IL-17 in synovial fluids from patients with rheumatoid arthritis is a potent stimulator of osteoclastogenesis

Abstract

IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.

Figures

Figure 1

OCL formation in cocultures of mouse bone marrow cells and osteoblasts in the presence of increasing concentrations of IL-17. After culture for 6 days, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. Experiments were repeated 10 times with similar results.

Figure 2

IL-17 induces OCL formation in cocultures of mouse bone marrow cells and osteoblasts in response to IL-17. (a) Control culture without IL-17. (b) Morphology of OCLs formed in coculture on day 6 in the presence of 1 ng/mL of IL-17. (c) Resorption pits formed on a dentine slice. Scale bars: 200 μm.

Figure 3

Effect of neutralizing antibody against IL-17 on OCL formation in cocultures of mouse bone marrow cells and osteoblasts in the presence of IL-17 (1 ng/mL), 1α,25(OH)2D3 (10–8 M), PTH (200 ng/mL), and IL-1β (10 ng/mL). Concentrations of antibody against IL-17 were 2.5 μg/mL for experiments with IL-17 and 5 μg/mL for those with 1α,25(OH)2D3, PTH, and PGE2. After culture for 6 days, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. Experiments were repeated 5 times with similar results.

Figure 4

Effect of NS398 (10–8 M) or indomethacin (10–8 M) on OCL formation in cocultures of mouse bone marrow cells and osteoblasts in the presence of IL-17 (1 ng/mL), 1α,25(OH)2D3 (10–8 M), and PGE2 (10–6 M). After culture for 6 days, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. Experiments were repeated 7 times with similar results.

Figure 5

Effect of NS398 (10-8 M) on OCL formation and PGE2 production in cocultures and separate cultures of osteoblasts and bone marrow cells in the absence or presence of IL-17 (1 ng/mL). After culture for 6 days, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. Experiments were repeated 5 times with similar results.

Figure 6

Effect of OCIF (1.5–50 ng/mL) on OCL formation in cocultures of mouse bone marrow cells and osteoblasts in the presence of IL-17 (1 ng/mL). After culture for 6 days, TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. Experiments were repeated 3 times with similar results.

Figure 7

Expression of ODF mRNA by IL-17. A blot loaded with 20 μg of total RNA from mouse osteoblasts cultured for 3 days in the absence or presence of various concentrations of IL-17 (0.1–10 ng/mL), NS398 (10–8 M), IL-1β (1 ng/mL), TNF-α (10 ng/mL), and 1α,25(OH)2D3 (10–8 M) was probed with ODF, OCIF, or β-actin. NS, NS398.

Figure 8

Concentrations of IL-17 in the synovial fluids (a) or culture media of synovial tissues (b) from RA, OA, trauma, and gout patients. Synovial fluids were obtained from 43 RA, 9 OA, 4 trauma, and 7 gout patients (a). Synovial tissues were obtained from 12 RA and 3 OA patients (b). A part of the tissue was dissected into small pieces (1 g), rinsed well with PBS (pH 7.4), and cultured in 1 mL of RPMI-1640 containing 100 U/mL of penicillin, 10 μg/mL of streptomycin, and 2 mM L-glutamine for 3 days in 5% CO2 in air at 37°C. Culture media were collected, and their concentrations of IL-17 were measured using a ELISA kit for human IL-17. The mean value of the concentration of IL-17 of 3 synovial pieces from a single joint was calculated. The results are expressed as box plots, as described in Methods. Significant differences between RA and OA: P < 0.0001 (a); P < 0.009 (b). Tr, trauma; G, gout.

Figure 9

Immunohistochemical staining for IL-17, CD4, CD8, and CD45RO in the synovial tissues of RA patients. Sections were prepared from frozen specimens obtained from RA patients. (a) Sections were stained with antibody against IL-17. The bottom panel of a shows a high-power view of a portion of the top panel of a. Sections from RA patients stained with antibody against IL-17 (right panels) were simultaneously stained with antibodies against CD4 (b), CD8 (c), or CD45RO (d), as viewed with an FITC filter (left panels). Single arrows show stained mononuclear cells. Original magnification: ×64 (top panel of a); ×320 (bottom panel of a); ×160 (b–d).

Figure 10

A possible mechanism of osteoclastogenesis by IL-17 in the affected joint of RA patients. IL-17 produced by T cells in the synovial tissues induces PGE2 synthesis in osteoblasts/stromal cells in the adjacent bone. PGE2 then induces ODF expression in osteoblasts. ODF transduces a signal for osteoclastogenesis through ODF receptor (RANK) expressed on osteoclast progenitors (18, 20). M-CSF produced by osteoblasts/stromal cells is also an essential soluble factor for osteoclast differentiation (7, 8, 18).