S Nijenhuis

Citrullinated proteins have increased immunogenicity and arthritogenicity and their presence in arthritic joints correlates with disease severity

Autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in patients with rheumatoid arthritis and have been suggested to be involved in the disease pathogenesis. The targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deiminase (PAD), which converts positively charged arginine to polar but uncharged citrulline. The aim of this study was to explore the effects of citrullination on the immunogenicity of autoantigens as well as on potential arthritogenicity. Thus, immune responses to citrullinated rat serum albumin (Cit-RSA) and to unmodified rat serum albumin (RSA) were examined as well as arthritis development induced by immunisation with citrullinated rat collagen type II (Cit-CII) or unmodified CII. In addition, to correlate the presence of citrullinated proteins and the enzyme PAD4 with different stages of arthritis, synovial tissues obtained at different time points from rats with collagen-induced arthritis were examined immunohistochemically. Our results demonstrate that citrullination of the endogenous antigen RSA broke immunological tolerance, as was evident by the generation of antibodies directed against the modified protein and cross-reacting with the native protein. Furthermore we could demonstrate that Cit-CII induced arthritis with higher incidence and earlier onset than did the native counterpart. Finally, this study reveals that clinical signs of arthritis precede the presence of citrullinated proteins and the enzyme PAD4. As disease progressed into a more severe and chronic state, products of citrullination appeared specifically in the joints. Citrullinated proteins were detected mainly in extracellular deposits but could also be found in infiltrating cells and on the cartilage surface. PAD4 was detected in the cytoplasm of infiltrating mononuclear cells, from day 21 after immunisation and onwards. In conclusion, our data reveal the potency of citrullination to break tolerance against the self antigen RSA and to increase the arthritogenic properties of the cartilage antigen CII. We also show that citrullinated proteins and the enzyme PAD4 are not detectable in healthy joints, and that the appearance and amounts in arthritic joints of experimental animals are correlated with the severity of inflammation.

Expression of PAD enzymes and occurrence of citrulline-containing proteins in human blood and synovial fluid cells

Antibodies directed against citrulline-containing antigens are extremely specific for RA. The amino acid citrulline is not incorporated into proteins during protein synthesis. It is generated by posttranslational modification of arginine residues by PAD (peptidylarginine deiminase) enzymes. We investigated the expression of PAD enzymes and the occurrence of citrullinated proteins in peripheral blood (PB) and synovial fluid (SF) cells. PAD types 1 and 3 were absent from the investigated cells, while PAD types 2 and 4 (also known as type 5) were present. In monocyte-derived macrophages PAD type 2 mRNA expression was at a similiar level as in monocytes, while PAD type 2 protein was increased. PAD type 4 mRNA expression was significant in monocytes and almost absent in monocyte-derived macrophages, while PAD type 4 protein levels were similar. In monocytes no citrullinated protein could be detected, while in monocyte-derived macrophages citrullinated vimentin, which is (part of) the Sa-antigen, was present. A similar pattern of mRNA and protein expression was observed in mononuclear cells in paired PB and SF samples of RA patients. These results suggest that PAD type 2 is involved in the citrullination of SF proteins during inflammation.

A rapid ELISA based method to determine peptidyl-arginine deiminase activity in biological samples

Peptidylarginine deiminases (PADs; EC 3.5.3.15) are a family of calcium-dependent enzymes that convert peptidylarginine into peptidylcitrulline. The recent finding that patients with rheumatoid arthritis (RA) produce autoantibodies against citrulline-containing epitopes greatly increased the interest in the PAD enzymes and their activities. It is not yet known whether there is a causative relationship between the generation of antibodies targeting citrullinated epitopes and the development of the disease. Characterisation of the structure and function of PADs may help to understand the production process of citrullinated antigens and possibly also the aetiology of RA. Several assays are known to monitor PAD activity in biological samples. However, these assays either have a low sensitivity or are laborious. Here, we describe the development of a simple, rapid method for the simultaneous analysis of many PAD-containing samples. This new method is based on the binding of an antibody specifically recognising a citrulline-containing epitope in a defined peptide. We show that this method is very sensitive and can be applied to monitor PAD activity in many types of biological samples, such as bacterial lysates, mammalian cell extracts and tissue extracts.

Citrullinated Proteins in Arthritis; their Presence in Joints and Effects on Immunogenicity

Autoantibodies directed against citrulline‐containing proteins have an impressive specificity of nearly 100% in RA patients and a suggestive involvement in the pathogenesis. The targeted epitopes are generated by a post‐translational modification catalysed by the calcium‐dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. The aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen‐induced arthritis in DA rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (RSA) and collagen type II (CII). Our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. Unimmunized animals or time points before clinical signs of arthritis were negative. By morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. A specific Cit‐RSA T‐cell response was observed in animals challenged by citrullinated RSA, no response was recorded when RSA was used as a stimulus. The IgG analysis reveals not only a response towards the modified protein but also cross‐reactivity to native RSA. No T‐cell or B‐cell response was noted in animals injected with unmodified RSA. Cit‐CII induced a disease with higher incidence and earlier onset than did the native counterpart. We conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. As inflammation proceeds, citrulline is detected specifically in the joints. All other organs investigated were negative. We also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties.