Background Anti citrullinated protein antibodies (ACPA) associate with bone destruction in RA
Objectives We aimed to characterize the molecular mechanisms responsible for APCAs effect.
Methods ACPA positive and negative IgGs were isolated from synovial fluid (SF, n=26) and peripheral blood (PB, n=38) samples of RA patients. CD14+ monocytes from PB of ACPA+ RA patients and healthy individuals were first cultured in the presence M-CSF to generate MΦ, and then further differentiated to OC in the presence of RANKL and M-CSF. In parallel, cells were grown on osteoassay surfaces and bone resorption area was quantified by computer assisted image analysis. In house generated anti-citrullinated monoclonal antibodies obtained from SF B-cells were also tested. Cytokines were measured by cytometric bead arrays in cultures supernatants. Immunohistochemistry (IHC) was used to stain the OCs with biotinylated ACPA IgG and monoclonal anti-citrullinated proteins antibodies. The effect of PAD inhibition (C.I.amidine), IL-8 and TNF inhibition was tested in the osteoclasts cultures.
Results Polyclonal SF-derived ACPA IgGs increased RANKL-driven osteoclastogenesis from a median of 238 OC/well, IQR 55.5-378.5 to a median of 333.5, IQR 88.8-489.3. PB derived ACPAs had similar effect and equally potency. These changes were paralleled by an increase of bone resorption areas by ACPA IgGs from a median of 4.9%, IRQ 1.9-8.5 to a median of 6.9%, IQR 2.7-10. No significant increase in either osteoclasts numbers or resorption areas was observed with the control ACPA-negative IgGs. Increased osteoclastogenesis was associated with significantly higher levels of IL-8 levels in cultures supernatants (fold increase of 2.0±0.5), while no changes were observed for either TNF or IL-6. Cell exposure to neutralizing anti-IL8 antibodies completely abolished ACPA-mediated osteoclastogenesis. Monoclonal antibodies against cit-vimentin and cit-enolase, but not cit-fibrinogen, had similar effects with the polyclonal ACPAs, promoting osteoclastogenesis. Fab fragments of these monoclonal antibodies retained similar effects. Binding of both polyclonal and monoclonal ACPAs on OC was confirmed using IHC.
Conclusions ACPA IgGs directly promote osteoclastogenesis and bone resorption, an effect that is dependent on antibody specificity and mediate through a novel IL-8 mediated mechanisms. IL-8 targeting might represent an alternative therapeutic approach to modulate antibody-mediated bone destruction.
Disclosure of Interest None declared
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