Background In rheumatoid arthritis (RA), anti-citrullinated protein antibodies (ACPA) are highly disease-specific biomarkers with suggested involvement in RA pathogenesis. Despite recent advances, their exact role in the inflammatory disease process and their effector mechanisms still have to be identified.
Objectives To gain insight in ACPA Fc-mediated effector functions, we studied the binding of ACPA immune complexes to various Fc gamma receptors.
Methods ACPA-IgG and non citrulline-reactive IgG (ncIgG) were isolated from plasma and synovial fluid samples of established RA patients by affinity chromatography using 2nd generation cyclic citrullinated peptide (CCP2) as an antigen. After further purification of the IgG1 subclass of both entities, immune complexes (IC) were generated using fluorescently labelled F(ab')2 fragments directed against the F(ab')2 part of IgG1 or by using citrullinated fibrinogen. The size of the IC was measured using asymmetric flow-field flow (AF4) molecular size/weight fractionation. ICs were incubated with various CHO cell lines stably transfected with different human Fc gamma receptors (FcγR). IC binding to these FcγR was analysed by flow cytometry. ACPA-IgG1 and IgG1 Fc glycosylation was measured by mass spectrometry.
Results ACPA-IgG1 and ncIgG1 ICs of comparable size bound predominantly to activating FcγRs whereas less binding of the ICs was detected to the inhibitory receptor FcγRIIB. Most IC binding was observed to FcγRI and FcγRIIIA (V176), whereas much lower binding signals were detected for FcγRIIA. Strikingly, ACPA-IgG1 showed reduced binding to FcγRIIIA when compared to ncIgG1. This corresponded to enhanced core fucosylation of the ACPA-IgG1 Fc tail. Finally, we observed enhanced binding of ACPA-IgG1 IC to IFN-γ activated neutrophils.
Conclusions The binding of ACPA-IgG1 to activating FcγR implies an enhanced immune activating effect with a particular role for FcγRI. Moreover, the reduced ACPA-IgG1 binding to FcγRIIIA suggests that the differential Fc glycosylation profile of ACPA could affect FcγR mediated effector functions.
Disclosure of Interest None declared
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