Background Autoimmunity in Rheumatoid arthritis (RA) patients is characterized by a spectrum of anti-modified protein antibodies (AMPA) directed against post-translationally modified (PTM) proteins. The best-known AMPA in RA are anti-citrullinated protein antibodies (ACPA). Much less is known about the occurrence and aetiology of other AMPA responses in RA such as autoantibodies directed to malondialdehyde-acetaldehyde (MAA) adducts, acetylated antigens, and carbamylated proteins. Anti-carbamylated protein (anti-CarP) autoantibodies recognize carbamylated proteins containing a homocitrulline, a PTM structurally similar to citrulline. With the presence of various AMPA responses in RA, PTM proteins have been implicated in the breach of autoreactive B-cell tolerance leading to the formation of these autoantibodies. At present it is unkown how AMPA are generated and how autoreactive B-cell responses against PTM proteins are induced.
Objectives To investigate how PTM proteins, more specifically carbamylated proteins, could contribute to a breach of B-cell tolerance.
Methods Serum reactivity towards five different carbamylated proteins was determined for 160 RA-patients and 40 healthy individuals. Anti-CarP antibody cross-reactivity was studied by inhibition experiments. Mass spectrometry was performed to identify carbamylated self-proteins in human rheumatic and osteoarthritic joint tissue. Mice were immunized with carbamylated- or non-modified foreign (OVA) and self-antigens (mouse Albumin). Sera of immunized mice and monoclonal antibodies were analyzed for antigen reactivity.
Results We show that anti-CarP antibodies in RA are highly cross-reactive towards multiple carbamylated proteins, including modified self- as well as modified non-self proteins. Immunization with carbamylated foreign proteins (Ca-OVA) induced a strong anti-CarP response against both carbamylated foreign- and self-proteins. Similar to murine 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, murine 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.
Conclusions Self-reactive AMPA-responses can be induced by exposure to foreign proteins containing PTM. Our findings show that autoreactive B-cell responses against PTM self proteins can be induced by exposure to PTM foreign proteins and provide new insights on the breach of autoreactive B-cell tolerance by foreign proteins.
Disclosure of Interest None declared