Study On Anti-CALR Autoantibody As A Potential Primary Sjogren’s Syndrome Specific Biomarker

Objective:Primary Sjogren’s Syndrome(pSS)is a chronic systemic disease,and characterized by functional impairment of exocrine glands such as salivary and lacrimal glands,leading to dry mouth and dry eyes.In addition to exocrine glands,other important organs may also be damaged,such as lung,kidney,brain and so on.Because of the strong concealment and complex pathogenesis of the disease,it is difficult to diagnose and get good treatment in the early stage,which seriously affects the life of patients.Although some autoantibodies have been used in the diagnosis of Sjogren’s syndrome(such as anti-SSA and anti-SSB autoantibodies),their specificity and sensitivity are not good enough,and the glandular damage may have occurred before the presence of anti-SSA and anti-SSB autoantibodies in some patients.Therefore,the purpose of this study is to find some new early gland-specific autoantibodies in serumnegative pSS patients(anti-SSA and anti-SSB negative),which can be used as a supplement to anti-SSA and anti-SSB autoantibodies in the clinical diagnosis and treatment of pSS.Methods:Immunofluorescence was used to analyze whether there were autoantibodies against the components of human salivary gland epithelial cell line A253 in the serum of patients with pSS.The protein components of A253 cells were separated by two-dimensional(2-D)gel electrophoresis and transferred to PVDF membrane,and then incubated with sera of patients with serum-negative pSS and healthy controls,respectively,to find possible pSS-specific autoantibodies.The corresponding proteins were identified by mass spectrometry(Liquid chromatographytandem mass spectrometry,LC-MS)on PAGE gel,and the possible pSS-specific autoantigens were analyzed.By reviewing the literatures,four potential antigens were selected to be further investigated by enzyme-linked immunosorbent assay(ELISA),and verified by Western blot to identify the new specific autoantibodies of pSS patients.Clinical significance of the new pSS specific autoantibodies identified was determined by ELISA in large simples of pSS patients.Furthermore,immunohistochemical method was used to detect the expression of corresponding autoantigens in labial glands of patients with pSS,and to explore its possible role in the pathogenesis of pSS.Results:There were autoantibodies against the protein components of A253 cell in the sera of serum negative pSS patients.2-D blot and mass spectrometry identified 6 autoantigens that may be associated with pSS.Investigated by ELISA,it was found that anti-calreticulin(CALR)autoantibody were significantly increased in the sera of pSS patients.Western blot confirmed the existence of anti-CALR autoantibody in the sera of pSS patients.The correlation between anti-CALR autoantibody and clinical characteristics of pSS patients were analyzed by enlarged samples.It was found that the titers of anti-CALR autoantibody in pSS patients were positively correlated with EULAR Sj(?)gren’s Syndrome Disease Activity Index(ESSDAI),ESR,IgG,IgG2,IgG3,KL-6,fibrinogen,D-dimer and fibrin degradation products,while no correlateion with complement C3,complement C4 and C-reactive protein.The incidence of vasculitis in pSS patients with positive anti-CALR autoantibody was significantly higher than that in pSS patients with negative anti-CALR autoantibody.The positivity of antinuclear antibody,anti-SSA antibody,anti-SSB antibody,anti-CENP B antibody and Rheumatoid factors(RF)were higher in pSS patients with positive anti-CALR autoantibody.The expression of IgG,IgG1,IgG3 in pSS patients with positive antiCALR autoantibody were higher than that in pSS patients with negative anti-CALR autoantibody,while the expression of IgM was lower.Anti-CALR autoantibody were found to be of significance in the diagnosis of pSS by using the method of receiver working characteristic(ROC)curve.The expression of CALR could be detected in labial glands of patients with pSS,but there was no significant correlation between the expression of CALR and the titer of anti-CALR antibody in serum and the severity of lymphocyte infiltration in labial glands.Conclution:The expression of anti-CALR autoantibody was significantly increased in pSS patients,which might be a new specific autoantibody for pSS and can be used as a supplementary diagnosis for pSS patients with negative anti-SSA and antiSSB autoantibodies.At the same time,anti-CALR autoantibody may also be used as a serum biomarker to evaluate the disease activity of pSS patients.In recent years,studies have found that CALR plays an important role in immune regulation,suggesting that it may become a new target in the study of the pathogenesis of pSS.