| Part1Effects of bone marrow-derived mast cells on the expression of type II collagen and glycosaminoglycan(GAG) of chondrocytesBackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by the chronic invasive arthritis. The major pathological change of the rheumatoid arthritis includes articular cartilage and bone damage.The pathogenesis of RA remains unclear at present The incidence of RA is about3/10000all over the world, the total prevalence of RA is about1%. Last decade, the treatment of RA developed rapidly, combined application of the new anti-rheumatic drugs and biological agents which maked the treatment of RA more efficient, such as infliximab, etanercept,Abatacept, anakinra, Tocilizumab and so on. Anti-TNF-a is an important advance of treatment of RA and spondyloarthropathy, but evidence-based medical research find that30-40%of patients’symptoms don’t significantly improved, the efficacy of other biological agents such as anti-IL-1or IL-6monoclonal antibody is even worse which indicate that except TNF-a,IL-1, IL-6and other external cytokines, some other mechanisms are involved in RA.The role of mast cells and their associated cytokines in the pathogenesis of RA is one of the hot spots recent years. Mast cells account for about3%of the total cells in normal synovium, its function may be involved in repairing the damage or clearing pathogens, studies have shown that the number of activated mast cells in normal joints is1%to5%, and in the synovium of RA patients, the number of activated mast cells increase strikingly to10%-15%. Mast cells often populate the pannus, with localization at the junction of pannus and cartilage and in areas where pannus is invading the cortical bone, and participante in the pathogenesis of RA via many ways, finally lead to the damage of cartilage and bone. Synovial mast cell interact with other cells which lead to the increasing of vascular permeability, induce activation of synovial cells, promote angiogenesis, matrix remodeling and destruction of cartilage and bone in turn.Synovial mast cells express a lot of basic fibroblast growth factor, Leukotriene C4and platelet-derived growth factor etc., which helped the hyperplasia of synovial fibroblasts and the proinflammatory activity of synovial fibroblasts. Such as the synovial fibroblasts express the cathepsins and the matrix metalloproteinases which involve in the damage of cartilage and bone, in addition, synovial mast cells also secreted a variety of cytokines involved in the pathogenesis of RA, such as TNF-α, IL-1β,IL-4, IL-5and IL-6, etc.One of the pathological features of rheumatoid arthritis is the destruction of articular cartilage. The cytological basis is the degradation of cartilage extracellular matrix. The two main ingredients of the extracellular matrix of cartilage are type II collagen and aggrecan, account for about90%of normal articular cartilage. Proteoglycans contains many ingredients, GAG is one of its main ingredients, type II collagen and other components constituted network skeleton of articular cartilage. Proteoglycans and hyaluronic acid crosslinked with each other to form brush-like structure which embedded cartilage. Type Ⅱcollagen and proteoglycan enable articular cartilage with good flexibility and pressure resistance. The loss and degradation of type II collagen and proteoglycan lead to the loss of elasticity of articular cartilage which result in damage to the articular cartilage. Therefore, we focus on the impact of mast cells on cartilage collagen type II and GAG to clarify the relationship between mast cells and the destruction of articular cartilage, and find the new targets for RA treatment.Method1. Experiment groups Primary cultured bone marrow-derived mast cells (BMMCs) and chondrocytes, there were four groups: chondrocytes (control group), chondrocytes+BMMCs group (BMMCs group), chondrocytes+BMMCs+sodium cromoglycate group (DSCG group), chondrocytes+BMMCs+Compound48/80group (C48/80group), three wells and two time points24h and48h.2. Primary cultured bone marrow-derived mast cells(1) A5weeks female C57BL/6mice was killed, sterilized in75%alcohol for three minutes.(2) Cut off the skin of hind legs of mice, taked down the hind femur, placed in the dish with3ml15%FBS RPMI-1640, removed the muscle tissue and connective tissue of femur under the stereo microscope, and then cut off the ends of the femur.(3) lml syringe was used to flush out the bone marrow until the bone marrow cavity turned white and cultured the cells with RPMI-1640medium in the clean bench, and the recombinant mouse IL-3and SCF was added in the concentration of10μg/ml.The culture medium was changed after3days, then changed the culture medium every4-7days. After4-6weeks, the cells were almost mast cells.3. Primary cultured chondrocytes of mouse(1) Two5weeks female C57BL/6mice were killed by cervical dislocation, sterilized in75%alcohol for three minutes.(2) Cut off the skin of hind legs of mice, taked down the whole hind legs, placed in the dish with serum-free DMEM.