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T CELL REGULATION OF OSTEOCLASTOGENESIS IN ASEPTIC LOOSENING

Introduction: The biological processes underlying osteolysis in aseptic loosening are not completely understood, but are believed to include factors such as hydrostatic pressure and wear debris. Characterisation of the pseudosynovial membrane from failed implants has revealed numerous cell types with well characterised roles in osteoclastogenesis and bone resorption. More recent work has demonstrated the presence of immunomodulatory cells, including T cells. IL-17 is a T cell product that is believed to be capable of inducing bone resorption. The aims of our study were to characterise the effects of IL-17 on the expression of RANKL and OPG by synovial fibroblasts and to evaluate its role in supporting osteoclastogenesis in vitro. Materials and Methods: Synovial fibroblasts (SFB) were isolated from tissue obtained at joint replacement surgery. SFB were expanded in culture and used in experiments between passage 4 and 5. Human SFB, and for comparison the human osteosarcoma cell line MG63, were treated with IL-17 (5 and 50ng/ml) for up to 48 hours. The expression and production of RANKL and OPG at 6, 24 and 48hours was assessed by RT-PCR, quantiative real-time PCR, Northern blot and Western blot analyses. To investigate osteoclastogenesis, peripheral blood mononuclear cells (PBMCs) were cultured with IL-17 (5 and 50ng/ml) either alone or with M-CSF (25 ng/ml). After 14–21 days, cultures were fixed and stained for tartrate-resistant acid phosphatase (TRAP) and multinucleated, TRAP positive cells counted. Experiments were repeated on ivory slices and resorption evaluated. Results: RT-PCR and QT-PCR analysis demonstrated that RANKL mRNA levels in SFB (4 of 5 patients) are enhanced by IL-17 in a biphasic manner. RANKL expression was elevated at 6 hours, returned to near control values at 24 hours before demonstrating increased levels at 48 hours. The expression of RANKL in MG63 cells was enhanced by IL-17 (5ng/ml) at 6 and 24 hours, and by IL-17 (50ng/ml) at 48 hours. The expression of OPG by SFB was upregulated by IL-17 (5 and 50ng/ml) at 6, 24 and 48 hours. The elevated expression of OPG in MG63 cells by IL-17 was time dependent, and this elevated expression was confirmed by Western blot. In cultures of PBMCs, IL-17 alone increased the numbers of TRAP+ve multinucleate cells dose-dependently. Similar levels of TRAP+ve cells were observed in the cultures treated with RANKL and M-CSF, but numbers of multinucleated cells were further increased when M-CSF was supplemented with IL-17. Resorption of ivory wafers was also observed in cultures treated with IL-17. Conclusions: These results suggest that IL-17 induced osteoclast formation could contribute to the bone loss associated with a wide range of pathological states involving osteolysis and aseptic loosening.