Peptidylarginine deiminase isoforms expressed in the synovial membrane of rheumatoid arthritis patients

Background

Antifilaggrin autoantibodies (AFAs) are highly specific for rheumatoid arthritis and are probably involved in its pathophysiology. We showed that they are synthesised in the rheumatoid synovial membrane and that their target antigens in the tissue correspond to variants of the α and β chains of fibrin. The variants are generated by deimination, i.e. transformation of their arginine into citrulline residues. Deimination, mediated by a peptidylarginine deiminase (PAD) activity, generates the epitopes recognised by AFA/antifibrin autoantibodies.

Four PAD isoforms (or types), have been identified and cloned in humans and rodents (mouse and rat). Expression of one or several of these isoforms has been reported in numerous tissues, but their targets are still poorly known.

Objective

Since fibrin deimination occurs in the rheumatoid synovial tissue, we undertook to identify which PAD types are expressed in the tissue.

Methods

By immunising rabbits with peptides situated in the most variable regions of the otherwise highly conserved PAD type sequences (three synthetic peptides per PAD), we produced antisera specific for each of the four PAD isoforms. The antisera were affinity-purified on the corresponding peptides. Each set of anti-peptide antibodies was confirmed to be specific for one isoform by immunoblotting on recombinant or purified PADs. Additional antisera or purified antibodies to whole human PADs II, III and V were used to confirm the results obtained with the anti-peptide antibodies.

The synovial tissue from seven patients with rheumatoid arthritis was analysed. In all the tissues, the presence of deiminated proteins and particularly of deiminated fibrin was demonstrated by immunoblotting and/or immunohistology. Then low-salt extracts of the tissues were analysed by immunoblotting with all the immunological tools to PADs.

Results

Expression of PAD type V was clearly detected in all seven patients. Expression of PAD type II was observed in six patients. No reactivities were observed with antibodies specific for PAD types I and III.

Conclusion

Of the four PAD isoforms, only the types II and V are significantly expressed in the synovial tissue of patients with rheumatoid arthritis. Their respective roles in deimination of fibrin in the tissue remain to be determined.