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，我们特别关注了人B细胞的细胞周期进程的调控以及细胞因子，包括IL-10和IL-2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Ig产生的相关性。在IL-10存在或不存在的情况下，用金黄色葡萄球菌Cowan I （SA）培养来自健康成人的高纯度B细胞。当IL-10存在于SA激活的B细胞初始活化时，IL-10促进SA激活的B细胞凋亡，从而导致IgM的产生非常适度。IL-2可以阻止il -10介导的SA活化B细胞在初始活化过程中的凋亡进程，从而恢复这些B细胞向Ig分泌细胞的进一步分化。而在培养72h后加入IL-10，在不受IL-2影响的情况下，IL-10反而能激活更多的B细胞免于凋亡，从而支持B细胞的分化。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。最近的临床试验已经证明了米佐比滨对类风湿关节炎和狼疮肾炎的疗效，这两种疾病也涉及B细胞功能异常。因此，我们研究了米佐利滨对人B细胞体外功能的影响。通过金黄色葡萄球菌（SA）和IL-2的刺激，从健康供体获得的高纯度B细胞诱导产生IgM。米佐瑞滨以剂量依赖的方式抑制其药理学上可达到的浓度（0.3-3mug/ml）的IgM的产生。时间动力学研究表明，米佐利滨需要在培养开始后的第一个120小时内存在，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表明，米佐利滨抑制活化B细胞的G1-S转变。米佐利滨对IgM产生的抑制作用不能通过补充GMP来补充GTP来逆转。虽然米佐利滨没有抑制B细胞中CD25和cdc2激酶的表达，但在SA激活的B细胞中，米佐利滨在mRNA水平上显著抑制了cyclin A的表达。这些结果表明，米佐利滨通过直接抑制B细胞而不干扰激活的初始阶段来抑制IgM的产生。此外，这些数据表明，米佐利滨通过抑制细胞周期蛋白A的表达，以一种不同于鸟嘌呤核糖核苷酸缺失的机制，抑制活化B细胞在细胞周期中的G1-S转变。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