REGULATION OF FAS-MEDIATED APOPTOSIS IN B CELLS

描述(改编自申请人的摘要)：B细胞无能是一种 清除自身反应性免疫球蛋白特异性的机制。 无能B细胞在抗原受体减弱的状态下存活 反应性，并被T诱导的Fas介导的细胞凋亡所删除 效应细胞。正常B细胞对Fas诱导的细胞死亡的敏感性 可由IL-4R和SIG信号调节，从而产生一种状态 Fas耐药。指导这项研究的假设是无能B细胞 作为特定免疫信号的结果，可能会对 Fas介导的细胞凋亡，从而避免消除和呈现 随后激活和产生自身抗体的可能性。元素 这一假设对B细胞免疫能力和 一般情况下对细胞凋亡的调控。出于这个原因，而且因为如此之少 已知触发Fas耐药的方式，以及 已建立，建议探索对Fas定向细胞的保护 死亡分为3个阶段。1)评估IL-4对抗Fas介导的能力 通过抗Ig诱导细胞凋亡，并确定其他T细胞来源的作用 淋巴因子，使用标准的细胞毒性分析。2)确定新陈代谢 IL-4和IL-4对Fas介导的细胞凋亡产生抵抗的水平 通过检测Fas触发的蛋白酪氨酸事件，通过抗Ig 磷酸化、神经酰胺生产和蛋白水解酶激活 印迹、放射性标记脂类的鉴定和酶分析 蛋白质消化，并通过基因和蛋白质检测评价Bclx的作用 蛋白质表达，以及过表达转基因动物的评价。3) 双核来源的无能B细胞对Fas耐药的诱导 转基因小鼠，并监测通过体细胞拯救的自身抗体 耐药的正常动物B细胞的杂交 是在体外产生的。这项工作应该会使我们更全面地理解 B细胞对Fas介导耐药的信号和机制 细胞凋亡，并验证或驳斥上述假设，该假说具有 对血清学自身免疫发病机制的重要意义。

DESCRIPTION (Adapted from the Applicant's Abstract): B-cell anergy is one mechanism utilized to expunge autoreactive immunoglobulin specificities. Anergic B-cells survive in a state of diminished antigen receptor responsiveness, and are deleted by Fas-mediated apoptosis induced by T effector cells. In normal B-cells sensitivity to Fas-directed cell death can be regulated by IL-4R and sIg signals that produce a state of Fas-resistance. The hypothesis guiding this study is that anergic B-cells may, as a result of specific immune signals, become resistant to Fas-mediated apoptosis, thereby avoiding elimination and presenting the potential for subsequent activation and autoantibody production. Elements of this hypothesis have ramifications for B-cell immunocompetence and the regulation of apoptosis in general. For this reason, and because so little is known about the means by which Fas-resistance is triggered and established, it is proposed to explore protection against Fas-directed cell death in 3 stages. 1) Evaluate the capacity of IL-4 to oppose Fas-mediated apoptosis by anti-Ig, and determine the role of other T-cell-derived lymphokines, using standard cytotoxicity assays. 2) Determine the metabolic level at which resistance to Fas-mediated apoptosis is produced by IL-4 and by anti-Ig, by examining Fas-triggered events of protein tyrosine phosphorylation, ceramide production, and protease activation, using Western blotting, identification of radiolabeled lipids, and enzymatic assays of protein digestion, and evaluate the role of Bcl-x, through tests of gene and protein expression, and evaluation of overexpressing transgenic animals. 3) Assess induction of Fas-resistance in anergic B-cells derived from double transgenic mice, and monitor autoantibodies rescued through somatic cell hybridization of B-cells from normal animals in which resistance is generated in vitro. This work should lead to a fuller understanding of the signals and mechanisms that produce B-cell resistance to Fas-mediated apoptosis, and validate or refute the hypothesis described above, which has important implications for the pathogenesis of serological autoimmunity.