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Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients
  1. M Chemel1,2,3,
  2. B Le Goff1,2,3,
  3. R Brion1,2,3,
  4. C Cozic1,2,3,
  5. M Berreur1,2,
  6. J Amiaud1,2,
  7. G Bougras1,2,
  8. S Touchais3,
  9. F Blanchard1,2,
  10. M F Heymann1,2,
  11. J M Berthelot1,2,3,
  12. F Verrecchia1,2,
  13. D Heymann1,2,3
  1. 1INSERM, UMR 957, France
  2. 2Université de Nantes, Nantes Atlantique Universités, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Nantes, France
  3. 3Pôle Ostéoarticulaire, CHU de Nantes, Nantes, France
  1. Correspondence to Professor Dominique Heymann, INSERM UMR-S 957, Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, Faculté de Médecine, 1 rue Gaston Veil, 44035 Nantes cedex 1, France; dominique.heymann{at}univ-nantes.fr

Abstract

Objectives Interleukin (IL) 34 is a new cytokine implicated in macrophage differentiation and osteoclastogenesis. This study assessed IL-34 expression in the tissue of patients with rheumatoid arthritis (RA).

Methods Immunohistochemistry was performed in synovial biopsies from patients with RA (n=20), osteoarthritis (n=3) or other inflammatory arthritis (n=4). IL-34 was detected in the synovial fluid by ELISA and its messenger RNA expression was studied by quantitative PCR in rheumatoid synovial fibroblasts after stimulation by tumour necrosis factor α (TNFα) and IL-1β. Wild-type, jnk1−/−–jnk2−/− and nemo−/− murine fibroblasts and pharmacological inhibition were used to determine the involvement of nuclear factor kappa B (NF-κB) and JNK in that effect.

Results IL-34 was expressed in 24/27 biopsies, with three samples from RA patients being negative. A significant association was found between IL-34 expression and synovitis severity. Levels of IL-34 and the total leucocyte count in synovial fluid were correlated. TNFα and IL-1β stimulated IL-34 expression by synovial fibroblasts in a dose/time-dependent manner through the NF-κB and JNK pathway.

Conclusion This work for the first time identifies IL-34 expression in the synovial tissue of patients with arthritis. This cytokine, as a downstream effector of TNFα and IL-1β, may contribute to inflammation and bone erosions in RA.

http://dx.doi.org/10.1136/annrheumdis-2011-200096

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    Rheumatoid arthritis (RA) is an autoimmune disease characterised by chronic inflammation of the synovial tissue that leads to progressive joint destruction. Among the cells located in the inflamed joint, synovial fibroblasts are important players driving inflammation and bone erosion.1 They are recognised as a source of cytokines such as interleukin (IL) 6 or RANKL, which activate immune response and osteoclastogenesis. There is evidence that colony-stimulating factors play a major role in inflammation and bone destruction in RA.2 Macrophage colony-stimulating factor (M-CSF) is the primary regulator of the biology of mononuclear phagocytes and is also essential for osteoclastogenesis.3 In the synovial tissue of RA patients, it is expressed by macrophages but also by synovial fibroblasts and is upregulated by inflammatory cytokines such as tumour necrosis factor alpha (TNFα).2 4 Furthermore, M-CSF knockout mice are protected against collagen-induced arthritis, and M-CSF administration exacerbates inflammation and joint destruction.2 5 In this context, therapeutic targeting of M-CSF is currently being developed.2

    IL-34 is a newly discovered cytokine that plays a role in macrophage differentiation and proliferation.6 IL-34 is expressed in various tissues and it is most abundant in the spleen. This cytokine shares numerous common features with M-CSF, especially its receptor, partly explaining their functional overlap. However, their interaction with M-CSF receptor, the signalling pathway activated and their expression patterns differ during embryonic development underlining their probable specific biological function.7 We have recently shown that IL-34 could be substituted for M-CSF to promote osteoclastogenesis in vitro.8 Its role in inflammation is also likely, as IL-34 increases IL-6 and chemokine levels in human whole blood.9 Although numerous studies have underlined the role of M-CSF in arthitis, no data are currently available on the expression of IL-34 in RA.

