CD11c, a characteristic marker for DC populations, and CD11b specific for myeloid lineage.

    Figure 1
    Figure 1 Surface phenotype and endocytosis assay of lipopolysaccharide (LPS)-treated dendritic cells (DCs). A. DCs were generated from DBA1/lacJ mice derived from bone marrow. After 7 days of culture in medium containing granulocyte macrophage colony-stimulating factor (GM-CSF), the expression of CD11c and CD11b was analysed by fluorescence-activated cell sorting (FACS). B. FACS analysis shows the expression of major histocompatibility complex class II and costimulatory molecules (CD40 and CD86). DCs loaded with bovine type II collagen (CII) were stimulated for 4 h (4hLPS/CII/DCs) or 24 h (24hLPS/CII/DCs) with LPS and compared with CII-loaded immature DCs (CII/DCs). Immature DCs (untreated DCs), 4-h and 24-h LPS-stimulated DCs, left unloaded (4hLPS/DCs and 24hLPS/DCs) were used as controls, respectively. Values are expressed as mean fluorescence intensity. C. Mean fluorescence intensity values for endocytically active immature DCs cultured for 7 days. DCs were differentially treated as mentioned in B. Values are expressed as endocytosis index (values obtained at 4°C/values obtained at 37°C). DCs were incubated with FITC-conjugated DEXTRAN (40 kDa). Data from a representative experiment (of three experiments performed) are shown.

    To study the response of DCs to various maturation stimuli, DCs obtained from DBA1/lacJ mice were previously loaded with bovine CII or left unloaded, and then subjected to LPS for 4 or 24 h as mentioned above. Untreated DCs were used as immature controls. After the different stimuli and incubation times, the cells were harvested and analysed by FACS for the expression of MHC class-II and costimulatory molecules (CD40 and CD86) (fig 1B). For all stimulatory or loading treatments, DCs showed equivalent levels of MHC class II. Interestingly, 4hLPS/CII/DCs displayed a lower level of CD40 and CD86 expression than 24hLPS/CII/DCs (p<0.01). However, these levels were similar to those of untreated DCs, which suggest that 4hLPS/CII/DCs correspond to a transition between immature and mature stages of phenotypic differentiation.

    The functional ability of DCs to take up the endocytosis marker FITC–dextran was also examined, although none of the cells analysed demonstrated significantly differential endocytosis abilities (fig 1C). Untreated DCs and CII/DCs displayed the highest levels of endocytosis compared with 4hLPS/CII/DCs, which showed a diminished endocytosis level, indicating a semimature transition state of these cells. 24hLPS/CII/DCs and 24hLPS/DCs both showed a diminished endocytotic ability, characteristic of functionally mature DCs. Thus, phenotypic and functional data indicate that 4hLPS/CII/DCs represent a semimature stage of DCs with the ability to present CII to naïve T lymphocytes.

    Cytokine determination

    Using non-purified DCs, we detected that 4hLPS/CII/DCs and 4hLPS/DCs showed a higher IL10 production than 24hLPS/CII/DCs (p<0.05) and untreated DCs (p<0.01) (fig 2A). All LPS-stimulated DCs showed a higher IL12 release when compared to untreated DCs (p<0.01), while no significant differences were detected between 4hLPS/DCs or 24hLPS/DCs loaded or unloaded with CII. In purified DCs, we observed that 4hLPS/DCs released higher IL10 levels than 24hLPS/DCs (p<0.001), while the IL12, IL6 and TNF levels released by 4hLPS/DCs were significantly lower than those released by 24hLPS/DCs (p<0.001) (fig 2B).

    Figure 2
    Figure 2 Cytokine detection in supernatants from bone marrow dendritic cells (DCs) from DBA1/lacJ mice. A. Non-purified DCs loaded with bovine type II collagen (CII) were stimulated for 4 h (4hLPS/CII/DCs) or 24 h (24hLPS/CII/DCs) with lipopolysaccharide (LPS) and compared with CII-loaded immature DCs (CII/DCs). Immature DCs (untreated DCs), 4-h and 24-h LPS-stimulated DCs, left unloaded (4hLPS/DCs and 24hLPS/DCs) were used as controls, respectively. Differentially stimulated DCs were washed and cultured for 24 h, and cytokine secretion was evaluated in supernatants. B. DCs, washed in phosphate buffered saline (PBS)/0.1% bovine serum albumin (BSA) and stained with fluorescein isothiocyanate (FITC)-labelled anti-mouse CD11c antibody, were sorted. Sorted CD11c+ cells (>98% as determined by flow cytometry) were incubated with medium (untreated DCs), with LPS for 4 h (4hLPS/DCs) or with LPS for 24 h (24hLPS/DCs). Cytokine contents in supernatants were assayed by ELISA. Data are the mean (SD) of three experiments performed in duplicate.

