Background/Purpose: Interleukin (IL)-32 and IL-17 play critical roles in pro-inflammatory responses and are highly expressed in the synovium of patients with rheumatoid arthritis (RA). We investigated not only the related induction of IL-17 and IL-32 in fibroblast-like synoviocytes (FLSs) from RA patients and T cells from healthy donors, but also the summative ability of the two cytokines to stimulate osteoclastogenesis.    

Methods: FLSs were isolated through surgical synovectomy obtained from patients with RA or osteoarthritis (OA) and peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors. Splenic CD4⁺ and CD4^– T cells and joint specimens were obtained from autoimmune arthritis mice.Real-time polymerase chain reactions were performed to evaluate the expression of IL-32, IL-17, orphan nuclear receptor Rorgt, tartrate-resistant acid phosphatase (TRAP), cathepsin K, calcitonin receptor (CTR), matrix metalloproteinase-9 (MMP9) mRNA. IL-17 protein was measured by enzyme-linked immunosorbent assay and the T helper (Th)17-expressing T cell count was detected by fluorescence-activated cell sorting. Immunohistochemical staining and TRAP staining were performed to determine the distribution of inflammatory cytokines and the presence of osteoclastogenesis.

Results: IL-17 induced the expression of IL-32 (4.3 fold) in the FLSs from RA patients, as assessed by microarray. The IL-32 and IL-17 levels in the FLSs from the RA patients were higher than those from the OA patients, and the expressions were colocalized. IL-32 production was increased by IL-17 through the nuclear factor (NF-¥êB)– kB and PI3 kinase signal pathways. When FLSs from RA patients and CD4+ T cells were cocultured, the CD4+ T cells differentiated into IL-17-producing Th17 cells, which stimulated the production of IL-32 in the FLSs. Moreover, IL-32 induced the production of IL-17 in human CD4+ T cells and also induced high expression levels of IL-17 in splenic CD4+ T cells of CIA mice. IL-32 and IL-17 were colocalized near TRAP-positive areas in joint specimens of autoimmune arthritis mouse models. IL-17 and IL-32 synergistically induced the differentiation of osteoclasts, as demonstrated by the expression of osteoclast-related genes such as TRAP, cathepsin K, CTR, and MMP9.

Conclusion: IL-17 affected the expression of IL-32 in FLSs of RA patients and IL-32 induced the production of IL-17 in CD4+ T cells. Both IL-17 and IL-32 cytokines can reciprocally influence each other’s production in RA and autoimmune arthritis models. Separately, IL-17 and IL-32 each stimulated osteoclastogenesis without RANKL. Together, the two cytokines synergistically amplified the differentiation of osteoclasts, independent of RANKL stimulation.