Effects Of Immune Complexes From Synovial Fluid Of Patients With Rheumatoid Arthritis On Fibroblast-like Snoviocytes

Backgroud:Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by persistent inflammation of the synovial tissues of the joints and infiltration of inflammatory cells, resulting in the loss of joint function, progressive joint destruction and deformity, even loss of function, but the mechanisms underlying the initiation and progression of RA are still not completely understood.Recently, many autoantigen, autoantibodies targeting cyclic citrullinated peptide (ACPA) and immune complexs (ICs) have been found in serum, synovial fluid and cartilage surface from RA patients. ICs can induce proinflammatory cytokines secretion by combinating with complement, Fc receptor and complement receptor, contributing to progressive cartilage and joint destruction and deformity. Osteoarthritis (OA) is a chronic disease characterized by articular cartilage degeneration and and bone hyperplasia. Knee osteoarthritis (KOA) and RA have similar clinical symptoms, so it has important diagnostic value for the differential diagnosis of KOA and RA.The synovial tissue is mainly composed of fibroblast-like synoviocytes (FLSs). There is a complicated interaction network between FLS, macrophages and lymphocytes in RA synovial inflammation. For now, the excessive proliferation and activation of FLSs is the real cause of synovial hyperplasia, not the infiltration of inflammatory cells in RA synovial joints. Moreover, FLSs can also generate cytokines (CKs) and matrix metalloproteinases (MMPs) by autocrine and paracrine, and play a pleiotropic role in the interaction with mononuclear macrophages, T cells, B cells, stromal cells and bone and cartilage cells, leading to RA synovial hyperplasia and cartilage and bone destruction. Therefore, it is important to study the function of synovial cells and further explore the pathogenesis of RA and screening anti-rheumatic drugs in vitro.Previously, we reported that the levels of anti-citrullinated peptide antibody specific immune complexes (ACPA-IC) from serum of RA patients were significantly higher than those in systemic lupus erythematosus (SLE) patients and healthy persons and were positively correlated with ACPA levels in serum. To further elucidate the clinical significance and pathogenic roles of the ACPA-IC played in RA, in the present study we isolated FLSs from the synovial tissues obtained from RA patients and exposed them to ACPA-IC or C-IC in vitro. The results showed that ACPA-IC enhanced the proliferation capacity and promoted the pro-inflammatory cytokines secretion in FLSs from RA patients, suggesting the pathogenic role of ACPA-IC in RA.So far, the research about the function ICs on RA synovial cells is only focused on mononuclear macrophages and cartilage cells. However, it has not yet been reported that the influence of ACPA-IC on RA FLSs proliferation and CKs, MMPs secretion in RA patients. In view of the fact that synovial fluid (SF) is one important target of RA immune inflammatory damage and ACPA, IC and FLSs are important effector molecules and cells in RA, researching the IC from SF can directly reflect the influence of IC and FLSs in synovial inflammatory injury. According to the preliminary research, we put forward the following hypothesis:local inflammation reaction in RA synovial joints results in the citrullination of related autoantigens, leading to the production of ACPA-IC, which comes from the combination of ACPA and citrullinated protein (Cit-P). The ACPA-IC contained a variety of Cit-P combine with FLSs induces the expression of related gene in the nuclei and activates the corresponding signal pathways, promoting signaling molecules activity and secretion of CKs and MMPs. Conversely, these inflammatory mediators can acts on various immune active cells in synovial tissue (ST), leading to more Cit-P production and more serious bone and cartilage destruction.The family of peptidylarginine deiminase (PADI) enzymes is responsible for the generation of the amino acid citrulline from arginine in proteins by post-translation modification. There is a close relationship between formation of citrullinated protein and generation of ACPA. These enzymes require Ca2+ to be enzymatically active and produce ammonia as a side product in the conversion of peptidylarginine to peptidylcitrulline. It is considered that citrullination interfer with the methylation of histone residues and PADI can reverse methylation of amino acid residues by transferring methylation of histone residues to citrulline. Inmammals, the family consists of five isotypes (PADI1-4 and PADI6). Each PADI enzyme has its own typical subcellular localization, tissue distribution and substrate specificity. PADI1 is mainly expressed in skin tissue, where it can citrullinate filaggrin and keratin, a process that contributes to skin cornification. The most ubiquitously expressed PADI enzyme is PADI2, the highest expression levels of which are found in the central nervous system, spleen, secretory glands and skeletal muscle tissue. Its known substrate proteins include myelin basic protein in the brain and spinal cord and vimentin in apoptotic monocytes and macrophages. The expression of PADI3 is restricted to hair follicles. Citrullination of trichohyalin contributes to directional hair growth. PADI4 is found mainly in white blood cells like granulocytes and macrophages and localizes to the nucleus. In the nucleus, its main protein substrates