    Modulation of CIA by immunisation with 4-h LPS-treated DCs loaded with collagen

    To evaluate the ability of the DCs to modulate CIA in an antigen-specific manner, they were pulsed with CII. After inducing active CIA, DBA1/lacJ mice were immunised at day 35 with differentially treated DCs. The progression and onset of the disease in the groups treated with CII/DCs, 4hLPS/DCs or 24hLPS/CII/DCs were similar to those observed in the control mice group immunised with CII/CFA alone (fig 3A,B) (p>0.05). By contrast, mice treated with 4hLPS/CII/DCs displayed significantly less-severe clinical disease as compared to the other groups over time (fig 3A,B) (p<0.001). Additionally, the injections with untreated DCs and 24hLPS/DCs were unable to modify the natural progression of CIA (data not shown). In addition, two injections (at days 28 and 35) with 4hLPS/CII/DCs resulted in significantly less severe clinical disease as compared to the other groups over time (p<0.001). Although two 4hLPS/CII/DC injections interfered slightly more with CIA progression than one injection, no significant differences were observed.

    Figure 3
    Figure 3 Modulation of active collagen-induced arthritis (CIA) in DBA1/lacJ mice by an injection of 4-h lipopolysaccharide (LPS)-stimulated immature dendritic cells (DCs). Once CIA was induced with bovine type II collagen (CII) added with Freund complete adjuvant (CFA), mice were inoculated intraperitoneally at day 35 after the first CII administration with 5×105 DCs as follows: CII DCs, immature DCs loaded with CII; 4hLPS/CII/DCs, 4-h LPS-stimulated DCs and loaded with CII; 24hLPS/CII/DCs, 24-h LPS-stimulated DCs loaded with CII; 4hLPS/DCs, 4-h LPS-stimulated DCs CII unloaded; CIA corresponds to control mice that did not receive any DCs injection (they were only immunised with CII and CFA for CIA induction). The mean arthritis severity score was determined until day 70. Arthritis severity was evaluated by two independent and blinded observers. A. Swollen joint severity was measure by a calliper in each group over time. Mice given 4hLPS/CII/DCs had lower scores compared with all other groups (*p<0.001). B. Joint score, shown on an index from 0 to 16 by animal. Treatment with 4hLPS/CII/DCs showed a lower mean joint score (*p<0.001). Data from a representative experiment of three experiments performed; n = 5–6 mice per group. Arrows indicate DC injection at day 35.

    Histological evaluation

    Histological examination of joints, as shown in fig 4A, revealed that control CIA mice had more severe inflammatory synovial microscopic changes. Morphometric analysis indicated a significantly lower (p<0.005) inflammatory cell infiltration index for 4hLPS/CII/DCs mice compared to joints from control CIA mice or from 24hLPS/CII/DCs mice (fig 4B). The group treated with 4hLPS/CII/DCs showed a similar leukocyte distribution in the inflammatory cell infiltrate including macrophages, lymphocytes and neutrophils as compared to CIA and 4hLPS/DCs groups (data not shown). The evaluation of cartilage damage indicated that mice treated with 4hLPS/CII/DCs showed significantly less damage than the CIA group, the 24hLPS/CII/DCs group or the group inoculated with 4hLPS/DCs (fig 4C) (p<0.01). The fibrosis damage analysis revealed that mice inoculated with 4hLPS/CII/DCs displayed a moderate fibrosis score when compared with the other groups (fig 4D) (p = 0.05).

