Rheumatoid and pyrophosphate arthritis synovial fibroblasts induce osteoclastogenesis independently of RANKL, TNF and IL-6

Abstract

Bone destruction is a common feature of inflammatory arthritis and is mediated by osteoclasts, the only specialized cells to carry out bone resorption. Aberrant expression of receptor activator of nuclear factor kappa β ligand (RANKL), an inducer of osteoclast differentiation has been linked with bone pathology and the synovial fibroblast in rheumatoid arthritis (RA). In this manuscript, we challenge the current concept that an increase in RANKL expression governs osteoclastogenesis and bone destruction in autoimmune arthritis. We isolated human fibroblasts from RA, pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients and analyzed their RANKL/OPG expression profile and the capacity of their secreted factors to induce osteoclastogenesis. We determined a 10-fold increase of RANKL mRNA and protein in fibroblasts isolated from RA relative to PPA and OA patients. Peripheral blood mononuclear cells (PBMC) from healthy volunteers were cultured in the presence of RA, PPA and OA synovial fibroblast conditioned medium. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP), vitronectin receptor (VNR), F-actin ring formation and bone resorption assays. The formation of TRAP(+), VNR(+) multinucleated cells, capable of F-actin ring formation and lacunar resorption in synovial fibroblast conditioned medium cultures occured in the presence of osteoprotegerin (OPG) a RANKL antagonist. Osteoclasts did not form in these cultures in the absence of macrophage colony stimulating factor (M-CSF). Our data suggest that the conditioned medium of pure synovial fibroblast cultures contain inflammatory mediators that can induce osteoclast formation in human PBMC independently of RANKL. Moreover inhibition of the TNF or IL-6 pathway was not sufficient to abolish osteoclastogenic signals derived from arthritic synovial fibroblasts. Collectively, our data clearly show that alternate osteoclastogenic pathways exist in inflammatory arthritis and place the synovial fibroblast as a key regulatory cell in bone and joint destruction, which is a hallmark of autoimmune arthritis.

Fig. 1

Immunohistochemistry of serial sections of synovial tissue from OA, RA and PPA patients showing RANKL and OPG expression. OA (a) control (b) RANKL (c) OPG, RA (d) control (e) RANKL (f) OPG and PPA (g) control (h) RANKL (i) OPG. (Immunoperoxidase: Original magnification (a, b, c, d, e, g, h, i), ×200 and (f) ×300.

Fig. 2

Immunohistochemistry of isolated synovial fibroblasts. Fibroblasts cultured on coverslips showing the (a–c) IgG control and lack of expression of (d–f) the T cell marker CD3 and (g–i) the B cell marker CD20 and positive expression of (j–l) vimentin and (m–o) proline-4-hydroxylase. Scale bar represents 50 μm.

Fig. 3

RANKL/OPG expression in OA, RA, and PPA. (a) Normalized average data of the mRNA RANKL/OPG ratio for OA, RA, and PPA. (b) Western blotting analysis of synovial fibroblast total cell lysates showed expression of RANKL and OPG protein. A human osteosarcoma cell line MG63 known to express RANKL and OPG was used as a positive control. (c) Densitometric analyses of bands for RANKL and OPG were obtained using ImageJ software, and results were normalized to basal level.

Fig. 4

TRAP cytochemical stain images of human PBMC cultured for 16 days in the presence of M-CSF and conditioned medium from a) OA, b) RA and c) PPA and results expressed d) graphically, (p < 0.05 Manne–Whitney nonparametric test, error bars standard error of the mean) data pooled from 3 experiments. Osteoclasts were also determined by the presence of multinucleated cells (e) expressing the specific osteoclast markers (f) VNR, and capable of (g) F-actin ring formation. (Representative pictures of at least three experiments M-CSF and CM OA, RA and PPA cultures that induced osteoclast formation).

Fig. 5

Scanning electron micrographs of dentine slices after PBMC culture for 21 days in the presence of M-CSF, OPG, and CM of cultured arthritic synovial fibroblasts. Fibroblasts isolated from (a) OA and (b) RA showed evidence of lacunar resorption formation. (Image representative of average lacunar resorption formation.) (c) Mean percentage area of lacunar resorption in OA, RA, and PPA conditioned medium in cultures of PBMCs incubated for 21 days in the presence of M-CSF, with or without OPG, anti-TNF, and anti-gp130. Scale bars represent 100 μm. (Representative data of at least three experiments.)