    In this study, we hypothesised that IL-34 could be expressed by the synovial fibroblasts of RA patients and this expression could be modulated by TNFα and IL-1β.

    Methods

    Synovial fluid and biopsies

    Synovial biopsies were obtained surgically at the time of arthroplasty and are summarised in supplementary table S1 (available online only). All patients enrolled have given their formal consent. The study was approved by the local ethics committee and by the French Research Ministry (no 2008-402). The mean (±SD) duration of RA was 11±5 years and the mean synovitis score was 4.4±1.9 (range 2–8). The mean scores of synovial hyperplasia, stroma activation and inflammatory infiltrates were 1.3±1.2, 1.5±0.6 and 1.6±0.5, respectively (supplementary table S1, available online only). IL-34 expression was detected by immunohistochemistry performed as previously described.8 The histopathological severity of synovitis was graded as described by Krenn et al.10 IL-34 levels were measured in synovial fluids by ELISA assay (antibodies-online-GmbH, USA) according to the manufacturer's recommendations.

    Cell cultures

    Synovial fibroblasts, obtained from the synovial tissue of RA patients, human fibroblasts (WI-26) and murine wild-type, jnk1−/−–jnk2−/− and nemo−/− fibroblast cell lines were cultured as previously described.11,–,14

    Reverse transcription PCR analysis

    Total RNA was extracted using the NucleoSpin RNAII kit (Macherey-Nagel, Hoerdt, France) and 1 μg total RNA was used for first strand complementary DNA synthesis using the Thermoscript kit (Invitrogen, Paris, France).8 Real-time PCR was performed using SYBRGreen Supermix (Biorad, Marnes-La-Coquette, France) and primers described in supplementary table S2 (available online only).

    Statistical analysis

    The Mann–Whitney test was use to look for an association between IL-34 expression and histological characteristics. Correlation between the level of IL-34 and the leucocyte count in synovial fluid was measured using the non-parametric Spearman rank order test. Student's t-test was used to assess the change in gene expression; p<0.05 was considered statistically significant.

    Results

    IL-34 is expressed in the synovial tissue of RA and osteoarthritis patients

    We first assessed the expression of IL-34 in synovial tissue in synovial biopsies from patients with RA (n=20), osteoarthritis (n=3) or other inflammatory arthritis (n=4) (see supplementary table S1, available online only). According to the grading system of all the biopsies, 11 had slight synovitis, nine had moderate synovitis and seven had strong synovitis. IL-34 was detected in 24 of the 27 biopsies, with three samples from RA patients being negative. In the synovial tissue, IL-34 was expressed in the synovial lining layer by synoviocytes and macrophages (figure 1A,B) as confirmed by the double immmunostaining for IL-34 and CD68 (see supplementary figure S1, available online only) and in the sublining layer by endothelial cells and fibroblasts (figure 1C,D). In RA patients, a significant association was found between IL-34 expression in the synovial lining layer and the histological severity of synovitis, with a mean score of synovitis of 5.8±1.9 and 3.7±1.6 in IL-34-positive and negative biopsies, respectively (p=0.022; figure 1E). IL-34 expression within the synovial lining layer is also associated with synovial hyperplasia/enlargement: the mean score of hyperplasia was 2.2±0.9 and 0.6±1 in biopsies with and without IL-34-positive cells, respectively (p=0.004; figure 1F). No significant association was found between IL-34 expression and the diagnosis. Interestingly, IL-34 levels were significantly higher in synovial fluids of RA patients compared with osteoarthritis patients (p<0.05; figure 1G), and were associated with inflammation intensity measured by the leucocyte counts (r=0.82, p<0.001; figure 1H).

