Activation of Peptidylarginine Deiminase in the Salivary Glands of Balb/c Mice Drives the Citrullination of Ro and La Ribonucleoproteins.

The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p < 0.001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren's syndrome.

Potential protein targets of the peptidylarginine deiminase 2 and peptidylarginine deiminase 4 enzymes in rheumatoid synovial tissue and its possible meaning.

OBJECTIVE: The molecular mechanism of citrullination involves the calcium-dependent peptidylarginine deiminase (PAD) family of enzymes. These enzymes induce a stereochemical modification of normal proteins and transform them into autoantigens, which in rheumatoid arthritis trigger a complex cascade of joint inflammatory events followed by chronic synovitis, pannus formation, and finally, cartilage destruction. By hypothesizing that PAD2 and PAD4 enzymes produce autoantigens, we investigated five possible synovial protein targets of PAD enzymes. MATERIAL AND METHODS: We measured PAD2, PAD4, and citrullinated proteins in 10 rheumatoid and 10 osteoarthritis synovial biopsies and then assessed the post-translational modifications of fibrinogen, cytokeratin, tubulin, IgG, and vimentin proteins using a double-fluorescence assay with specific antibodies and an affinity-purified anti-citrullinated peptide (CCP) antibody. The degree of co-localization was analyzed, and statistical significance was determined by ANOVA, Fisher's exact test, and regression analysis. RESULTS: The principal results of this study demonstrated that citrullinated proteins, such as fibrinogen, IgG, and other probed proteins, were targets of PAD2 and PAD4 activity in rheumatoid synovial biopsies, whereas osteoarthritis biopsies were negative for this enzyme (p<0.0001). An analysis of citrullination sites using the UniProtKB/Swiss-Prot data bank predicts that the secondary structure of the analyzed proteins displays most of the sites for citrullination; a discussion regarding its possible meaning in terms of pathogenesis is made. CONCLUSION: Our results support the conclusion that the synovial citrullination of proteins is PAD2 and PAD4 dependent. Furthermore, there is a collection of candidate proteins that can be citrullinated.

Autoimmune vitiligo in rheumatic disease in the mestizo Mexican population.

Vitiligo is a chronic disease characterized by the dysfunction or destruction of melanocytes with secondary depigmentation. The aim of the present study was to determine the prevalence of vitiligo associated with autoimmune rheumatic diseases. The clinical records from a 10-year database of patients with rheumatic diseases and associated vitiligo was analysed, with one group of patients having autoimmune rheumatic disease and another non-autoimmune rheumatic disease. Available serum samples were used to assess the anti-melanocyte antibodies. A total of 5,251 individual clinical files were archived in the last 10 years, and these patients underwent multiple rheumatology consultations, with 0.3% of the group presenting with vitiligo. The prevalence of vitiligo in the autoimmune rheumatic disease group was 0.672%, which was mainly associated with lupus and arthritis. However, patients with more than one autoimmune disease had an increased relative risk to develop vitiligo, and anti-melanocyte antibodies were positive in 92% of these patients. By contrast, the prevalence was 0.082% in the group that lacked autoimmune rheumatic disease and had negative autoantibodies. In conclusion, the association between vitiligo and autoimmune rheumatic diseases was relatively low. However, the relative risk increased when there were other autoimmune comorbidities, such as thyroiditis or celiac disease. Therefore, the presence of multiple autoimmune syndromes should be suspected.

An Animal Model Using Metallic Ions to Produce Autoimmune Nephritis.

Autoimmune nephritis triggered by metallic ions was assessed in a Long-Evans rat model. The parameters evaluated included antinuclear autoantibody production, kidney damage mediated by immune complexes detected by immunofluorescence, and renal function tested by retention of nitrogen waste products and proteinuria. To accomplish our goal, the animals were treated with the following ionic metals: HgCl2, CuSO4, AgNO3, and Pb(NO3)2. A group without ionic metals was used as the control. The results of the present investigation demonstrated that metallic ions triggered antinuclear antibody production in 60% of animals, some of them with anti-DNA specificity. Furthermore, all animals treated with heavy metals developed toxic glomerulonephritis with immune complex deposition along the mesangium and membranes. These phenomena were accompanied by proteinuria and increased concentrations of urea. Based on these results, we conclude that metallic ions may induce experimental autoimmune nephritis.

Posttranslational Protein Modification in the Salivary Glands of Sjögren's Syndrome Patients.

The present study investigated posttranslational reactions in the salivary glands of patients with Sjögren's syndrome. We analysed the biopsies of primary Sjögren's patients using immunohistochemistry and a tag-purified anticyclic citrullinated protein (CCP) antibody to detect citrullinated peptides, and the presence of peptidylarginine deiminase 2 (PAD2) was assessed simultaneously. The present work demonstrated the weak presence of the PAD2 enzyme in some normal salivary glands, although PAD2 expression was increased considerably in Sjögren's patients. The presence of citrullinated proteins was also detected in the salivary tissues of Sjögren's patients, which strongly supports the in situ posttranslational modification of proteins in this setting. Furthermore, the mutual expression of CCP and PAD2 suggests that this posttranslational modification is enzyme dependent. In conclusion, patients with Sjögren's syndrome expressed the catalytic machinery to produce posttranslational reactions that may result in autoantigen triggering.