Objective: To demonstrate the effects of disease-modifying antirheumatic drugs and antiinflammatory cytokines on human osteoclastogenesis through their effects on receptor activator of nuclear factor kappaB (RANK), osteoprotegerin (OPG), and RANK ligand (RANKL).
Methods: Peripheral blood mononuclear cells (PBMCs) and rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were cocultured in the presence of macrophage colony-stimulating factor, 1,25-dihydroxyvitamin D(3), and various concentrations of methotrexate (MTX), sulfasalazine (SSZ), hydroxychloroquine (HCQ), anti-tumor necrosis factor alpha monoclonal antibody (infliximab), interleukin-4 (IL-4), and IL-10. Osteoclast formation was assayed by counting cells after staining for tartrate-resistant acid phosphatase. RANKL expression in RA FLS and RANK expression in PBMCs were assayed by Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), and real-time PCR. OPG expression was measured by enzyme-linked immunosorbent assay, RT-PCR, and real-time PCR in cultures of RA FLS.
Results: MTX, SSZ, infliximab, and IL-4, but not IL-10 and HCQ, each inhibited osteoclast formation in a dose-dependent manner. We observed no evidence of synergistic inhibition of osteoclast formation by IL-4 and IL-10. High doses of infliximab suppressed the expression of RANK in PBMCs. MTX, SSZ, infliximab, and IL-4 each inhibited the expression of RANKL in RA FLS in a dose-dependent manner, and also increased the secretion of OPG in RA FLS supernatants.
Conclusion: MTX, SSZ, infliximab, and IL-4 inhibit human osteoclastogenesis by modulating the interaction of RANKL, RANK, and OPG. These results are indicative of the underlying mechanisms of the antiresorptive effects of these 4 agents.