    Figure 1
    Figure 1

    IL-34 is expressed in the synovial tissue of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Representative immunohistochemical staining for IL-34 (red staining) (ab75723, Abcam, France) in synovial biopsy samples from patients with RA. (A–D) IL-34 was expressed in the synovial lining layer by synoviocytes (open arrow) (A), multinucleated giant cells (open triangle) and macrophages (asterisk) (B). In the sublining layer, endothelial cells (dotted arrow), inflammatory cells (triangle) and fibroblasts (arrow) were also positive for IL-34 (C, D). Comparison of the mean synovitis score (from 0, slight synovitis to 9, strong synovitis) (E) and mean hyperplasia of the lining layer score (from 0, no hyperplasia to 3, major hyperplasia) (F) according to the expression of IL-34 in the synovial lining layer; *p<0.05; **p<0.01 compared with the IL-34− group. (G) Comparative levels of IL-34 measured by ELISA (ref ABIN455583) in synovial fluids in osteoarthritis and RA patients, each plot being an individual sample, ***p<0.001. (H) Correlation between IL-34 levels and total leucocyte counts in the synovial fluids of patients with RA and osteoarthritis, r=0.82, p<0.0001.

    TNFα and IL-1β increase IL-34 gene expression in rheumatoid synovial fibroblasts

    We next assessed the expression of IL-34 by synovial fibroblasts in vitro and its regulation by TNFα and IL-1β. IL-34 messenger RNA was detectable in non-stimulated cells. Stimulation with TNFα resulted in a significant dose-dependent induction of IL-34 mRNA, with a plateau from 25 ng/ml and a maximum of induction after 6 h with 10 ng/ml (figure 2A). The time course study using 10 ng/ml TNFα shows that this effect remained stable until 24 h (data not shown). Similarly, IL-1β also dose-dependently increased IL-34 mRNA expression, with a peak reached at 6 h then decreasing quickly thereafter (figure 2B). Confocal microscopy (figure 2C) and flow cytometry (see supplementary figure S2, available online only) analyses confirmed that TNFα and IL-1β upregulated the expression by synovial fibroblasts of IL-34 at the protein level compared with untreated cells.

    Figure 2
    Figure 2

    Tumour necrosis factor alpha (TNFα) and IL-1β induce IL-34 mRNA expression in rheumatoid arthritis (RA) synovial fibroblasts. Synovial fibroblasts from RA patients (n=3) were stimulated with (A) increased dose of TNFα (1–50 ng/ml) for 6 h or with TNFα 10 ng/ml for 2 h, 6 h, 10 h and 24 h, and (B) increased dose of IL-1β (1–25 ng/ml) for 6 h or with IL-1β 10 ng/ml for 2 h, 6 h, 10 h and 24 h. After incubation, IL-34 mRNA levels were determined by real time reverse transcription PCR, normalised to GAPDH. (C) Synovial fibroblasts cultured on labtek chamber slides (Millipore, France) were treated or not with TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 24 h. IL-34 expression (green), actin filaments detected by alexa fluor 546-conjugated phalloidin (red), and nuclei stained by DAPI (blue) were observed by confocal microscopy. A representative experiment is shown.

    JNK and NF-κB activities are required for TNFα and IL-1β to stimulate IL-34 mRNA levels in fibroblasts and synoviocytes

    TNFα and IL-1β treatment of WI-26 fibroblasts led to a time and dose-dependent increase in IL-34 mRNA levels (figure 3A,B). Rapid and persistent induction of IL-34 mRNA levels was observed in response to TNFα (10 ng/ml; figure 3A) and IL-1β (10 ng/ml; figure 3B) peaking at 6 h and 10 h, respectively, and remaining at levels significantly higher than their basal expression state up to 24 h and 48 h. To understand the mechanisms by which TNFα and IL-1β are able to stimulate IL-34 mRNA levels, the role played by the JNK and nuclear factor kappa B (NF-κB) pathways was examined, respectively, in immortalised jnk−/− and nemo−/− fibroblasts (figure 3B,C) and in synoviocytes (figure 3D), with or without specific inhibitors of the JNK and NF-κB pathways. Results shown in figure 3C (left panel) indicate that the effects of TNFα on IL-34 gene expression was significantly inhibited both in nemo−/− and jnk−/− cells after 6 h. Finally, treatment of synoviocyte cultures with the specific IKKβ inhibitor or JNK inhibitor significantly inhibited (approximately 88% in the presence of 10 µM IKKV and 28% in the presence of 10 µM SP600125) the effect of 10 ng/ml TNFα (figure 3D) after treatment of the cells with 10 ng/ml TNFα (figure 3C) or IL-1β (data not shown). Confocal microscopy analyses confirmed the effects of signalling pathway inhibitors at the protein level (see supplementary figure S3, available online only).

