免疫突触对类风湿关节炎巨噬细胞凋亡的影响及亲环素A在其中的作用

Immunological synapse formation inhibits the apoptosis of macrophages in rheumatoid arthritis

目的 探讨类风湿关节炎（RA）患者外周血诱导分化的巨噬细胞作为抗原递呈细胞参与免疫突触的形成后对其自身凋亡的影响,并进一步研究亲环素A在此过程中的作用.方法 将人单核细胞系（THP-1）诱导分化的巨噬细胞经葡萄球菌肠毒素（SEB）（100 ng/ml）包被后,与活化的Jurkat T细胞共培养,促免疫突触形成后置于无血清RPMI-1640培养液中诱导凋亡,16 h后以Annexin V-碘化丙啶染色,流式细胞术检测巨噬细胞的凋亡情况.以单独无血清RPMI-1640培养液培养的巨噬细胞作为对照.同时,观察加或不加亲环素A（200 ng/ml）对突触调控的巨噬细胞凋亡的影响.临床收集10名健康对照及10例确诊的活动期RA患者外周血,采用免疫磁珠法分离CD4＋T细胞,将其与诱导分化的巨噬细胞进行共培养.数据采用两样本t检验进行分析.结果 在细胞株、健康者及RA患者外周血参与免疫突触形成的巨噬细胞凋亡率分别为（32.9±2.8）%、（24.7±1.6）%、（14.5±1.2）%,较单独RPMI-1640培养液培养的巨噬细胞凋亡率（61.4±2.4）%、（45.5±2.6）%、（22.9±1.5）%显著降低（P〈0.05）.经亲环素A作用后,上述来源巨噬细胞的凋亡率分别进一步降至（27.2±2.1）%、（20.1±1.1）%、（12.9±1.0）%,与相应参与免疫突触形成的巨噬细胞的凋亡率比较差异有统计学意义（P〈0.05）.结论 RA患者体内参与免疫突触形成的巨噬细胞的凋亡率较单独培养的巨噬细胞的凋亡率显著降低,亲环素A能进一步促进该效应,以上结论 为RA患者体内巨噬细胞的生存时间延长从而分泌大量的细胞因子并导致炎症加剧和骨破坏提供了新的理论依据.

Objective To determine whether macrophages can behave as antigen presenting cells participating the formation of immunological synapse in rheumatoid arthritis （RA） and whether this process can affect the apoptosis. Moreover, this study was aimed to observe the function of cyclophilin A （CypA） in immunological synapse formation and its role in regulating the apoptosis of macrophages. Methods human acute monocytic leukemia cell line （THP-1） induced macrophages were coated with staphylococcal enterotoxin B（SEB） （100 ng/ml） and co-cultured with activated Jurkat T cells （human acute T-cell leukemia cell line）, then incubated in the RPMI-1640 for 16 hours to induce apoptosis. The apoptosis of the macrophages were analyzed by flow cytometry by Annexin V-PI staining. The macrophages cultured in the RPMI-1640 alone were used as control. Meanwhile, CypA （200 ng/ml） were added to or not added in order to observe the apoptosis of macrophages. The function of CypA and the apoptosis of macrophages isolated from RA peripheral blood were also investigated through co-culture with CD4＋T cells isolated by immunomagnetic beads. Comparisons between groups were performed by two-sample t-tests. Results In the peripheral blood of healthy people and RA patients, the apoptosis of macrophages which participated immunological synapse was （32.9±2.8）%, （24.7±1.6）%, （14.5±1.2）% respectively, which was significantly lower than the apoptosis of macrophages cultured alone [ respectively for （61.4±2.4）%, （45.5±2.6）%, （22.9±1.5）%, （P〈0.05） ]. After CypA was added, the apoptosis of macrophages in cell lines, healthy people and RA patients decreased to （27.2±2.1）%, （20.1±1.1）%, （12.9±1.0）%, lower than the apoptosis of macrophages which participated immunological synapse formation （P〈0.05）. Conclusion In RA, the macrophages participate in the formation of immunological synapse by interacting with CD4＋ T cells. They can significantly reduce the apoptosis on themselves. CypA can enhance this effect. These results provide a new theoretical foundation for prolonged survival of macrophages in RA, which can secrete a variety of cytokines to enhance inflammation and joint destruction.