Objectives Smoking has been connected to citrullination of antigens and formation of anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Since smoking can modify proteins by carbamylation (formation of homocitrulline), this study was conducted to investigate these effects on vimentin in animal models and RA.
Methods The efficiency of enzymatic carbamylation of vimentin was characterised. B-cell response was investigated after immunisation of rabbits with different vimentin isoforms. Effects of tobacco smoke exposure on carbamylation of vimentin and formation of autoantibodies were analysed in mice. The antibody responses against isoforms of vimentin were characterised with respect to disease duration and smoking status of patients with RA.
Results Enzymatic carbamylation of vimentin was efficiently achieved. Subsequent citrullination of vimentin was not disturbed by homocitrullination. Sera from rabbits immunised with carbamylated vimentin (carbVim), in addition to carbVim also recognised human IgG-Fc showing rheumatoid factor-like reactivity. Smoke-exposed mice contained detectable amounts of carbVim and developed a broad immune response against carbamylated antigens. Although the prevalence of anti-carbamylated antibodies in smokers and non-smokers was similar, the titres of carbamylated antibodies were significantly increased in sera of smoking compared with non-smoking RA. CarbVim antibodies were observed independently of ACPAs in early phases of disease and double-positive patients for anti-mutated citrullinated vimentin (MCV) and anti-carbVim antibodies showed an extended epitope recognition pattern towards MCV.
Conclusions Carbamylation of vimentin is inducible by cigarette smoke exposure. The polyclonal immune response against modified antigens in patients with RA is not exclusively citrulline-specific and carbamylation of antigens could be involved in the pathogenesis of disease.
Trial registration number ISRCTN36745608; EudraCT Number: 2006-003146-41.
- Rheumatoid Arthritis
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Handling editor Tore K Kvien
KG and GRB have equally contributed to the work.
Contributors CO performed the analysis of the mouse experiments and contributed in writing and reviewing the manuscript; HB advised vimentin carbamylation, animal immunisation, HeLa cell fractionation, Western blot analysis, ELISA kits developing and contributed in writing and reviewing the manuscript; EF designed the study, collected the results and contributed in writing and reviewing the manuscript; GC and SK performed and designed the analysis of the mouse experiments and contributed in reviewing the manuscript; JD collected samples and clinical data of RCT HIT HARD and contributed in reviewing the manuscript; AK developed and performed the analysis of the ELISA kits, immunoblot experiments and contributed in reviewing the manuscript; SG contributed to the study design and in reviewing the manuscript; KG performed and designed the analysis of ELISA experiments, collected clinical data and contributed in writing and reviewing the manuscript and GRB contributed to the study design and in writing and reviewing the manuscript.
Funding The study was supported by the FP7 HEALTH programme under the grant agreement FP7-HEALTHF2-2012-305549, HIT HARD study (German Federal Ministry of Education and Research (BMBF, grant number: 01KG0602) and EU IMI grant BeTheCure, contract no 115142-2; ArthroMark of the German Federal Ministry of Education and Research (BMBF, grant number: 01EC1401A).
Competing interests HB is an employee of Orgentec Diagnostika GmbH, Mainz, Germany; test kits were kindly provided by Orgentec.
Patient consent Obtained.
Ethics approval Charité-Universitätsmedizin Berlin, Germany.
Provenance and peer review Not commissioned; externally peer reviewed.