    Figure 3
    Figure 3

    JNK and nuclear factor kappa B (NF-κB) activities are required for tumour necrosis factor alpha (TNFα) and IL-1β to stimulate IL-34 mRNA levels in fibroblasts and synoviocytes. WI-26 fibroblast cells were treated either with 10 ng/ml TNFα (A) or 10 ng/ml IL-1β (B) for 2 h, 6 h, 10 h, 24 h and 48 h (right panels) or with various concentrations of TNFα or IL-1β (1, 5, 10, 25 and 50 ng/ml) for 24 h, as indicated. After incubation, IL-34 mRNA steady-state levels were determined by real time reverse transcription (RT)-PCR. The expression of the houskeeping gene GAPDH was used as control. (C) Wild-type nemo−/− and jnk−/− fibroblasts were cultured in the presence or the absence of 10 µM JNK inhibitor (JNK II) or 10 µM IKKβ inhibitor V (IKKV). One hour later 10 ng/ml TNFα or IL-1β were added for 6 h. IL-34 mRNA steady-state levels were then determined by real time RT-PCR. Bars indicate mean±SD of two independent experiments performed, each with duplicate samples. *p<0.05; ***p<0.001 compared with the control. (D) Human synovial fibroblasts were treated with with or without 10 µM JNK inhibitor or IKKβ inhibitor V. One hour later 10 ng/ml TNFα or IL-1β were added for 6 h. After incubation, IL-34 mRNA steady-state levels were determined by real time RT-PCR. A representative experiment is shown.

    Discussion

    Pro-inflammatory cytokines, promoting inflammation and osteoclastogenesis in the arthritic joint, are fundamental to RA pathophysiology.15 In this study we demonstrated that a newly discovered cytokine, IL-34, is expressed by synovial fibroblasts and that TNFα and IL-1β stimulate its expression. The present report shows for the first time that IL-34 is expressed in the synovial tissue of patients with arthritis, mainly by the cells of the synovial lining layer and to a lesser extent by fibroblasts and endothelial cells in the sublining layer. Our data support synovial fibroblasts being a source of this cytokine as revealed by IL-34 mRNA and protein expression in vitro. Importantly, our data showed that the expression of IL-34 in the synovial lining layer was associated with the severity of synovitis. IL-34 may have a pro-inflammatory effect, promoting macrophage differentiation and proliferation in synovial tissue. On the other hand, we have shown that pro-inflammatory cytokines are able to increase IL-34 expression by synovial fibroblasts, explaining this association.

    Pro-inflammatory cytokines are known to induce the expression of a large range of cytokines, chemokines or metalloproteases in synovial fibroblasts.1 We have shown that TNFα and IL-1β are able to enhance IL-34 expression in RA synovial fibroblasts. These results were confirmed in a human lung fibroblast cell line (WI-26), showing that the expression of IL-34 may extend to other fibroblastic cells. We have shown that JNK and NF-κB activities, the two main signalling pathways activated by TNFα and IL-1β,16 17 are required for these cytokines to stimulate IL-34 mRNA levels in fibroblasts and synoviocytes. IL-34 could thus be a downstream effector of IL-1β and TNFα, mediating their effect on inflammation and osteoclastogenesis.

    In view of the role of IL-34 in osteoclastogenesis and inflammation, this cytokine is likely to play a role in the pathogenesis of RA and therefore could constitute a new therapeutic target. Further explorations are needed to delineate the exact role of IL-34 in the inflammatory process associated with RA.

    Acknowledgments

    The authors would like to thank the MicroPICell platform (Nantes University, IFR26) for confocal microscopy and Philippe Hulin for his technical assistance.

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    Footnotes

    • MC and BLG contributed equally to this work.

    • Funding This work was supported by the ARTHRITIS Fondation Courtin (to JMB).

    • Competing interests None.

    • Patient consent Obtained.

    • Ethics approval The experimental procedures followed were carried out in accordance with the ethical standards of the responsible institutional committee on human experimentation and the Helsinki Declaration. The study was approved by the institutional ethics committee.

    • Provenance and peer review Not commissioned; externally peer reviewed.