    Figure 4
    Figure 4 Histopathological analysis of joints after inoculation with 4-h lipopolysaccharide (LPS)-stimulated immature dendritic cells (DCs). Collagen-induced arthritis (CIA) was induced in DBA1/lacJ mice, which at day 35 were inoculated intraperitoneally with differentially treated 5×105 DCs. A. Normal: joint with a conserved synovial space; bovine type II collagen (CII) CIA: control mice with pannus infiltration, diminished synovial space and cartilage damage; CIA DCs +4-h LPS: massive cellular infiltration and severe synovitis; CIA DCs CII +4-h LPS: moderate cellular infiltration and mild synovitis. The specimens were stained with haematoxylin and eosin and represent original magnification 40× and 200×. Paw sections were examined by two independent observers who were blinded to the experimental group. B. Cellular infiltration index in the synovial membrane. Normal mice show absence of cell infiltration while the group of mice treated with 4-h LPS-stimulated and CII-loaded DCs showed significant lesser cellular infiltration than CIA and 4-h LPS-treated CII-unloaded DCs (**p<0.005). C. Cartilage damage score was assigned using values between 0–5. Mice inoculated with 4-h LPS-stimulated and CII-loaded DCs showed less cartilage damage (*p<0.01). D. Fibrosis damage score. Mice treated with 4-h LPS-stimulated and CII-loaded DCs displayed moderate fibrosis (*p = 0.05).

    Inhibition of splenocyte IFNγ production by 4-h LPS-treated DCs loaded with collagen

    In order to evaluate the inhibitory activity of 4hLPS/CII/DCs on the effector cells involved in CIA induction, splenocytes from CIA mice were co-cultured with differentially stimulated DCs and the IFNγ secretion was assessed. As depicted in fig 5, splenocytes co-cultured with 4hLPS/CII/DCs showed a dramatically diminished IFNγ production when compared to splenocytes co-cultured with 24hLPS/CII/DCs, CII/DCs or untreated DCs (p<0.001). All naïve DCs were able to induce IFNγ production by splenocytes (including 4hLPS/DCs and 24hLPS/DCs). However, DCs/CII and 24hLPS/CII/DCs were able to induce a higher IFNγ production than CII-unloaded DCs (p<0.05) and than 24hLPS/DCs (p<0.01), unlike 4hLPS/CII/DCs.

    Figure 5
    Figure 5 Collagen-specific interferon (IFN)γ production inhibition in splenocytes co-cultured with 4-h lipopolysaccharide (LPS)-stimulated immature dendritic cells (DCs). Splenocytes (spl) from mice with established collagen-induced arthritis (CIA) (day 70) were co-cultured with 4-h LPS-stimulated DCs and loaded with type 2 collagen (CII) (4hLPS/CII/DCs), 24-h LPS-stimulated DCs loaded with CII (24hLPS/CII/DCs), immature DCs loaded with CII (CII/DCs) or untreated DCs (DCs) for 7 days. A total of 100 000 cells, isolated from the co-cultures, were placed into a 96-well, flat-bottomed enzyme-linked immunosorbent spot (ELISPOT) assay plate, coated with 2 μg/ml of anti-mouse IFNγ monoclonal antibody. Then, 20 000 differentially treated DCs were added, and the plate was incubated with a secondary biotinylated monoclonal antibody specific for bound IFNγ. After alkaline phosphatase colour development, spots representing IFNγ secreted by a single cell were counted.

    Additionally, in order to investigate whether the injection with differentially treated DCs could affect the mouse antibody immune response, we measured CII-specific IgG1, IgG2a and IgG2b mouse antibodies in serum obtained from mice at day 70, after CIA induction. No differences were detected for IgG1, IgG2a or IgG2b mouse subclasses (data not shown).

    Finally, in order to determine whether CD4+CD25+Foxp3+ regulatory T lymphocytes (Treg) were responsible for the effect of 4hLPS/CII/DCs injection on CIA progression, intracellular Foxp3 expression was assessed by FACS at day 36, 38, 42 and 49 after the injection of DCs treated with the different stimuli. Although not significant, we observed that the percentage of Treg cells was greater in spleen and lymph nodes from mice treated with 4hLPS/CII/DCs than other groups at day 42 and 49 (data not shown).

