Effects of overexpression of PAD4 enzyme in mouse synovium

Autoantibodies directed against citrullinated proteins (e.g. anti-CCP) can be detected in rheumatoid arthritis (RA) patients with very high specificity. The antibodies are present already years before the first clinical symptoms and their presence predicts the development of erosive disease. Citrullinated proteins, the target of anti-CCP antibodies, are formed by post-translational deimination of arginine residues, catalyzed by peptidylarginine deiminase enzymes (PADs). PAD enzymes are expressed by cells present in the inflamed joints of RA patients. These data are the basis of our hypothesis that protein citrullination by PAD is intimately involved in the development of RA.

To investigate the effect of PAD4 overexpression in synovium of naive mice and in mouse models of arthritis.

An adenoviral delivery tool for local expression of mouse PAD4 was successfully constructed. Using this adenoviral construct, PAD4 overexpression was effectuated in cells present in the synovial cavity after intraarticular injection. Effects of PAD4 overexpression in mice with or without co-induction of arthritis (streptococcal cell wall arthritis) were investigated using routine scoring of arthritic features (e.g. swelling) and histology. The presence of citrulline-specific antibodies was measured by ELISA using a synthetic citrullinated peptide (CCP1) and a noncitrullinated control peptide.

In all naive mice (C57/Bl6, n = 12) we were able to effectively overexpress PAD4 in the synovial tissue, lasting at least 1 week. This overexpression resulted in the generation of citrullinated proteins in vivo. In mice receiving a control adenovirus no citrullinated proteins were observed. The overexpression did not induce inflammation. Similar experiments in streptococcal cell wall (SCW)-induced mice (C57/Bl6 + SCW, n = 8) showed a prolonged expression of citrullinated proteins when the knees were preloaded with PAD4 (compared with control adenovirus-treated SCW mice, n = 8), but this did not affect the level of inflammation or tissue damage. Overexpression of PAD4 did not lead to the production of citrulline-specific autoantibodies in these mice.

Adenoviral expression constructs can successfully be used to introduce PAD enzymes in mouse synovium. Overexpression of PAD4 results in the production of synovial citrullinated proteins, but did not cause additional inflammation in mice nor did it cause an immune response to citrullinated proteins. Generating anti-CCP-positive mice will be crucial in studying the effect of specific immune complexes on arthritic phenomena in mouse models of arthritis.

This work was financially supported by the Netherlands Foundation for Chemical Research and the Netherlands Technology Foundation, the Dutch League against Rheumatism and the Netherlands Foundation for Medical Research.

Affiliations

1. Department of Biochemistry 161, Radboud University Nijmegen, The Netherlands
   • AJW Zendman
   • , WAM Horstman
   • , ER Vossenaar
   • , WJ van Venrooij
   •  & GJM Pruijn
2. Department of Experimental Rheumatology, University Medical Center St Radboud, Nijmegen, The Netherlands
   • AJ Arntz
   • , MB Bennink
   • , WB van den Berg
   •  & FAJ van de Loo

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