PROGRAMMED CELL DEATH IN LYMPHOCYTES

该项目对T淋巴细胞程序化细胞的两个方面进行了研究 死亡：(1)钙蛋白酶和活性氧中间体在死亡中的作用 成熟T细胞“激活诱导”死亡中的TCR触发事件 细胞，这涉及TCR诱导的Fas配体上调和 随后的Fas交联；(2)ICE家族蛋白酶的作用 (半胱氨酸天冬氨酸氨基转移酶)作为多个途径的共同下游调节因子 T淋巴细胞发育各阶段的凋亡性死亡。上一首 结果表明，TCR诱导的杂交瘤和 成纤维细胞同时被蛋白酶抑制剂和抗氧化剂阻断，我们 继续定义这些抑制的机制。特定的 钙依赖性半胱氨酸蛋白酶的抑制剂 选择性阻断TCR诱导的FasL基因表达上调。这些 抑制剂阻断激活诱导的FasL启动子活性 通过用荧光素酶报告构建的转染进行评估。 抗氧化剂的作用类似于钙蛋白酶抑制剂，因为它最能阻止 TCR诱导Fas配体在转录水平上调。 检测到激活诱导的活性氧中间体 双氢罗丹明氧化杂交瘤的流式细胞术检测。 我们已经讨论了ICE家族蛋白水解酶(Caspase)的作用 通过检测T淋巴细胞凋亡性死亡的下游介质 半胱氨酸氨基转移酶抑制剂阻断各种T细胞死亡的能力 通过不同的刺激。我们主要使用了 多肽-氟甲基酮半胱氨酸酶抑制剂zVAD-FMK(一种抑制剂 ICE和CPP32的抑制剂)和BD-FMK(CPP32的抑制剂，但不是ICE的抑制剂)。两者都有 化合物完全和特异性地阻断胸腺细胞的所有读数 四条独立的路径导致死亡。静息外周T细胞时 经检验，zVAD-FMK的效果略逊于BD-FMK，而 对于T细胞母细胞，zVAD-FMK的抑制作用要弱得多 而不是BD-FMK。对CTLL-2细胞的凋亡性抑制作用 BD-FMK，而不是通过zVAD-FMK。这些结果证明半胱氨酸酶确实发挥了作用。 在所有T细胞凋亡性死亡中起关键作用，但 对这些抑制剂的不同敏感性表明，不同的 Caspase家族成员在不同的T细胞和刺激中起关键作用。

This project has examined two aspects of T lymphocyte programmed cell death: (1)The roles of calpain and reactive oxygen intermediates in the TcR-triggered events in the "activation-induced" death of mature T cells, which involves TcR-induced Fas ligand upregulation and subsequent Fas crosslinking; (2) the role of ICE-family proteases (caspases) as common downstream mediators of multiple pathways of apoptotic death in all stages of T lymphocyte development. Previous results had shown that the TcR-induced death pathway in hybridomas and blasts is blocked by both protease inhibitors and antioxidants, and we have continued to define the mechanisms of these inhibitions. Specific inhibitors of the calcium-dependent cysteine protease calpain selectively blocked TcR induced FasL mRNA upregulation. These inhibitors blocked activation-induced FasL promoter activity as assessed by transfection with a luciferase reporter construct. Antioxidants behave like calpain inhibitors in that most block the TcR-induced Fas ligand upregulation at the mRNA transcription level. Activation-induced reactive oxygen intermediates were detected in hybridomas by dihydrorhodamine oxidation as detected by flow cytometry. We have addressed the role of ICE-family proteases (caspases) as downstream mediators of apoptotic death in T lymphocytes by testing the ability of caspase inhibitors to block death in various T cells induced by different stimuli. We have principally used the peptide-fluoromethyl ketone caspase inhibitors ZVAD-FMK (an inhibitor of ICE and CPP32) and BD-FMK (an inhibitor of CPP32 but not ICE). Both compounds completely and specifically block all readouts of thymocyte death by four independent pathways. When resting peripheral T cells were examined, ZVAD-FMK was somewhat less effective than BD-FMK, while with T cell blasts, ZVAD-FMK was considerably less potent an inhibitor than BD-FMK. The apoptotic death of CTLL-2 cells was inhibited by BD-FMK but not by ZVAD-FMK. These results argue that caspases do play a critical functional role in all T cell apoptotic death, but the differential sensitivity to these inhibitors suggests that different caspase family members are critical in different T cells and stimuli.