    DISCUSSION

    Different approaches for modulating DCs in an antigen-specific manner have been successfully developed, including the establishment of human and murine-modified DCs with potent immunoregulatory properties.21–23 In autoimmune disease models, vaccination with TNF-modulated DCs has been shown to prevent EAE and CIA.13 14 The use of VIP has succeeded in reducing the progression of CIA and EAE15 and significantly ameliorating the clinical and histopathological severity of murine colitis.24 More recently, the administration of LF 15-0195-treated DCs, a nuclear factor (NF)-κB inhibitor, has been shown to interfere with CIA, improving clinical score and reducing antibody response to the CII.25

    In this study, we have demonstrated that DCs can affect the outcome of CIA in an antigenic-specific manner. Inoculation of mice with a single injection of 4hLPS/CII/DCs, after established CIA, led to less severe arthritis as determined by swollen joint severity and joint score. The histological evaluation allowed us to conclude that mice treated with 4hLPS/CII/DCs revealed significantly less inflammatory synovitis, pannus formation, cartilage damage and fibrosis compared with the other study groups. Additionally, the ex vivo antigen-specific IFNγ production blockade by 4hLPS/CII/DCs suggests that the inhibition of arthritis progression observed in mice treated with 4hLPS/CII/DCs could be a consequence of in vivo IFNγ production inhibition, responsible for damage observed in CIA. In addition to the report of Morita et al, who with a single injection of IL4-transduced DCs, reduced the incidence and severity of CIA, ours is the first report ever published using the inoculation of regulatory antigen-pulsed DCs as a therapeutic approach.26

    Sorted 4hLPS/DCs secreted lower levels of IL12p70, IL6 and TNF than those detected in 24hLPS/DCs, suggesting that 4hLPS/DCs could have the ability to polarise the immune response in a pro-tolerogenic fashion. However, 4hLPS/DCs and 4hLPS/CII/DCs showed a higher secretion pattern of IL10 (in sorted and non-sorted cells), a key cytokine in modulating and regulating in vivo and ex vivo immune responses, indicating that this mechanism may be responsible for CIA blockade observed in mice injected with 4hLPS/CII/DCs as a consequence of the IL10 inhibitory effect on IFNγ-producing T cells, responsible for CIA damage. These data agree with previous reports involving specific DC subsets and IL10 as mediators of tolerance by the induction of regulatory T cells or by anergy.27 28

    Thus, in the EAE model, Menges et al found that vaccination with peptide-pulsed TNF-stimulated DCs led to the induction of IL10-producing CD4+ T cells and an inhibited generation of autoantigen-specific Th1 cells.13 Chorni et al demonstrated that the progressive reduction observed in EAE and CIA, after the inoculation of VIP-treated DCs, was mediated by IL10-producing T lymphocytes (Tr1).15 Additionally, Charbonnier et al have reported that repeated injections with immature DCs trigger the expansion of a novel CD4+ α-galactosylceramide/CD1d-nonrestricted/IL10-producing T cell regulatory population (CD49b+), able to protect mice from CIA.29 In our case, the evaluation of Treg after 4hLPS/CII/DCs injection showed that the percentage of CD4+CD25+Foxp3+ cells was greater in spleen and lymph nodes from mice treated with 4hLPS/CII/DCs than other groups at day 42 and 49, suggesting that Treg may be involved in the 4hLPS/CII/DCs-mediated CIA modulation. These results, together with our observation that 4hLPS/DCs expressed intermediate CD86 levels, are consistent with the finding reported by Yamazaki et al who demonstrate that DCs induced antigen-specific Treg from Foxp3(–) precursors. During this process, the CD86 expression proved to be essential for inducing IL2 production by T cells stimulated by DCs.30

    Alternatively, the effect of 4hLPS/CII/DCs injection could have been mediated by a clonal anergy mechanism triggered by the 4hLPS/DCs, displaying regulatory properties. This hypothesis is consistent with the fact that 2 months after the DC injection, the 4hLPS/CII/DCs-inoculated mouse group showed CIA progression re-establishment in 100% of the animals (data not shown). There is previous evidence of the induction of anergic and regulatory T cells by plasmacytoid DCs and other DC subsets.31 32 Thus, immature human IL10 DCs have shown to induce antigen-specific anergy by the interaction of CTLA-4 on T lymphocyte33 and indoleamine 2,3 dioxygenase production by DC subsets.27

    Although LPS has been identified as a maturation agent for DCs, there are reports demonstrating that, depending on the stimuli used to terminally mature BM-DCs, DCs synthesise a different pattern of cytokines and exhibit distinct T helper cell-driving potential.34 Thus, the combination of low doses of LPS and cholera toxin is able to induce IL10 production of immature DCs, characterised by enhanced expression of CD80 and CD86 but inhibited CD40 expression and inflammatory cytokines production.35 Therefore, our findings describing a phenotypic and functional intermediate stage for 4hLPS/DCs, in addition to their high IL10 production and in vivo modulating abilities, are in agreement with data previously reported.

