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A2.05 Carbamylated autoantigens facilitate the break of tolerance: A novel mechanism in the pathogenesis of autoimmune arthritis
  1. JS Dekkers1,
  2. MK Verheul1,
  3. JN Stoop1,
  4. PA van Veelen2,
  5. GMC Janssen2,
  6. TW Huizinga1,
  7. LA Trouw1,
  8. RE Toes1
  1. 1Department of Rheumatology, Leiden University Medical Center; Leiden, The Netherlands
  2. 2Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands

Abstract

Background and objectives Rheumatoid arthritis is characterised by an immune response against posttranslational modified proteins, in particular citrullinated and homo-citrullinated (carbamylated) proteins. Posttranslational protein modification can result in the generation of neo-epitopes that may subsequently trigger auto-immune responses. Antibodies recognising carbamylated proteins (anti-CarP antibodies) are present in sera of RA-patients as well as in different animal models of arthritis. It is currently unknown how responding B cells interact with homo-citrullinated proteins or whether carbamylated self-proteins induce a breach of tolerance. We hypothesise that not only carbamylation of foreign, but also self-proteins can induce anti-carbamylated protein responses both at the T-cell and B-cell level, enabling T-cell dependent antibody production against carbamylated autoantigens.

Materials and methods Mice were immunised with carbamylated antigens (ovalbumin/murine albumin/murine fibrinogen) or non-modified antigens. T cell responses were studied by proliferation assays and cytokine ELISAs. Murine anti-CarP hybridomas were sequenced using single cell PCR-based antibody cloning technology. Reactivity of human anti-CarP antibodies towards CaFCS and albumin was determined for by ELISA 160 RA-patients of the Leiden Early Arthritis Cohort and controls.

Results After Ca-mAlb immunisation, mice developed high titers of anti-CarP antibodies, recognising different carbamylated foreign- and auto-antigens. In contrast, no anti-CarP antibodies were detected in mAlb immunised mice. Similar responses (i.e. anti-CarP antibody responses against carbamylated human albumin) were observed in RA-patients. Murine monoclonal anti-CarP antibodies show a similar pattern of cross-reactivity towards carbamylated antigens, together indicating that homo-citrulline is seen in a “hapten-like manner” by responding B cells. Analysis of Ig gene sequences revealed high numbers of somatic mutations, indicative of antigen-driven selection for antibody generation. T cell cultures derived from Ca-mAlb immunised mice responded to stimulation with Ca-mAlb, but not to the native protein.

Conclusions Carbamylation of both foreign as self-proteins can facilitate a breach of tolerance, resulting in formation of carbamylated protein-specific T-cell and B-cell responses in mice. In mice, homo-citrulline-residues are detected by anti-CarP-antibodies in a “hapten” like manner. Interestingly, carbamylated albumin, which is able to break tolerance in mice, is also recognised by anti-CarP antibodies in RA-patients. These data provide first evidence explaining the abundance of carbamylated self-proteins recognised by anti-CarP antibodies.

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