Suppressing anti-citrullinated protein antibody-induced osteoclastogenesis in rheumatoid arthritis using anti-CD64 and PAD-2 inhibitors.

ABSTRACT

OBJECTIVES: To evaluate the role of Fcγ receptors (FcγR) and peptidyl arginine deiminase (PAD) in anti-citrullinated protein antibody (ACPA)-induced fibroblast-like synoviocytes (FLSs)-mediated osteoclastogenesis in patients with rheumatoid arthritis (RA). METHODS: FLSs and peripheral blood mononuclear cells were collected from patients with RA. We stimulated RA-FLS with ACPA (100 ng/ml) with and without anti-cluster of differentiation (CD)32a/CD64 (FcγRIIA/FcγRI) antibody and PAD-2/4 inhibitors. Flow cytometry and enzyme-linked immunosorbent assay were also performed. CD14+ monocytes were cultured with receptor activator of nuclear factor kappa beta (RANKL) and macrophage colony-stimulating factor, and ACPA-stimulated RA-FLSs were added. These cells were cultured for 14 days, and osteoclastogenesis was quantified using tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: ACPA increased RANKL+ and tumour necrotic factor-alpha (TNF-α+) FLS, which decreased dose-dependently by adding 5 and 10 ug/mL anti-CD64 antibody rather than anti-CD32a antibody. In PAD inhibitor experiments, the proportion of RANKL+ and TNF-α+ FLS decreased in 50 µM condition containing PAD-2 inhibitor rather than PAD-4 inhibitor. The co-culture of ACPA-stimulated RA-FLSs and osteoclast precursors increased the TRAP+ multinucleated osteoclast count, which was decreased by anti-CD64 antibody and PAD2 inhibitor. CONCLUSIONS: The present study showed that ACPA increased RANKL and pro-inflammatory cytokine expression in RA-FLSs, and ACPA-activated RA-FLSs could augment osteoclastogenesis. These processes were inhibited by treatment with anti-CD64 antibody and PAD-2 inhibitors. These results show that CD64 and PAD-2-induced pathways may be involved in ACPA-induced FLS activation and osteoclastogenesis in patients with RA. Therefore, regulating the CD64 and PAD-2 pathways may improve RA treatment.