    Differentiation of naïve CD4+ T cells into IFNγ-producing Th1 cells is critical for the generation of IgG2a antibodies in mice. We showed that 4hLPS/CII/DCs injection was unable to modify the established Th1 pattern in CIA mice. Since no Th1/Th2 cytokine profiles were determined, this was only shown by the absence of differences in the IgG2a CII-specific antibodies in the different mouse groups, reflecting that our strategy failed to shift the established Th1 response, unlike treatments to prevent CIA.

    Finally, while 4hLPS/DCs expressing a regulatory phenotype may display a transient stage of maturation with the ability to interfere in the cellular processes operating in autoimmune processes such as arthritis, the molecular mechanisms involved remain to be elucidated.

    REFERENCES

    1. ↵
      1. Santiago-Schwarz F,
      2. Anand P,
      3. Liu S,
      4. Carsons SE
      . Dendritic cells (DCs) in rheumatoid arthritis (RA): progenitor cells and soluble factors contained in RA synovial fluid yield a subset of myeloid DCs that preferentially activate Th1 inflammatory-type responses. J Immunol 2001;167:1758–68.
      OpenUrlAbstract/FREE Full Text
    2. ↵
      1. Harrington LE,
      2. Mangan PR,
      3. Weaver CT
      . Expanding the effector CD4 T-cell repertoire: the Th17 lineage. Curr Opin Immunol 2006;18:349–56.
      OpenUrlCrossRefPubMedWeb of Science
    3. ↵
      1. Hirota K,
      2. Hashimoto M,
      3. Yoshitomi H,
      4. Tanaka S,
      5. Nomura T,
      6. Yamaguchi T,
      7. et al
      . T cell self-reactivity forms a cytokine milieu for spontaneous development of IL-17+ Th cells that cause autoimmune arthritis. J Exp Med 2007;204:41–7.
      OpenUrlAbstract/FREE Full Text
    4. ↵
      1. Rosloniec EF,
      2. Brand DD,
      3. Myers LK,
      4. Whittington KB,
      5. Gumanovskaya M,
      6. Zaller DM,
      7. et al
      . An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen. J Exp Med 1997;185:1113–22.
      OpenUrlAbstract/FREE Full Text
    5. ↵
      1. Brand DD,
      2. Kang AH,
      3. Rosloniec EF
      . Immunopathogenesis of collagen arthritis. Springer Semin Immunopathol 2003;25:3–18.
      OpenUrlCrossRefPubMedWeb of Science
    6. ↵
      1. Lopez M,
      2. Aguilera R,
      3. Perez C,
      4. Mendoza-Naranjo A,
      5. Pereda C,
      6. Ramirez M,
      7. et al
      . The role of regulatory T lymphocytes in the induced immune response mediated by biological vaccines. Immunobiology 2006;211:127–36.
      OpenUrlCrossRefPubMedWeb of Science
    7. ↵
      1. Banchereau J,
      2. Steinman RM
      . Dendritic cells and the control of immunity. Nature 1998;392:245–52.
      OpenUrlCrossRefPubMedWeb of Science
    8. ↵
      1. Veeraswamy RK,
      2. Cella M,
      3. Colonna M,
      4. Unanue ER
      . Dendritic cells process and present antigens across a range of maturation states. J Immunol 2003;170:5367–72.
      OpenUrlAbstract/FREE Full Text
    9. ↵
      1. Hawiger D,
      2. Inaba K,
      3. Dorsett Y,
      4. Guo M,
      5. Mahnke K,
      6. Rivera M,
      7. et al
      . Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med 2001;194:769–79.
      OpenUrlAbstract/FREE Full Text
    10. ↵
      1. Steinman RM,
      2. Hawinger D,
      3. Nussenzweig MC
      . Tolerogenic dendritic cells. Annu Rev Immunol 2003;21:685–711.
      OpenUrlCrossRefPubMedWeb of Science
    11. ↵
      1. Hackstein H,
      2. Thomson AW
      . Dendritic cells: emerging pharmacological targets of immunosuppressive drugs. Nat Rev Immunol 2004;4:24–34.
      OpenUrlCrossRefPubMedWeb of Science
    12. ↵
      1. Steinbrink K,
