LAUSR

Introduction Pain and bone loss are cardinal features of rheumatoid arthritis (RA), which can be triggered by anti-citrullinated protein antibodies (ACPAs). Objectives We aimed to investigate whether ACPA fine specificities could influence osteoclast (OC) induction and we studied the contribution of ACPA Fc and Fab regions in the regulation of OCs. We also investigated whether in vivo targeting of OCs by bis-phosponate could influenceACPA-induced pain in mice. Methods Polyclonal ACPAs and control IgGs were purified from the peripheral blood of RA patients using protein G and anti-cyclic citrullinated peptide (CCP)−2 affinity chromatography. Monoclonal ACPA and rheumatoid factor (RF) IgGs were generated from single synovial plasma cells or antigen tetramer-sorted peripheral blood memory B-cells. OCs were generated from CD14 +monocytes of healthy individuals or bone marrow cells of FcgRIII or FcRg chain knockout mice in the presence of the various antibodies. OC differentiation was monitored by counting TRAP-positive multinucleated cells or in bone erosion assays. Mechanical hypersensitivity was assessed over time by Von Frey filaments and the up-down method in female adult wild type or FcRg chain knockout mice injected i.v. with polyclonal or monoclonal ACPA or control antibodies. Bone density was measured by micro-CT. Zoledronate (100 ug/kg) was injected i.p every third day to examine if blocking osteoclast activation alter ACPA-induced pain. Results Polyclonal and two out of the nine tested monoclonal ACPAs increased osteoclastogenesis. Addition of a monoclonal RF antibody to OC cultures could not influence osteoclastogenesis in itself, but it significantly increased the effect of ACPAs. Dimeric Fab fragments prepared from polyclonal ACPAs could increase OC numbers similarly as the intact antibodies, suggesting a crucial role of the cell surface antigens triggered by ACPA binding in mediating the effects of these antibodies. On the other hand, whereas ACPAs increased osteoclastogenesis from bone marrow precursors of wild type mice, no stimulatory effects could be observed when bone marrow cells of FcgRIII or FcRg chain knockout mice were used, suggesting that Fc receptors might also be important for ACPA-mediated OC stimulation. Polyclonal and monoclonal ACPA induced pronounced mechanical hypersensitivity lasing for at least 3 weeks and injection of polyclonal and certain combinations of monoclonal ACPA lead to bone erosion detectable by micro-CT- Bisphosphonate treatment and FcRg chain depletion prevented development of pain-like behaviour in those groups. Conclusions We demonstrated that ACPAs with certain specificities have the capacity to increase osteoclastogenesis whereas most of the tested clones showed no effect on OCs. The mechanism triggered by ACPA binding to developing OCs was mediated through both Fc-dependent and independent signals. ACPA-mediated pain hypersensitivity was dependent both on osteoclast activity and Fcg receptors. Disclosure of interest None declared