(3) Separated the cartilage cap of femoral head, fibula and tibia head and shinbone under the stereoscopic microscope, removed the synovial tissues and connective tissues. Cut the cartilage into lmm3fragments, digested the fragments with0.25%trypsin at37℃shaking (200/min) for30minutes,washed fragments with PBS twice. Digested it with0.3%of the type IV collagenase in the37℃oscillation incubator (200beats/min) for1hour,300g centrifugation harvested supernatant. (4) Cultured the cells with DMEM in the clean bench. Changed the medium three days later, then the medium was changed every5-7days. When cells grownd more than80%, cells were digested with trypsin according to1:2-3passage.4. Expression of type II collagen of chondrocytes of each groups200μl lysis buffer was added to each well of different groups, put the buffer on the ice for30minutes transferred the cell lysate to a1.5ml EP tube, centrifuged at14,000rpm and harvested supernatant. Western Blotting and ELISA were used to detecet the expression of chondrocytes of differert groups.5. Expression of glycosaminoglycan (GAG) of chondrocytes of each groups200μl lysis buffer was added to each well of each groups, put the buffer on the ice for30minutes, transferred the cell lysate to a1.5ml EP tube, centrifuged at14,000rpm and harvested supernatant.1,9dimethylmethylene blue(DBM PH=3.0,16mg/L) was used to detect the expression of glycosaminoglycan (GAG) of different groups.6. StatisticsAll values are mean±SEM. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was P＜0.05.Result1. Primary cultured bone marrow-derived mast cellsFlow cytometry was used to detecte the expression of CD117of bone marrow-derived mast cells cultured for four weeks, the purity of bone marrow-derived mast cell cultured for four weeks was94%.2. Primary cultured chondrocytesAfter one week, chondrocytes showed typical cobblestone growth,The toluidine blue staining of chondrocytes were purple, Alcian blue staining of chondrocytes were blue.3. The effects of mast cells on the expression of type Ⅱ collagen of chondrocytes After treatment for24hours,The BMMCs group and DSCG group had no significant difference in the expression of type II collagen of chondrocytes compared with the control group (p>0.05), Compared with the control group and BMMCs group,the expression of type II collagen in C48/80group was significantly lower (p＜0.01), The results of48hours were consistent with24hours’.There was no significant difference in the expression of type II collagen of chondrocytes between the24hours and48hours.4. The effects of mast cells on the expression of GAG of chondrocytesAfter treatment for24hours,The BMMCs group and DSCG group had no significant difference in the expression of GAG of chondrocytes compared with the control group (p>0.05), Compared with the control group and BMMCs group,the expression of GAG in C48/80group was significantly lower (p＜0.01), The results of48hours were consistent with24hours’, There was no significant difference in the expression of GAG of chondrocytes between the24hours and48hours.Conclusion: C48/80activated BMMCs can reduce the expression of type II collagen and GAG of chondrocytes.PartⅡEffect of mast cells on the expression of cathepsin S in collagen antibody-induced arthritis (CAIA)BackbroundMast cell is a kind of nonspecific immune cells which widely distribute over the body tissues and organs, previous studies almost focus on the role of mast cell in acute allergic reaction.Not until2002, Studies found that the incidence of arthritis depended on the presence of mast cells in animal experiments which confirmed that mast cells played a critical role in the pathogenesis of RA, especially in the immune complex-mediated arthritis, but how mast cells involved in the pathogenesis of RA, is not yet entirely clear.Cathepsin S is a lysosomal enzyme that belongs to the papain family of cysteine proteases. This protein is expressed by antigen presenting cells including macrophages, B-lymphocytes, dendritic cells and microglia. This enzyme has the catalytic activity of a variety of proteins, especially MHC II constant chain and extracellular matrix (ECM) proteins. The list of proposed cathepsin S substrates includes laminin, fibronectin elastin, osteocalcin and some collagens. It has been proved that cathepsin S was involved in many diseases, such as atherosclerosis, asthma, chronic obstructive pulmonary disease, tumor, osteoarthritis and several autoimmune diseases. Now, the role for cathepsin S in the pathogenesis of RA is less. Past researchs showed that the mice knocked out cathepsin S had a low sensitivity to CIA. The level of cathepsin S in synovial fluid is highly increased in RA patients. Indicated that cathepsin S may be involved in the pathogenesis of RA. Past research has shown that, Overexpression of cathepsin S induces atopic dermatitis in mice, mast cells accumulated in the skin lesion area. In abdominal aortic aneurysm mouse model, the expression of cathepsin S was significantly reduced in mast cell-deficient mice, these studies suggest that mast cells may regulate or secreted cathepsin S in the development process of the disease.Collagen antibody-induced arthritis (CAIA) a simple mouse model of rheumatoid arthritis that can be used to address questions of pathogenic mechanisms and to screen candidate therapeutic agents. Arthritis is induced by the systemic administration of a cocktail of monoclonal antibodies that target various regions of collagen type II, which is one of the major constituents of articular cartilage matrix proteins, together with lipopolysaccharide (LPS). Administration of LPS after the antibody cocktail reduces the amount of monoclonal antibody required