Background/Purpose: Autoantibodies are an important hallmark of Rheumatoid Arthritis (RA). Approximately 50% of RA patients harbor anti-carbamylated protein (CarP) antibodies. These autoantibodies target proteins that are modified through a posttranslational modification named carbamylation or homocitrullination. It has been postulated that posttranslational modifications can play a role in the breach of tolerance towards self-antigens. These anti-carbamylated protein (CarP) antibodies can recognize both carbamylated foreign- and self-proteins. It is currently unknown whether carbamylated foreign-proteins can evoke an anti-CarP autoimmune response or whether this can only be facilitated through carbamylation of self-proteins. Therefore we studied whether carbamylation of self- and foreign-proteins can drive loss of B-cell tolerance to carbamylated proteins.

Methods: Mice were immunized with carbamylated- or non-modified (auto)antigens and analyzed for autoantibody responses. Anti-CarP hybridomas were sequenced using single cell PCR-based antibody cloning technology. Mass spectrometry was used to identify carbamylated self-proteins in rheumatic joint tissue. Serum reactivity towards Ca-human serum albumin and Ca-FCS was determined for 100 RA-patients of the Leiden Early Arthritis Cohort and 40 healthy subjects.

Results: Immunization with carbamylated self-proteins (Albumin) resulted in a potent anti-CarP antibody response against both carbamylated foreign- and self-proteins. Likewise, immunization with carbamylated foreign-proteins (OVA) induced a strong anti-CarP response against both carbamylated foreign- and self-proteins. Similar to serum anti-CarP antibodies, murine monoclonal anti-CarP antibodies were highly specific and cross-reactive to multiple carbamylated (auto)antigens. Although citrulline greatly resembles homocitrulline residues in structure, anti-CarP antibodies differ in antigen recognition profile from ACPA as they are able to discriminate between citrullinated and homocitrullinated forms of the same protein. Interestingly, we were able to identify carbamylated-albumin, in RA synovial tissue, indicating that albumin is present in carbamylated form locally in the inflamed joint. Moreover, we detected antibody responses against carbamylated-human albumin were observed in 38% of early RA patients from the Leiden Early Arthritis Clinic.

Conclusion: These results show that not only carbamylated self-, but also carbamylated non-self-proteins can lead to a breach of B-cell tolerance leading to B-cell responses against carbamylated self-proteins. These results indicate that auto-reactive B-cells to carbamylated proteins as well as it is underlying T-cell response could find its origin in the exposure to carbamylated foreign-proteins.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/exposure-to-carbamylated-self-and-non-self-proteins-can-lead-to-a-break-of-tolerance-and-the-induction-of-autoimmunity/