PROGRAMMED CELL DEATH IN LYMPHOCYTES

为了验证淋巴细胞程序性死亡的假设 涉及内源性蛋白酶的激活，我们已经测试了 几种半胱氨酸蛋白酶抑制剂，以阻止TcR诱导的死亡， CD 4 + T细胞杂交瘤2B 4。 两个化学性质不同的家族 钙蛋白酶和组织蛋白酶的抑制剂，E-64和亮抑酶肽家族， 通过台盼蓝测定发现， 在固定化α-CD 3上过夜培养后碘化丙啶摄取 或α-TcR抗体。 流式显微荧光法测定DNA断裂 也被封锁了。 这些药物通过增强TcR诱导的IL-2分泌， 多达9倍，表明它们不通过干扰 信号转导 淋巴细胞中的其他程序性细胞死亡系统 测试的是类固醇诱导的CD 4 + CD 8+胸腺细胞死亡， 和CH 31 B细胞系，其 这些抑制剂不影响mIgM诱导的死亡。 我们还 发现短期培养的淋巴细胞可以进行细胞程序化， TcR交联后死亡。 在激活纯化的静息后， 小鼠淋巴结T细胞（或PNA+胸腺细胞）通过固定化α-CD 3或 α-TcR抗体，连续分裂的细胞被维持在 IL 2持续数天至数周的时间。 当重新暴露于表面结合的 针对TcR或CD 3的抗体，这些细胞被发现经历了 细胞分裂和线粒体代谢减少，伴有 大量的细胞死亡 当纯化的CD 4+和CD 8+细胞群体 以这种方式培养，细胞死亡效应主要见于 CD 4群体，在CD 8群体中的所有缓解中的影响更温和 人口 在CD 4+细胞中，可以清楚地看到DNA断裂， 当通过流动显微荧光测定法分析时伴随细胞死亡。 这种效果 被环孢菌素阻断，与脾巨噬细胞共培养，或 半胱氨酸蛋白酶抑制剂。

In order to test the hypothesis programmed cell death in lymphocytes involves activation of an endogenous protease, we have tested the ability of several cysteine protease inhibitors to block the TcR-induced death of the CD4+ T cell hybridoma 2B4. Two chemically distinct families of inhibitors of calpains and cathepsins, the E-64 and leupeptin families, were found to rescue 2B4 cells from death measured by trypan blue or propidium iodide uptake after overnight culture on immobilized alpha-CD3 or alpha-TcR antibodies. DNA breakdown measured by flow microfluorimetry was also blocked. These drugs enhanced the TcR-induced IL-2 secretion by as much as 9-fold, showing that they do not act by interfering with signal transduction. Other programmed cell death systems in lymphocytes tested were the steroid-induced death of CD4+CD8+ thymocytes, which was blocked by leupeptin family inhibitors, and the CH31 B cell line, whose mIgM-induced death was unaffected by these inhibitors. We have also found that short-term-cultured lymphocytes can undergo programmed cell death after TcR cross-linking. After activation of purified resting mouse lymph node T cells (or PNA+ thymocytes) by immobilized alpha-CD3 or alpha-TcR antibodies, continuously dividing cells were be maintained in IL2 for periods of days to weeks. When re-exposed to surface-bound antibodies against TcR or CD3, these cells were found to undergo a decrease in cell division and mitochondrial metabolism, accompanied by substantial cell death. When purified populations of CD4+ and CD8+ cells were cultured this way, the cell death effect was principally seen in the CD4 population, with more modest effects in all responses in the CD8 population. In the CD4+ cells DNA breakdown was clearly seen to accompany cell death when analyzed by flow microfluorimetry. This effect was blocked by cyclosporin, co-culture with splenic macrophages, or cysteine protease inhibitors.