      2. Wolfl M,
      3. Jonuleit H,
      4. Knop J,
      5. Enk AH
      . Induction of tolerance by IL-10-treated dendritic cells. J Immunol 1997;159:4772–80.
      OpenUrlAbstract
    13. ↵
      1. Menges M,
      2. Rossner S,
      3. Voigtlander C,
      4. Schindler H,
      5. Kukutsch NA,
      6. Bogdan C,
      7. et al
      . Repetitive injections of dendritic cells matured with tumor necrosis factor α induce antigen-specific protection of mice from autoimmunity. J Exp Med 2002;195:15–21.
      OpenUrlCrossRefPubMedWeb of Science
    14. ↵
      1. van Duivenvoorde LM,
      2. Louis-Plence P,
      3. Apparailly F,
      4. van der Voort EI,
      5. Huizinga TW,
      6. Jorgensen C,
      7. et al
      . Antigen-specific immunomodulation of collagen-induced arthritis with tumor necrosis factor-stimulated dendritic cells. Arthritis Rheum 2004;50:3354–64.
      OpenUrlCrossRefPubMedWeb of Science
    15. ↵
      1. Chorny A,
      2. Gonzalez-Rey E,
      3. Fernandez-Martin A,
      4. Pozo D,
      5. Ganea D,
      6. Delgado M
      . Vasoactive intestinal peptide induces regulatory dendritic cells with therapeutic effects on autoimmune disorders. Proc Natl Acad Sci USA 2005;102:13562–7.
      OpenUrlAbstract/FREE Full Text
    16. ↵
      1. Lutz MB,
      2. Kukutsch N,
      3. Ogilvie AL,
      4. Rossner S,
      5. Koch F,
      6. Romani N,
      7. et al
      . An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 1999;223:77–92.
      OpenUrlCrossRefPubMedWeb of Science
    17. ↵
      1. Winzler C,
      2. Rovere P,
      3. Rescigno M,
      4. Granucci F,
      5. Penna G,
      6. Adorini L,
      7. et al
      . Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures. J Exp Med 1997;185:317–28.
      OpenUrlAbstract/FREE Full Text
    18. ↵
      1. Lutz MB,
      2. Rovere P,
      3. Kleijmeer MJ,
      4. Rescigno M,
      5. Assmann CU,
      6. Oorschot VM,
      7. et al
      . Intracellular routes and selective retention of antigens in mildly acidic cathepsin D/lysosome-associated membrane protein-1/MHC class II-positive vesicles in immature dendritic cells. J Immunol 1997;159:3707–16.
      OpenUrlAbstract
    19. ↵
      1. Escobar A,
      2. Lopez M,
      3. Serrano A,
      4. Ramirez M,
      5. Perez C,
      6. Aguirre A,
      7. et al
      . Dendritic cell immunizations alone or combined with low doses of interleukin-2 induce specific immune responses in melanoma patients. Clin Exp Immunol 2005;142:555–68.
      OpenUrlPubMedWeb of Science
    20. ↵
      1. Bacus JW,
      2. Grace LJ
      . Optical microscope system for standardized cell measurements and analyses. Appl Optics 1987;26:3280–93.
      OpenUrlCrossRefPubMed
    21. ↵
      1. Sato K,
      2. Yamashita N,
      3. Baba M,
      4. Matsuyama T
      . Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells. Blood 2003;101:3581–9.
      OpenUrlAbstract/FREE Full Text
      1. Jonuleit H,
      2. Schmitt E,
      3. Schuler G,
      4. Knop J,
      5. Enk AH
      . Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J Exp Med 2000;192:1213–22.
      OpenUrlAbstract/FREE Full Text
    23. ↵
      1. Dhodapkar MV,
      2. Steinman RM,
      3. Krasovsky J,
      4. Munz C,
      5. Bhardwaj N
      . Antigen-specific inhibition of effector T cell function in humans after injection of immature dendritic cells. J Exp Med 2001;193:233–8.
      OpenUrlAbstract/FREE Full Text
    24. ↵
      1. Gonzalez-Rey E,
      2. Delgado M
      . Therapeutic treatment of experimental colitis with regulatory dendritic cells generated with vasoactive intestinal peptide. Gastroenterology 2006;131:1799–811.
