Analysis of the mechanism of abnormal B cell activation characteristic of various autoimmune diseases

本研究旨在探讨自身免疫性疾病中B细胞异常活化的机制。为此，特别关注人B细胞的细胞周期进程的调节以及包括IL-10和IL-2在内的细胞因子的影响。首先，我们详细研究了IL-10对人外周血B细胞存活的调节及其与免疫球蛋白产生的关系。用含或不含IL-10的金黄色葡萄球菌Cowan I(SA)培养健康成人的高纯度B细胞。在SA激活B细胞的初始阶段，当存在IL-10时，IL-10促进SA激活的B细胞的凋亡，从而导致非常少量的IgM产生。IL-2在SA激活的B细胞的初始激活过程中阻止了IL-10介导的B细胞的凋亡进程，从而恢复了这些B细胞向Ig分泌细胞的进一步分化。相比之下，IL-10反而被SA激活的…激活培养72小时后加入IL-2，可使B细胞免于凋亡，从而支持B细胞的分化，而不受IL-2的影响。这些结果表明，IL-10的作用取决于抗原受体结扎后B细胞的激活状态。最近的临床试验表明，咪唑立宾对类风湿性关节炎和狼疮性肾炎的疗效是不同的，这些疾病也涉及B细胞功能的异常。因此，我们研究了咪唑立宾对人B细胞体外功能的影响。在金黄色葡萄球菌Cown I(SA)和IL-2的刺激下，从健康供者获得的高纯度B细胞中诱导产生IgM。咪唑立宾以剂量依赖的方式抑制药物可获得的浓度(0.3-3微克/毫升)的IgM的产生。时间动力学研究表明，咪唑立宾必须在培养开始后的第一个120小时内出现，以发挥其对B细胞反应的抑制作用。碘化丙啶染色细胞周期分析显示，咪唑立宾抑制活化的B细胞的G1-S转变。Mizoribine对IgM生成的抑制作用不能被补充GMP的GTP逆转。虽然Mizoribine不能抑制B细胞CD25和cdc2蛋白的表达，但在mRNA水平上，Mizoribine显著抑制SA激活的B细胞中细胞周期蛋白A的表达。这些结果表明，咪唑立宾通过直接抑制B淋巴细胞而不干扰激活的初始阶段来抑制IgM的产生。此外，数据表明，咪唑立宾通过抑制细胞周期蛋白A的表达来抑制细胞周期中活化的B细胞的G1-S转变，其机制与鸟嘌呤核苷酸耗竭不同。较少

The current studies were undertaken to explore the mechanisms of abnormal B cell activation in autoimmune disease. For this purpose, special attention was paid to the regulation of cell cycle progression of human B cells and the influences of cytokines, including IL-10 and IL-2. First, we examined in detail the regulation of the survival of human peripheral blood B cells by IL-10 and its relevance to the Ig production. Highly purified B cells from healthy adult individuals were cultured with Staphylococcus aureus Cowan I (SA) in the presence or absense of IL-10. When IL-10 was present during the initial activation of B cells with SA,IL-10 faciliated the apoptosis of SA activated B cells, thus resulting in the very modest IgM production. IL-2 prevented the IL-10-mediated progression of the apoptosis of SA activated B cells during the initial activation, and thus restored the further differentiation of these B cells into Ig secreting cells. By contrast, IL-10 rather rescued SA activated … More B cells from apoptosis and thus supported the differentiation of these B cells without any influences of IL-2, when it was added after 72h of cultures. These results indicate that the effects of IL-10 are different depending on the state of activation of B cells after ligation of antigen receptors.Recent clinical trials have demonstrated the efficacy of mizoribine in rheumatoid arthritis and lupus nephritis, in which abnormalities of B cell functions are also involved. We therefore examined the effects of mizoribine on the in vitro function of human B cells. IgM production was induced from highly purified B cells obtained from healthy donors by stimulation with Staphylococcus aureus Cown I (SA) plus IL-2. Mizoribine suppressed the production of IgM at its pharmacologically attainable concentrations (0.3-3mug/ml) in a dose-dependent manner. Time kinetics study revealed that mizoribine was requied to be present within the first 120 hr after the initiation of cultures to exert its suppressive effects on B cell responses. Cell cycle analysis by staining with propidium iodide disclosed that mizoribine suppressed the G1-S transition of activated B cells. The suppressive effects of mizoribine on the IgM production was not reversed by repletion of GTP with supplementation of GMP.Althogh mizoribine did not suppress the expression of CD25 and cdc2 kinase in B cells, mizoribine markedly suppressed the expression of cyclin A in SA activated B cells at the mRNA levels. These results indicate that mizoribine suppresses the production of IgM by directly inhibiting B clls with out interfering with the initial phase of activation. Moreover, the data demonstrate that mizoribine inhibits the G1-S transition of activated B cells in the cell cycle by suppressing the expression of cyclin A by a mechanism distinct from guanine ribonucleotide depletion. Less