Autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage.

Autoimmune responses against posttranslationally modified antigens are a hallmark of several autoimmune diseases. For example, antibodies against citrullinated protein antigens (ACPA) have shown their relevance for the prognosis and diagnosis of rheumatoid arthritis (RA), and have been implicated in disease pathogenesis. It is conceivable that other autoantibody systems, recognizing other posttranslationally modified proteins, are also present in RA. Here, we describe the presence of an autoantibody system that discriminates between citrulline- and homocitrulline-containing antigens in the sera of RA-patients. IgG antibodies recognizing carbamylated (homocitrulline-containing) antigens were present in sera of over 45% of RA-patients. Likewise, anticarbamylated protein (anti-CarP) IgA antibodies were observed in 43% of RA-sera. ACPA and anti-CarP antibodies are distinct autoantibodies because, in selected double-positive patients, the anti-CarP antibody binding to carbamylated antigens could be inhibited by carbamylated antigens, but not by control or citrullinated antigens. Similarly, ACPA-binding to citrullinated antigens could only be inhibited by citrullinated antigens. In line with this observation, 16% of ACPA-negative RA-patients, as measured by a standard ACPA assay, harbored IgG anti-CarP antibodies, whereas 30% of these patients tested positive for IgA anti-CarP antibodies. The presence of anti-CarP antibodies was predictive for a more severe disease course in ACPA-negative patients as measured by radiological progression. Taken together, these data show the presence of a unique autoantibody system recognizing carbamylated, but not citrullinated, protein antigens. These antibodies are predictive for a more severe clinical course in ACPA-negative RA-patients, indicating that anti-CarP antibodies are a unique and relevant serological marker for ACPA-negative RA.

Cross-reactivity of IgM anti-modified protein antibodies in rheumatoid arthritis despite limited mutational load.

BACKGROUND: Anti-modified protein antibodies (AMPA) targeting citrullinated, acetylated and/or carbamylated self-antigens are hallmarks of rheumatoid arthritis (RA). Although AMPA-IgG cross-reactivity to multiple post-translational modifications (PTMs) is evident, it is unknown whether the first responding B cells, expressing IgM, display similar characteristics or if cross-reactivity is crucially dependent on somatic hypermutation (SHM). We now studied the reactivity of (germline) AMPA-IgM to further understand the breach of B cell tolerance and to identify if cross-reactivity depends on extensive SHM. Moreover, we investigated whether AMPA-IgM can efficiently recruit immune effector mechanisms. METHODS: Polyclonal AMPA-IgM were isolated from RA patients and assessed for cross-reactivity towards PTM antigens. AMPA-IgM B cell receptor sequences were obtained by single cell isolation using antigen-specific tetramers. Subsequently, pentameric monoclonal AMPA-IgM, their germline counterparts and monomeric IgG variants were generated. The antibodies were analysed on a panel of PTM antigens and tested for complement activation. RESULTS: Pentameric monoclonal and polyclonal AMPA-IgM displayed cross-reactivity to multiple antigens and different PTMs. PTM antigen recognition was still present, although reduced, after reverting the IgM into germline. Valency of AMPA-IgM was crucial for antigen recognition as PTM-reactivity significantly decreased when AMPA-IgM were expressed as IgG. Furthermore, AMPA-IgM was 15- to 30-fold more potent in complement-activation compared to AMPA-IgG. CONCLUSIONS: We provide first evidence that AMPA-IgM are cross-reactive towards different PTMs, indicating that PTM (cross-)reactivity is not confined to IgG and does not necessarily depend on extensive somatic hypermutation. Moreover, our data indicate that a diverse set of PTM antigens could be involved in the initial tolerance breach in RA and suggest that AMPA-IgM can induce complement-activation and thereby inflammation.