Although the etiology of rheumatoid arthritis remains unclear, it seems that immunological, genetic and environmental factors are involved in pathogenesis. In the recent years, the role of anticitrullinated peptide antibodies (ACPA) has been studied extensively. Anti-citrullinated peptideantibodies are directed against citrullinated epitopes of native proteins as a result of conversion ofarginine to citrullin mediated by the action of specific enzymes called peptidyl arginine deiminases(PADs). Citrullination is considered a post-translational process occurring under certain conditionssuch as inflammation, apoptosis and smoking that induce the action of PADs. Human cells have 5isoforms of PAD with isoform 4 (PADI4) being involved in autoimmunity. Anti-citrullinated peptide antibodies are detected in serum of RA patients several years before the appearance of clinical manifestations and have been associated with erosive and severe disease. In clinical practice, anti-cyclic citrullinated petptide (anti-CCPs) antibodies have been used to reveal ACPA reactivity in sera of RA patients, although the sequence of CCPs remains unknown. Sensitivity of anti-CCPs for RA approximates 80% while specificity exceeds 95%. Anti-CCP antibodies are considered a collection of ACPA reflecting ACPA positivity and are used as a biomarker for RA diagnosis and response to treatment. Previous studies have shown that serum and/or joint fluid from RA patients react against citrullinated epitopes of different proteins such as vimentin, fibrinogen, aenolase, filaggrin and collagen. However no predominant B cell epitope has been identified yet. The aim of this study was to investigate whether ACPA constitute a homogenous population and if possible to identify the major citrullinated epitopes. We tested the reactivity of sera form 141 RA patients, against six distinct linear citrullinated peptides derived from the previous mentioned human proteins, by applying an ELISA protocol. The sequences of citrullinated peptides were carefully chosen based on data from previous studies. Citrullinated PADI4 was also included as anti-PADI4 antibodies have been found in the serum of RA patients while non-citrullinated PADI4 was used as a peptide control. Sera from 60 SLE patients, 54 Sjogren’s patients and 100 healthy volunteers were used as controls. We found that among RA patients, PAD211-30 displayed 29.08% sensitivity, vim60-75 29.08%, enol5-21 37.59%, fibrin617-31 31.21%, col-II358-75 29.97% and filaggrin306-24 28.37% while control ctrlPAD621- 40 showed no reactivity. Specificity for all citrullinated peptides ranged between 91-97%. The percentage of RA patients with reactivity against 1, 2, 3, 4, 5 or 6 citrullinated peptides was 61%, 45%, 31%, 24%, 17% and 5% respectively. Sensitivity for anti-CCP3 and the equamolar mixture containing all six citrullinated peptides was 60.78% and 46.08% respectively while specificity was found 94.12% and 82.22% respectively. By choosing two highly positive anti-CCP3 sera from RA patients, we tested specificity and cross reactivity of ACPA against the six citrullinated peptides. For both sera and almost all the peptides, homologous inhibition was found over 85%. Cross reactivity was minimal for both sera although a significant cross reaction (>70%) was observed mainly between filaggrin and the majority of anti-citrullinated-peptide antibodies. On the contrary, antifilaggrin and anti-alpha-enolase antibodies exhibited the least cross-reactivity and therefore might be considered the most specific for these two sera. We concluded that ACPA in RA constitute a heterogeneous population with limited cross reactivity and without a predominant epitope. Diversity of ACPA reactivity can be attributed to many factors including genetic features, epitope spreading, T cell immunity as well as the interference of the anti-idiotype antibodies. Future studies should be focused on different citrullinated epitopes mixtures and evaluate the contribution of cellular immunity in ACPA responses.
Protein Biomarkers of Periodontitis in Saliva