are nucleophosmin and histones. The most recently identified PADI enzyme, PADI6, was first discovered in egg cells and embryos and has its highest expression levels in ovary and testis tissue, although expression of the PAD6 mRNA can also be found in leukocytes. Its function and enzymatic activity remain unclear. Among them, PADI2 and PADI4 have close relationship with RA.Objective:1. To study possible effects of ACPA-IC on proliferation capacity of FLSs, RA FLSs was stimulated with different concentrations of ACPA-IC exacted respectively from RA and OASF.2. To study possible effects of ACPA-IC on pro-inflammatory cytokine and MMPs secretion of FLSs, RA FLSs was stimulated with different concentrations of ACPA-IC exacted respectively from RA and OA SF. The levels of IL-6, IL 8,GM-CSF and MMP-3 in supernatants was detected by ELISA methods.3. Proteins was extracted respectively from synovial tissue (ST) of patients with RA and OA, and then identified respectively.4. To analyse the possible difference of PADI4 expression in ST from RA and OA patients, extracted protein was detected by western blot method. In order to study the possible correlation between PADI4 expression in ST and clinically relevant indexes and further explore the pathogenic role of PADI4 in RA, citrulline peptide antibody (CCP), anti-streptolysin (ASO), RF and C-reactive protein (CRP) in serum of RA patients were tested.Methods:1. ICs from the SF of RA patients and OA patients were purified by affinity chromatography with immobilized protein G and ICs concentration were detected by BCA. The levels of ACPA-IC were determined by immunochemiluminescence assays.2. FLSs were isolated and cultured from ST from RA patients who undergoing surgery. Passages 4-7 of the FLSs were used in subsequent experiments, at which time they were a homogeneous population of fibroblasts.3. Purified ICs from the SF of the RA and OA groups were used to stimulate RA FLSs, and CCK-8 method was employed to examine the proliferation of FLSs after stimulation.4. Different concentrations of ACPA-IC after purification were used to stimulate RA FLSs and ELISA method was adopted to examine the levels of IL-6, IL-8,GM-CSF and MMP-3 in supernatants after stimulation.5. Proteins were extracted respectively from ST of patients with RA and OA by liquid nitrogen. The expression of PADI4 was detected by western blot method.Results:1. Influence of RA-IC from SF on FLS proliferation capacity.Compared with the control group treated with IC and blank control group, RA-IC stimulation had an obvious impact on inhibiting proliferation of the RA FLSs at concentrations of 25,50,100 and 200μg/ml respectively, especially at the concentration of 25,50,100μg/ml, with significant difference(P<0.01).The higher of concentrations, the weaker of inhibitory effect. Compared with BC group,OA-IC has no effect on the proliferation of FLSs.2. Influence of RA-IC from SF on CKs and MMPs secretion in FLSs.After FLS cultures were treated withRA-IC for 24 to 72 hours, the IL-6, IL-8 and GM-CSF levels in the culture supernatants were remarkably higher than those treated with IC in ACPA-IC(-) OA group(P<0.05). Additionally, when FLSs cultures were treated with RA SF at concentrations of 50,100 and 200μg/ml, the levels of GM-CSF and IL-6, which were secreted by FLSs, were significantly higher than those in OA group and blank control (BC) group. The levels of IL-8 which came from FLSs stimulated by four different concentrations of IC were significantly higher than those of the OA group and BC group (P<0.01). However, only when FLSs cultures were treated with at concentrations of 100 and 200μg/ml, the levels of MMP-3 were significantly higher than those in OA group and BC group(P<0.01), suggesting IC from RA SF affects weakly on the capacity of MMP-3 secretion at low concentrations. There is no significant difference between OA-IC group and BC group, except for low concentrations. At the same time, there was a positive correlation between levels of IL-6, IL-8,GM-CSF and MMP-3 in three group and time3. The identification of protein from ST.Proteins extracted from RA and OA ST were mixed with an equal volume and quantified by BCA method. SDS-PAGE results showed ICs from RA group contained bands that not existed in the OA group. Two bands located in 25KDa and 55KDa represented respectively the light chain and heavy chain of antibody Ig.4. PADI4 expression levels of ST from RA and OA patients.The PADI4 expression level of ST from RA was obviously higher than that of OA (P<0.05), but not found the correlation between it and anti-CCP, RF, CRP and ASO levels in serum.Conclusion:1. Relative high levels of ACPA-IC from RA SF had an impact on inhibiting proliferation of the FLSs. The inhibitory effect decreased with the increase of concentrations, but the effect of low and higher concentrations of ACPA-IC was needed to confirm.2. After FLS cultures were stimulated with ACPA-IC from RA SF, the IL-6, IL-8, GM-CSF and MMP-3 levels in the culture supernatants were remarkably higher, suggesting that ACPA-IC contained Cit-P or ACPA might have an important role in FLSs secretion.3. SDS-PAGE results showed ICs from RA group contained bands that not existed in the OA group, which suggested that ICs might contain Cit-P and antigens that bonds with mouse-anti-human PADI4 antibody.4. The expression level of PADI4 in ST from RA patients was higher than that in OA patients, but there might not be a correlation between its level and anti-CCP, RF, CRP and ASO levels in serum.