Interleukin 34 expression is associated with synovitis severity in rheumatoid arthritis patients

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.

Figure 1. IL-34 is expressed in the synovial tissue of patients with OA and RA

Representative immunohistochemical staining for IL-34 (red staining) (ab75723, Abcam, France) in synovial biopsies 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 (Asterix) (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 to the IL-34^- group. (G) Comparative levels of IL-34 measured by ELISA assay (ref. ABIN455583) in synovial fluids in OA and RA patients, each plot being an individual sample, *** p<0.001. (H) Correlation between IL-34 levels and total leukocyte counts in the synovial fluids of patients with RA and OA, r = 0.82, p<0.0001.

Figure 2. TNF–α and IL-1β induce IL-34 mRNA expression in RA synovial fibroblasts

Synovial fibroblasts from RA patients (n=3) were stimulated with (A) increased dose of TNF-α (1 to 50 ng/mL) for 6h hours or with TNF–α 10 ng/mL for 2h, 6h, 10h and 24h and (B) increased dose of IL-1β (1 to 25 ng/mL) for 6 hours or with IL-1β 10 ng/mL for 2h, 6h, 10h and 24h. After incubations, IL-34 mRNA levels were determined by real time RT-PCR, normalized to GAPDH (C) Synovial fibroblasts cultured on labtek chamber slides (Millipore, France) were treated or not with TNF–α (10 ng/mL) or IL-1β (10 ng/mL) for 24h. 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 was shown.

Figure 3. JNK and NF-κB activities are required for TNF-α and IL-1β to stimulate IL-34 mRNA levels in fibroblasts and synoviocytes

WI-26 fibroblast cells were treated either with 10 ng/mL TNF-α (A) or 10 ng/mL IL1β (B) for 2h, 6h, 10h, 24h and 48h (right panels) or with various concentrations of TNF-α or IL-1β (1, 5, 10, 25 and 50 ng/mL) for 24h, as indicated. After incubations, IL-34 mRNA steady-state levels were determined by real time RT-PCR. The expression of the houskeeping gene GAPDH was used as control. (C) Wild type (wt) 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 TNF-α or IL-1β were added for 6h. IL-34 mRNA steady-state levels were then determined by real time RT-PCR. Bars indicate mean ± S.D. of two independent experiments performed, each with duplicate samples. *p<0.05; ***p<0.001 compared to 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 TNF-α or IL-1β were added for 6h. After incubations, IL-34 mRNA steady-state levels were determined by real time RT-PCR. A representative experiment was shown.