| Rheumatoid arthritis (RA) is a systemic inflammation disease characterized by chronic and invasive synovitis that causes cartilage destruction and subchondral bone erosion. Its a chronic inflammatory function disorder which mainly contains synovial lining layer hypertrophy caused enhanced invasive of macrophage-like synovial cells and fibroblast-like synovial cells. There is abundant evidence that there are numerous macrophages which produce large amounts of proinflammatory factors and chemokines in the inflamed synovial membrane and excessed synovial fluid .These macrophages are closely related to the pathogenesis of RA, and ultimately, lead to increased inflammation and joint destruction. Besides, in RA, they can be used as antigen presenting cells to interaction with T lymphocytes to promot generating supramolecular structure- the Immunological synapse which can lead the activation of T lymphocytes in the body, at the same time, macrophages will also be activated. Activated macrophages secrete large amounts of IL-1, TNF-α, IL-6 and other cytokines,helping promote the progression of rheumatoid arthritis. Macrophage Activation Syndrome Which endanger the lives of patients will appear when large numbers of macrophages and T cells over-activation or excessive proliferation.Therefore, macrophage treatment of rheumatoid arthritis as the main therapeutic target, researching how to inhibit activation and promote apoptosis way of macrophage will play a role-oriented path to the treatment of rheumatoid arthritis.A study have reported that immunological synapse formation in the human body can inhibit apoptosis of participated antigen-presenting cells, so whether the large number of macrophages in RA patients will reduce the rate of apoptosis of themselves when involving in immunological synapse formation ? No relevant reports at home and abroad. At the same time, studies have shown immunosuppressant Cyclosporin A commonly used in the treatment of RA can inhibit immunological synapse formation in monocytic cell line THP-1,but whether Cyclosporin A can also inhibit the apoptosis macrophages participated immunological synapse have no related researches,too, that is why the contents of this experimental design the following experiment:【Objective】This subject is to study:1、To compare apotosis of THP-1 monocytic cell line induced macrophage when cocultured with Jurkat T cells to form immunological synapse with apotosis of macrophage cultured alone.2、To observe the apotosis of macrophages abound in RA when involving in immunological synapse formation.3、To explore the regulation effect of high expressed Cyclophilin A in RA to the apotosis of macrophage which participated the formation of immunological synapse.4、Further explore the effect of immunosuppressant Cyclosporin A which is receptor of Cyclophilin A and commonly used in the treatment of rheumatoid arthritis,to the apotosis of macrophage participated immunological synapse.【Methods】Mainly divided into two parts:1、THP-1 induced macrophages were coated with SEB(100ng/ml)and cocultured with activated Jurkat T cells,then incubated the co-culture system in the RPMI-1640 for 16 hours to induce apopotosis. The apopotosis of the macrophages were analyzed by flow cytometry by Annexin V-PI staining or observed by TUNEL staining.The macrophages cultured in the RPMI-1640 alone were used as control.Meanwhile, CypA(200ng/ml)or CsA(1 ug/ml) were added or not to observe the apoptosis of macrophages.2、10 healthy controls and 10 confirmed cases of active RA patients were collected. CD4+T cells isolated by immunomagnetic beads were transfered to anti-CD3 antibody-coated 24-well plate to induce activation for 24-hours,then actived CD4+T cells were co-cultured with induced macrophages to promote the immunological synapse formation.Adding CypA(200ng/ml)、CsA(1 ug/ml) or not. After that,co-culture system was transfered into serum-free RPMI-1640 medium 16 hours to indce apoptosis. The apopotosis of the macrophages were analyzed by flow cytometry by Annexin V-PI staining and observed by TUNEL staining. Differences between groups were examined for statistical significance using the Student,s t-test.【Results】1、Apoptosis rate of THP-1 macrophages observed by Annexin V-PI flow cytometry assay show:the apoptosis of macrophages which participated immunological synapse is (32.9±2.8)%,significantly lower than the apoptosis rate of macrophages cultured alone (61.4±2.4)% (P <0.05).After adding the CypA, the apoptosis rate of macrophages decreased to(27.2±2.1)%,lower than the apoptosis of macrophages which participated immunological synapse formation(P <0.05).However, adding CsA in the process of synapse formation can increased the rate of apoptosis of macrophages to (48.8±2.0)% (P <0.05).2、Apoptosis of macrophages in RA and healthy controls observed by Annexin V-PI flow cytometry assay show:In RA patients,the apoptosis rate of macrophages cultured alone in RPMI-1640 is (24.1±9.7)%,significantly lower than the apoptosis rate of macrophages cultured alone in healthy controls (37.1±12.5)% (P <0.05).When participating the formation of Immunological synapse,the apoptosis rate of macrophages in RA droped to (15.2±5.5)%(P <0.001).After adding the CypA, the apoptosis rate of macrophages decreased to (13.7±3.9)%,lower than the apoptosis rate of macrophages which participated in immunological synapse formation(P<0.05).But adding CsA can reverse the rate of apoptosis of macrophages in the time of synapse formation (15.2±5.5)% to (19.3±6.1)%(P <0.01).In healthy controls,macrophages apoptosis was protected by synapse formation, but the effect of CsA/CypA to the process of synapse formation is no statistically significant.3、TUNEL apoptosis detection demonstrate that the percentage of TUNEL + cells rate of macrophages cultured alone in RPMI-1640 in RA is (26.5±4.4)%, however,the rate decreased to (19.0±5.1)% when macrophages participated the formation of immunological synapse(P <0.01)After adding CypA in the process of synapse formation , the TUNEL + cells rate of macrophages droped to (18.2±2.6)% (P <0.05).But Adding CsA in the process of synapse formation can increase TUNEL + cells rate of macrophages back to (30.8±4.2)%(P <0.05). In addition,percentage of TUNEL + cells rate of macrophages cultured alone in RPMI-1640 in healthy controls is (35.0±7.3)%, Significantly higher than TUNEL + cells rate of macrophages cultured alone in RA(P <0.05). At the same time,when participating the formation of Immunological synapse,percentage of TUNEL + cells rate of macrophages in healthy controls and cell lines can decrease TUNEL + cells rate compare with macrophages cultured alone,the difference has statistically significant.However,the difference between other groups in healthy controls or cell lines have no statistically significant.【Conclusion】1、The immunological synapse formed between superantigen SEB coated THP-1 derived macrophages and Jurkat T cells can significantly reduce the apoptosis rate of macrophages,indacateing that the formation of synapses can inhibit the apoptosis rate of macrophages.2、In RA,the immunological synapse formation between macrophage and T cells can significantly inhibit the apoptosis of macrophages,that is, prolong survival of them.3、High expressed Cyclophilin A in RA can promote the protection effect of the immunological synapse in inhabiting the apoptosis of macrophages.4、Cyclosporin A can suppress protection effert of immunological synapse on macrophages apoptosis through weaken immunological synapse formation,that is,CsA can helping promote the apoptosis of macrophage .This effect provide a theoretical basis to CsA reduceing the secretion of inflammatory cytokines and decreasing joint destruction . |
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