to induce arthritis.The monoclonal antibody-induced arthritis model offers several key advantages over conventional CIA. First, arthritis is induced within only a few weeks rather than the several months that are required to induce arthritis by immunization with type II collagen. Second, unlike the CIA model, which requires autoantibody generation, CAIA can be generated in a wider spectrum of mice, including gene-deficient mice, transgenic mice and strains that are resistant to classic CIA. Moreover, the CAIA model has a high uptake rate. The pathogenic features of the CAIA model have striking similarities with human rheumatoid arthritis, including synovitis with infiltration of polymorphonuclear and mononuclear cells, pannus formation, cartilage degradation and bone erosion. CAIA is an extension of the classical collagen-induced arthritis (CIA) model, which has been used extensively in rats, mice and so on.Therefore, we established collagen antibody-induced arthritis (CAIA) model, investigate the effects of mast cells on the expression of cathepsin S in collagen antibody-induced arthritis (CAIA) model, explore the role of mast cells in the pathogenesis of RA.1. Experiment groupsThere were four groups:control group, CAIA model group, CAIA model+DSCG group (sodium cromoglycate,one kind of mast cell stabilizer, intraperitoneal injection25mg/kg/d), CAIA model+C48/80group (Compound48/80,one kind of mast cell activator,intraperitoneally4mg/kg/d).2. Establish of collagen antibody induced arthritis (CAIA) modelCAIA model group:on day0, administer150μl(1.5mg) i.v. per mouse of five anti-type Ⅱcollagen monoclonal antibody complex,on day3, administer of70μl (35μg) LPS i.p. per mouse; CAIA model+C48/80groups: the same with the CAIA model, in addition, administer4mg/kg/d i.p. per mouse of C48/80; CAIA model+DSCG groups: the same with CAIA model, in addition, administer25mg/kg/d i.p. per mouse of DSCG; the control group (control):On day0, administer150μli.v. per mouse of0.9%saline, on day3, administer of70μl0.9%saline i.p. per mouse.3. Arthritis scores of mice in each groupsThe standard of Arthritis scores:0point:normal;1point:mild wrist or ankle swelling;2points:moderate wrist or ankle swelling;3:severe swelling, including toe, foot, ankle;4points:Max inflammation.4. Collection and processing of mice of lung and ankle(1) Anesthetized by intraperitoneal injection in mice: intraperitoneal injection of3%sodium pentobarbital and fixed on the mouse stage with supine position. (2) Quickly opened the chest, the gavage needle puncture from the left ventricle into the aorta, and cut the right atrial appendage, used4℃ice-cold saline (containing heparin20U/ml)200ml quickly rinse, seeing the kidney, liver, lung, skin and muscle quickly paled, the clear colorless liquid from right atrial appendage flow out.(3) Rapidly separated the lung and ankle, placed in a cuture dish containing4℃precooled1x PBS (pH7.4) and cleaning. Put the tissues in4%paraformaldehyde solution for fixation at4℃in24h. Rinse Lung tissue with water in2h, Graded alcohol dehydration, xylene, dipped wax embedded, ankles were treated with10%neutral decalcification EDTA solution, Rinse ankle tissue with water in2h, graded alcohol dehydration, xylene, dipped wax embedded.4. HE staining of lung tissue and ankle of mice in each group.5. Expression of CD117and catepsin S of mast cells in ankle and lung tissue of mice in each group.6. StatisticalAll values are mean±SEM. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was P＜0.05.Result1. Arthritis scores of each group of miceAfter injection antibody and LPS, the control group and the CAIA model+DSCG group were0point, The Arthritis scores of CAIA+C48/80group and CAIA model began to appear on day4-5, reach peak on day8-9days,on day10-13decreased, but The Arthritis scores of CAIA model+C48/80group was significantly higher than the other three groups (p<0.05),on day10-13,There is no significantly difference among the other three groups.2. HE staining of lung tissue and ankleHE staining show that CAIA model group, CAIA+DSCG group, CAIA+C48/80groups have different levels of synovial hyperplasia and cartilage and bone damage, And cartilage and bone and damage synovial hyperplasia of CAIA+C48/80 groups is significantly higher than the other two groups, HE staining of lung tissue showed no significant change of four groups.3. Expression of CD117of mast cells in ankle and lung tissue of mice in each groupsExpression of CD117of mast cells in ankle and lung tissue: Compared to CAIA+DSCG groups and control group, expression of CD117of mast cells in ankle and lung tissue of CAIA group were significantly increased (p<0.05), the expression of CD117of mast cells in ankle and lung tissue of CAIA+C48/80group ankle was significantly increased compared to the other three groups (p<0.05).4. Expression of cathepsin S in ankle of mice in each groupsExpression of cathepsin S in ankle:Compared to CAIA+DSCG groups and control group, expression of cathepsin S in ankle of CAIA group were significantly increased (p<0.05), the expression of cathepsin S in ankle of CAIA+C48/80group ankle was significantly increased compared to the other three groups (p<0.05).Conclusion: Mast cells may participate in damage of the joint in of CAIA model via regulating or secreting cathepsin S. |
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