Study On Anti-citrullinated Vimentin Autoantibody And Its ICs In Sera And Synovial Tissues Of Patients With RA

Background and objective:Rheumatoid arthritis(RA)is a common and systemic autoimmune disease characterized with chronic joint inflammatory.The pathogenesis of RA remains unknown,however,abundant researches have showed that autoimmune responses,especially the responses to citrullinated protein(CitP),play a key role in the development of RA.Anti-citrullinated protein autoantibodiy(ACPA)has proved to be the specific biomarker in RA patients.Recent investigations indicated that multiple antigen specific ACPAs could be detected in the patients with RA,including anti-citrullinated fibrinogen(Cit-Fib)antibody and anti-mutated citrullinated vimentin(AMCV)antibody.Moreover,AMCV antibody had been applied in the early diagnosis of RA with high specificity and sensitivity.However,the expressions of AMCV antibody and corresponding immune complexes(ICs)in the serum and synovial tissues of RA and the effects of AMCV-IC on FLSs were still unclear.Immunohistochemistry(IHC),immunocytochemistry(ICC),western blot,enzyme-linked immunosorbent assay(ELISA)and chemiluminescent immunoassay(CLIA)assays had been implied in this research to localize or quantify the CitP,Vim,AMCV antibody,AMCV-IC in the synovial tissues,FLSs and serum of RA patients.ICs from the serum of ACCP+,AMCV+ RA patients,systemic lupus erythematosus(SLE)patients and healthy controls were purified,and the effects of AMCV-IC on FLSs proliferation were investigated.Materials and methods:(1)Synovial tissues from RA patients who undergoing surgery were collected.Moderate synovial tissues were fixed by 4%paraformaldehyde for further immunohistochemistry experiments and fresh synovial tissues were digested by collagenase to extract FLSs.Immunocytochemistry was employed to detect the expressions of CitP and Vim in FLSs and immunohistochemistry was adopted to examine the CitP,Vim,Ig and C3 expressions in RA synovial tissues.(2)Detection of AMCV antibody and ACCP antibody.Serum of diagnosed RApatients,SLE patients and healthy controls were collected,the levels of ACCP antibody were examined by CLIA.Serum of ACCP+ RA patients(n=100),SLE patients(n=42)and healthy controls(n=50)were enrolled in this study,the concentrations of AMCV antibody were tested by ELISA technique.(3)Affinity chromatography with immobilized protein G was utilized to purity the ICs from the serum of ACCP+/AMCV+,ACCP+/AMCV-RA patients,ACCP-/AMCV-SLE patients and healthy controls.The concentrations of purified ICs were determined by BCA method,and ELISA and CLIA were used to detect the levels of AMCV antibody and ACCP antibody.The expressions of CitP and Vim in ICs were determined by western blot assay.(4)Purified ICs from the serum of the above four groups were used to stimulate RA FLSs,and CCK-8 method was employed to detect the proliferation after stimulation.(5)Different concentrations of AMCV-IC formed in vitro were used to stimulate RA FLSs and CCK-8 method was adopted to examine the proliferation of FLSs after stimulation.Results:(1)Immunohistochemistry results showed that 7 out of 9 synovial tissues expressed varying CitP degrees ranging from + to +++,which were mainly existed in the synovial lining layer and were significant higher than OA patients.Ig,C3 and Vim were widely expressed in both RA and OA patients.Immunocytochemistry assay showed that except specific Vim,CitP was also expressed in the cytoplasm of RA FLSs.(2)The positive rate of AMCV antibody in the serum of ACCP+ RA patients(n=100)was 80%.The positive rate of AMCV antibody in SLE patients was 4.8%and the ACCP antibody positive rate was 7.1%.The AMCV antibody and ACCP antibody in healthy control group were negative.(3)ICs purified from ACCP+/AMCV+,ACCP+/AMCV-RA patients,ACCP-/AMCV-SLE patients and healthy controls by affinity chromatography with immobilized protein G were identified by SDS-PAGE,and the result showed that ICs contained high purified antigens and antibodies.Western blot results indicated that ICs from ACCP+/AMCV+,ACCP+/AMCV-RA patients contained bands that not existed in the SLE and healthy control groups,which suggested that ICs from the above two groups might contain CitP or Cit-Vim.(4)CCK-8 assay results showed that ICs(AMCV-IC)from the serum of ACCP+/AMCV+ group could stimulate the proliferation of RA FLSs,and the proliferation activity was significant higher that of ACCP+/AMCV-,SLE and healthy control groups.(5)AMCV-IC formed in vitro could also stimulate the proliferation of RA FLSs in a concentration-dependent manner examined by CCK-8 assay.Conclusions:(1)CitP were widely expressed in the synovial tissues of RA patients and FLSs.(2)AMCV-IC from serum or formed in vitro could both stimulate the proliferation of RAFLSs.