Strong Inflammation Is Essential For The Expression Of Articular Cartilage-Specific Citrullinated Antigens

BACKGROUND:Rheumatoid arthritis(RA)is a chronic,invasive autoimmune disease that is characterized by cartilage destruction and bone erosion in affected joints,and with intractable joint pain.At present,5%of the world’s population is susceptible to autoimmune diseases.About 0.5-1%of global population is affected by RA.An important step in autoimmune diseases is the loss of immune tolerance to its own antigen and thus producing autoantibodies.Many antigens are available for binding autoantibodies in RA that leading to formation of pathogenic immune complexes(ICs).ICs deposits of synovial tissue activates complement system,inducing the persistence of synovitis in RA patients.Anti-citrullinated protein/peptide antibodies(ACPAs)can be detected on synovial fluid and serum in patients with RA,which has highly specificity and sensitivity to RA and are an important hallmark for the serological diagnosis.ACPAs is present decades before the onset of arthritis,The production of ACPAs is associated with the PAD enzyme.and the target antigens of this kind of autoantibodies contain citrullinated epitopes,it binds to citrullinated proteins and peptides.In the inflammatory environment,apoptosis or autophagy may increase the concentration of intracellular calcium and then PAD enzyme is activated,proteins in synovial tissues are modified,producing citrullinated antigen,activating autoimmune B cells which express and secrete ACPAs,and then inducing autoimmune reaction.the pathogenicity of ACPAs has been reported in a large of studies.However,ACPAs could also have protective effect on arthritis.Type Ⅱ collagen(CⅡ),an important component of articular cartilage,is one of the major sources of RA autoantigens,which is the main target for the attack of inflammatory reaction.Collagen induced arthritis(CIA)is a classic animal model that in many ways resembles RA.CIA can be induced in susceptible animals by immunization with CⅡ.Our team have prepared a series of monoclonal antibodies that against the citrullinated C1 epitope of CⅡ,including the monoclonal antibodies ACC1 and ACC4.These antibodies can bind to citrullinated and non-citrullinated proteins in synovial tissue and cartilage,and also can induce arthritis in mice.OBJECTIV:In order to treat RA in the early stages,it is important to fully understand its pathogenesis and how the joint target tissue is attacked?Therefore,the purpose of this study was to investigate under which condition citrullination can be induced in the cartilage.Establish the animal model of rheumatoid arthritis,to investigate the expression of citrullinated epitopes in articular cartilage protein and find whether expression of citrullinated epitopes is sufficient to induce ACPA response.METHODS:1.Cultured ACC1/ACC4/L243 monoclonal hybridoma cells,inoculated with hybridoma cells into mice,and produced ascites,purified and biotinylated the monoclonal antibodies.2.ELISA was used to detect the secretion of antibodies from monoclonal hybridoma,and the level of anti-CⅡ antibodies in mice.3.Induced protein citrullination experiments with LPS,heat-killed bacteria(E.Coli,S.aureus)and Ⅱ-specific monoclonal antibodies in vitro.4.Induced protein citrullination experiments by injecting LPS into joints of rats.5.Established CIA model in different strains of mice.6.Histology of RA patient serum to detect the staining of the articular cartilage.7.Detected of citrullinated expression in mouse articular cartilage by histology.8.Detected the anti-citrullinated protein antibody responses and CⅡ specific B cell dominant antigen epitopes in mouse serum by Luminex.RESULTS:1.LPS and inactivated bacteria were used to induce citrullination of cartilage but are insufficient to induce citrullination of articular cartilage proteins in vitro.2.A combination of LPS and anti-CⅡ mAbs was used but it also did not induce citrullination of cartilage proteins in vitro.3.To test this in vivo,LPS was injected intra-articularly,which can induce acute(mild)inflammation.This was also cannot induce citrullination of articular cartilage proteins.4.Citrullinated epitope can be detected in strong inflammation when using CⅡinduced in vivo.5.Antibodies from CIA mice were analyzed by Luminex and the results show that more signals are needed to induce ACPA response.CONCLUSIONS:1.Strong but not weak inflammation is needed for citrullination expression in vivo.2.Murine ACC1 monoclonal antibody can bind to CⅡ of articular cartilage from different species.3.Inflammatory articular cartilage proteins in CIA express citrullinated epitopes.4.Expression of citrullinated epitopes is not enough to induce ACPA response.5.H2d haplotype has dominantly suppressed H2q haplotype in arthritis susceptibility and severity,which depends on non-MHC genes.6.Anti-CⅡ(GFS-15JV(F4),GFS-17JV(U1))antibodies appear in the early stages of collagen-induced arthritis in B10Q.Ncfl and BD mice and may serve as early biomarkers for experimental arthritis.