Semaphorin 4D Contributes to Rheumatoid Arthritis by Inducing Inflammatory Cytokine Production: Pathogenic and Therapeutic Implications

Abstract

Objective: Semaphorin 4D (Sema4D)/CD100 has pleiotropic roles in immune activation, angiogenesis, bone metabolism, and neural development. We undertook this study to investigate the role of Sema4D in rheumatoid arthritis (RA).

Methods: Soluble Sema4D (sSema4D) levels in serum and synovial fluid were analyzed by enzyme-linked immunosorbent assay. Cell surface expression and transcripts of Sema4D were analyzed in peripheral blood cells from RA patients, and immunohistochemical staining of Sema4D was performed in RA synovium. Generation of sSema4D was evaluated in an ADAMTS-4-treated monocytic cell line (THP-1 cells). The efficacy of anti-Sema4D antibody was evaluated in mice with collagen-induced arthritis (CIA).

Results: Levels of sSema4D were elevated in both serum and synovial fluid from RA patients, and disease activity markers were correlated with serum sSema4D levels. Sema4D-expressing cells also accumulated in RA synovium. Cell surface levels of Sema4D on CD3+ and CD14+ cells from RA patients were reduced, although levels of Sema4D transcripts were unchanged. In addition, ADAMTS-4 cleaved cell surface Sema4D to generate sSema4D in THP-1 cells. Soluble Sema4D induced tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) production from CD14+ monocytes. IL-6 and TNFα induced ADAMTS-4 expression in synovial cells. Treatment with an anti-Sema4D antibody suppressed arthritis and reduced proinflammatory cytokine production in CIA.

Conclusion: A positive feedback loop involving sSema4D/IL-6 and TNFα/ADAMTS-4 may contribute to the pathogenesis of RA. The inhibition of arthritis by anti-Sema4D antibody suggests that Sema4D represents a potential therapeutic target for RA.

Figure 1

Levels of soluble semaphorin 4D (sSema4D) in serum and synovial fluid samples from patients with rheumatoid arthritis (RA), and Sema4D protein expression in RA synovium. A, Soluble Sema4D levels in serum samples from healthy individuals and from 101 patients with RA, 10 patients with osteoarthritis (OA), 10 patients with ankylosing spondylitis (AS), and 34 patients with systemic lupus erythematosus (SLE). B, Levels of sSema4D in synovial fluid samples from 7 RA patients and 10 OA patients. Values in A and B are the mean ± SD. ND = not detectable. * = P < 0.05; ** = P < 0.01; ***= P < 0.001. C, Hematoxylin and eosin (H&E) staining and immunohistochemical staining for Sema4D, CD3, CD20, and CD68 in synovial tissue samples from patients with OA or RA. Images shown are representative of samples from 7 RA patients and 10 OA patients. Original magnification × 40. Ab = antibody.

Figure 2

Correlations of serum levels of soluble semaphorin 4D (sSema4D) with markers of rheumatoid arthritis disease activity. A, Positive correlation of serum sSema4D levels with the Disease Activity Score in 28 joints (DAS28), the C‐reactive protein (CRP) level, the rheumatoid factor (RF) titer, and the level of urinary deoxypyridinoline (u‐DPD). B, Serum sSema4D levels before and after biologic disease‐modifying antirheumatic drug (DMARD) treatment in 9 good responders and 8 moderate responders or nonresponders according to the European League Against Rheumatism response criteria. *** = P < 0.001. NS = not significant. C, Correlation of change ratio in serum sSema4D levels with change ratio of DAS28 after treatment with biologic DMARDs (n = 17 samples).

Figure 3

Semaphorin 4D (Sema4D) expression, and soluble Sema4D (sSema4D) production with ADAMTS‐4 as the sheddase. A, Histograms of cell surface expression of Sema4D in peripheral blood CD3+, CD19+, and CD14+ cells. Results shown are representative of findings from 5 rheumatoid arthritis (RA) patients and 5 healthy individuals. B, Expression of mRNA for Sema4D in peripheral blood CD3+, CD19+, and CD14+ cells. Results shown are from 5 RA patients and 5 healthy individuals. C, Levels of sSema4D in culture supernatant of THP‐1 cells cultured with recombinant matrix metalloproteinase 3 (MMP‐3), MMP‐9, ADAM‐17, and ADAMTS‐4 (n = 5 samples per group). Results are representative of 3 independent experiments. D, Serum levels of ADAMTS‐4 in 20 RA patients and 16 healthy individuals. E, Synovial fluid levels of ADAMTS‐4 in 7 RA patients and 10 osteoarthritis (OA) patients. Values in B–E are the mean ± SEM. ** = P < 0.01. NS = not significant.

Figure 4

Inflammatory cytokine production induced by semaphorin 4D (Sema4D), and elevated ADAMTS‐4 expression by inflammatory cytokines. A, Tumor necrosis factor α (TNFα) and interleukin‐6 (IL‐6) levels in culture supernatant of CD14+ monocytes from rheumatoid arthritis (RA) patients after stimulation with naturally cleaved soluble Sema4D (sSema4D) for 72 hours. Results shown are representative of 3 independent experiments. B, TNFα and IL‐6 levels in culture supernatant of CD14+ monocytes from RA patients after stimulation with naturally cleaved sSema4D for 48 hours with or without anti‐Sema4D antibody. Results shown are representative of 3 independent experiments. C and D, Elevated expression of ADAMTS‐4 mRNA (C) and elevated ADAMTS‐4 protein levels (D) in primary cultures of TNFα‐ and IL‐6–stimulated synovial cells from RA patients. Data were compiled from 5 independent experiments. Values are the mean ± SEM. * = P < 0.05; ** = P < 0.01. non = not stimulated.

Figure 5

Blocking of semaphorin 4D (Sema4D) ameliorates severity of collagen‐induced arthritis (CIA) in mice. A, Average arthritis scores of mice with CIA. Anti‐Sema4D or control antibody (Ab) (50 mg/kg) was administered intraperitoneally on days 28 and 35 (arrows) (n = 6 mice per group). Data are representative of 3 independent experiments. B, Sections of a mouse ankle joint on day 42 after first immunization. Sections were stained with hematoxylin and eosin (H&E) or Safranin O (SO). Original magnification × 100. C, Average pathologic scores of paw sections on day 42 (n = 6 mice per group). D, Serum levels of tumor necrosis factor α (TNFα) and interleukin‐6 (IL‐6) on day 42 (n = 6 mice per group). Values are the mean ± SEM and are representative of 3 independent experiments. * = P < 0.05; ** = P < 0.01.