The Screening Of Citrullinated Autoantigenes And Research Of Low-expressed Protein In Rheumatoid Arthritis

BACKGROUND: Rheumatoid arthritis (RA) is a chronic systematic autoimmunedisease, featured with synovitis, inflammatory cell infiltration, abnormal proliferation ofsynoviocytes, angiogenesis, and destruction of articular cartilage and bone.Serum fromthe patients contains autoantibodies such as rheumatoid factor (RF), anti filaggrinautoantibody (AFA), anti keratin antibody (AKA), anti perinuclear factor (APF), antivimentin autoantibody, and anti cyclic citrullinated peptide antibody (anti-CCP). B cellepitopes of the most RA autoantibodies contain citrulline converted from arginine residueby citrullination. Although several proteins such as filaggrin, fibrin and vimentinfollowing citrullination were demonstrated to induce autoimmunity of RA, citrullinatedproteins as autoantigens had not been systematically investigated. In previous study, wescreened the citrullinated autoantigens with two-dimensional western blotting (2-D WB).As results, Alpha-1-antitrypsin (A1AT), dynein heavy chain3(DYH3), fibrinogen betachain (FIBB), keratin type II cuticular Hb4(KRT84), Cytokeratin4(KRT4),lumican(LUM), tubulin beta chain (TUBB) and vimentin (VIME) were detected by both RA serum and anti-citrulline antibody using2-D WB. Although2-D WB had preliminarilyscreened citrullinated proteins as autoantigens, the results remain to be confirmed byfurthermore studies.OBJECTIVE: In present study,we aimed to investigate the expression levels ofscreened Candidate autoantigensKRT84（keratin type II cuticular Hb4）、 KRT4（Cytokeratin4）、TUBB（tubulin beta chain）in synovial tissues and synovial fluids ofRA. Furthermore, the expression levels of autoantibodies in RA blood were detected toidentified the novel autoantigens of RA.METHODS: Immunohistochemistry was applied to detect expression of KRT84、KRT4and TUBB in synovial tissues of RA compared to the samples of OA. For quantitativeanalysis,Western blot analysis was applied to detect expression of KRT84、KRT4andTUBB in synovial tissues of RA compared to the samples of OA. ELISA was appliedto detect expression of the candidate proteins in synovial fluids. ELISA was performedto measure the levels of autoantibodies against KRT84，KRT4and TUBB in sera fromRA patients with the health as controls.RESULTS: Immunohistochemistry indicated that KRT84、TUBB had significantlyhigher expression in synovial tissues of RA than in the samples of OA. No expression ofKRT4was found in synovial tissues of RA,neither in the samples of OA. Western-blotindicated that KRT84、TUBB had significantly higher expression in synovial tissues ofRA than in the samples of OA(P=0.030，P=0.041and P=0.013).KRT4had almost noexpression both in synovial tissues of RA and in the samples of OA Sandwich-ELISAindicated significantly higher expression of KRT84in blood and synovial fluid of patientswith RA than in samples of OA (p=0.0031). Level of anti KRT84autoantibody wassignificantly elevated in blood of RA (p=0.00025) compared with the samples from thehealth. The average levels of anti-KRT4autoantibody and anti-TUBB autoantibody werenot significantly alternated between blood samples of RA patients and the health.However, one third of RA patients had relatively high expression of TUBB autoantibodyin their blood. CONCLUSION: Significantly increased expression of KRT84was detected in RAsynovial membranes and synovial fluids.Autoantibody to KRT84was detected in RAblood. Present study identified KRT84as a novel autoantigens of RA. It is hypnotizedthat the citrullinated KRT84in synovial tissues participated the pathological process ofRA by breaking tolerance of the immune system and driving the auto immune response.About one third of RA patients had relatively high levels of anti-TUBB autoantibody insera of RA patients, indicating that TUBB may be also involved in autoimmunity of RA. OBJECTIVE：This study used a proteomic approach to screen the proteins withdecreased expression in the synovial tissues of rheumatoid arthritis (RA) patients bycomparing their expression profiles to that of osteoarthritis(OA). and ankylosingspondylitis (AS) patients. The result was complemented by a SNP analysis.METHODS: Proteins extracted from the synovial membranes (n=10for each disease)were separated by2D electrophoresis. The proteins with significantly decreasedexpression in the RA samples were subjected to MALDI-TOF/TOF MS. The result wasverified using western blotting. Tag SNPs located in the targeted gene were assessedusing the Taqman assay in a cohort of267Chinese patients with RA and160healthycontrols. The genotyping result was confirmed in a large cohort of389patients with RAand371healthy controls.RESULTS: The proteomic approach detected significantly decreased expression ofvitamin D-binding protein (VDBP) in the synovial membranes from patients with RA,which was confirmed with western blot analysis. SNP rs2282679was significantlyassociated with RA and AS (p=0.026794). The result was confirmed in a large cohort ofRA (OR=0.678639,95%CI=[0.541113~0.851118],p=0.000776).CONCLUSION:1,25-dihydroxyvitamin D3inhibits cell proliferation, immunoglobulinproduction and the release of cytokines through binding to VDBP. VDBP also mediates bone resorption by activating osteoclasts. The decreased expression and the genetic effectof VDBP in RA suggest a novel pathogenic pathway that vitamin D contributes to thearthritic process of the disease.