      OpenUrlCrossRefPubMedWeb of Science
    25. ↵
      1. Popov I,
      2. Li M,
      3. Zheng X,
      4. San H,
      5. Zhang X,
      6. Ichim TE,
      7. Suzuki M,
      8. et al
      . Preventing autoimmune arthritis using antigen-specific immature dendritic cells: a novel tolerogenic vaccine. Arthritis Res Ther 2006;8:R141.
      OpenUrlCrossRefPubMed
    26. ↵
      1. Morita Y,
      2. Yang J,
      3. Gupta R,
      4. Shimizu K,
      5. Shelden EA,
      6. Endres J,
      7. et al
      . Dendritic cells genetically engineered to express IL-4 inhibit murine collagen-induced arthritis. disease. J Clin Invest 2001;107:1275–84.
      OpenUrlCrossRefPubMedWeb of Science
    27. ↵
      1. Munn DH,
      2. Sharma MD,
      3. Baban B,
      4. Harding HP,
      5. Zhang Y,
      6. Ron D,
      7. et al
      . GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 2005;22:633–42.
      OpenUrlCrossRefPubMedWeb of Science
    28. ↵
      1. Dillon S,
      2. Agrawal S,
      3. Banerjee K,
      4. Letterio J,
      5. Denning TL,
      6. Oswald-Richter K,
      7. et al
      . Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance. J Clin Invest 2006;116:916–28.
      OpenUrlCrossRefPubMedWeb of Science
    29. ↵
      1. Charbonnier LM,
      2. van Duivenvoorde LM,
      3. Apparailly F,
      4. Cantos C,
      5. Han WG,
      6. Noel D,
      7. et al
      . Immature dendritic cells suppress collagen-induced arthritis by in vivo expansion of CD49b+ regulatory T cells. J Immunol 2006;177:3806–13.
      OpenUrlAbstract/FREE Full Text
    30. ↵
      1. Yamazaki S,
      2. Bonito AJ,
      3. Spisek R,
      4. Dhodapkar M,
      5. Inaba K,
      6. Steinman RM
      . Dendritic cells are specialized accessory cells along with TGF-β for the differentiation of Foxp3+ CD4+ regulatory T cells from peripheral Foxp3- precursors. Blood 2007;110:4293–302.
      OpenUrlAbstract/FREE Full Text
    31. ↵
      1. Kuwana M
      . Induction of anergic and regulatory T cells by plasmacytoid dendritic cells and other dendritic cell subsets. Hum Immunol 2002;63:1156–63.
      OpenUrlCrossRefPubMedWeb of Science
    32. ↵
      1. Mandrekar P,
      2. Catalano D,
      3. Dolganiuc A,
      4. Kodys K,
      5. Szabo G
      . Inhibition of myeloid dendritic cell accessory cell function and induction of T cell anergy by alcohol correlates with decreased IL-12 production. J Immunol 2004;173:3398–407.
      OpenUrlAbstract/FREE Full Text
    33. ↵
      1. Steinbrink K,
      2. Graulich E,
      3. Kubsch S,
      4. Knop J,
      5. Enk AH
      . CD4(+) and CD8(+) anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity. Blood 2002;99:2468–76.
      OpenUrlAbstract/FREE Full Text
    34. ↵
      1. Morelli AE,
      2. Zahorchak AF,
      3. Larregina AT,
      4. Colvin BL,
      5. Logar AJ,
      6. Takayama T,
      7. et al
      . Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation. Blood 2001;98:1512–23.
      OpenUrlAbstract/FREE Full Text
    35. ↵
      1. Lavelle EC,
      2. McNeela E,
      3. Armstrong ME,
      4. Leavy O,
      5. Higgins SC,
      6. Mills KH
      . Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation. J Immunol 2003;171:2384–92.
      OpenUrlAbstract/FREE Full Text

    Footnotes

    • Funding: This study was supported by Fondecyt-Chile (104-0860 and 107-0553).

    • Competing interests: None declared.

    • Ethics approval: All protocols were approved by the University of Chile Bioethics Committee.

    • LS, OA, AE, JC-L, N R-C, DC, AA, RZ, F S-O and JCA are members of the Millennium Nucleus on Immunology and Immunotherapy – Chile (P04/030-F).