Anti–citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). However, the molecular basis for ACPA production is still unclear. The purpose of this study was to determine if circulating plasmablasts from RA patients produce ACPAs and whetherPorphyromonas gingivalisfacilitates the generation of ACPAs.Methods.
Using a single-cell antibody cloning approach, we generated 217 and 110 monoclonal recombinant antibodies from circulating plasmablasts from 7 RA patients and 4 healthy controls, respectively. Antibody reactivity with citrullinated antigens was tested by a second-generation anti–cyclic citrullinated peptide (anti-CCP) kit and by enzyme-linked immunosorbent assays (ELISAs) against citrullinated human antigens. Antibody reactivity withP gingivaliswas tested by ELISAs against outer membrane antigens (OMAs) and citrullinated enolase fromP gingivalis.Results.
Approximately 19.5% of plasmablast-derived antibodies from anti-CCP–positive RA patients, but none from 1 anti-CCP–negative RA patient or the healthy controls, specifically recognized citrullinated antigens. The immunoglobulin genes encoding these ACPAs were highly mutated, with increased ratios of replacement mutations to silent mutations, suggesting the involvement of active antigen selection in ACPA generation. Interestingly, 63% of the ACPAs cross-reacted with OMAs and/or citrullinated enolase fromP gingivalis. The reactivity of ACPAs against citrullinated proteins fromP gingivaliswas confirmed by immunoblotting and mass spectrometry. Furthermore, some germline-reverted ACPAs retained their reactivity withP gingivalisantigens but completely lost their reactivity with citrullinated human antigens.Conclusion.
These results suggest that circulating plasmablasts in RA patients produce ACPAs and that this process may be facilitated by anti–P gingivalisimmune responses.