Background/Purpose: Rheumatoid arthritis patients produce a variety of anti-citrullinated protein antibodies (ACPAs) as well as antibodies that bind homocitrullinated and malondialdehyde-acetaldehyde adducted proteins leading to the theory that rheumatoid arthritis antibodies target a range of post-translationally modified proteins. Further, protein modifications may contribute to the loss of immune tolerance in rheumatoid arthritis. However, most rheumatoid arthritis patients also generate antibodies against immunoglobulin G (IgG), called rheumatoid factor (RF), with a high incidence of co-positivity for RF and anti-cyclic citrullinated peptide (CCP) antibodies. While native proteins can be targeted in rheumatoid arthritis, their citrullinated counterparts are often preferentially targeted. IgG is citrullinated in rheumatoid arthritis, but it is not known if native, homocitrullinated, or citrullinated IgG is preferentially bound by autoantibodies. The purpose of this study is to determine if antibodies in rheumatoid arthritis bind to citrulline-containing, homocitrulline-containing, and/or native IgG peptides.

Methods: A high density peptide array was designed to include every possible 12 amino acid peptide in the constant region of the heavy chain of human IgG1, IgG2, IgG3, and IgG4. Serum from control as well as CCP+RF+, CCP-RF+, CCP+RF-, and CCP-RF- rheumatoid arthritis subjects (n=12) was obtained from our biorepository and subjected to the array. Average raw signal intensity for binding of serum IgM and IgG for each category of rheumatoid arthritis subjects was compared to controls to generate a fold increase value for each peptide. Native and citrulline-containing versions of 3 peptides with very high serum IgG binding and also high homology among IgG isotypes were used to detect serum IgG binding for control and CCP+RF+ rheumatoid arthritis subjects (n=40) by enzyme linked immunosorbent assay (ELISA). ELISA results were compared by t test with p<0.05 considered significant.

Results: RF+ subjects (RF+CCP- and RF+CCP+) demonstrate a small elevation in binding of IgM to many native, homocitrulline-containing, and citrulline-containing peptides of the constant region of IgG1, IgG2, IgG3, and IgG4. Serum from the CCP+RF+ group, and no other rheumatoid arthritis group, demonstrated high IgG binding predominantly to citrulline-containing peptides in the constant region of IgG1, IgG2, IgG3, and IgG4. When measured by ELISA, CCP+RF+ subjects had more IgG binding than controls to only 1 of the 3 native IgG peptides, whereas CCP+RF+ subjects had more IgG binding than controls to all 3 of the citrulline-containing versions of the same IgG peptides. Moreover, for the 1 native peptide with increased binding in CCP+RF+ subjects compared to controls, the binding was 3x higher to the citrulline-containing version of that peptide than the native version.

Conclusion: IgG from CCP+RF+ rheumatoid arthritis patients has high binding to citrulline-containing IgG peptides suggesting that rheumatoid factor of the IgG isotype may be an ACPA. These findings support a unified view of autoantibodies in rheumatoid arthritis and may explain how tolerance is broken against IgG.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/serum-immunoglobulin-g-targets-citrulline-containing-immunoglobulin-g-peptides-in-rheumatoid-arthritis-patients-who-test-positive-for-rheumatoid-factor-and-anti-cyclic-citrullinated-peptide-antibodies/