TARGET CELL DAMAGE BY IMMUNE MECHANISMS

为了确定CTL介导的细胞毒作用的分子途径，我们 之前测试了半胱氨酸天冬氨酸酶前体 (内源性“死亡蛋白酶”)是生理上相关的颗粒酶。 导致靶向裂解的底物。通过使用两种类型的caspase 抑制剂，我们发现caspase是FasL/Fas所必需的 细胞毒性途径及颗粒剂对核损伤的影响 胞吐途径，但后者不是靶向裂解所必需的 路径。我们现在已经测试了其他未解体的非核目标 颗粒胞吐途径造成的损伤是caspase依赖的。 使用流式细胞仪和脂质探针标记目标细胞，我们有 发现靶细胞磷脂酰丝氨酸迅速暴露在 靶细胞外膜对CTL或NK细胞损伤的反应 同时利用颗粒胞吐和FasL/Fas途径。PS曝光通过 前者不受多肽-FMK caspase抑制剂的影响， 而通过后者的PS暴露被特别和完全地阻止。 我们用DiOC5测量了线粒体电位psi，发现 CTL诱导损伤的两条途径都会使psi迅速丧失。半胱氨酸酶 抑制剂再次阻断通过FasL/Fas途径的丢失，但不能阻止颗粒 胞吐途径。我们还对靶细胞进行了显微镜检查 对于膜泡的出现，发现CTL诱导迅速 在两条损伤路径上都发出了呜咽声。半胱氨酸天冬氨酸酶抑制剂再次阻断 由FasL/Fas途径诱导的气泡，而不是颗粒胞吐 路径。因此，对于目标损害的三种不同的非核措施 在这两条损伤通路中，caspase抑制剂共同阻断FasL/Fas 而不是颗粒胞吐途径。这些发现支持 我们之前提出的颗粒酶和半胱氨酸天冬氨酸酶共有的模型 导致靶细胞死亡的底物，而颗粒酶诱导的细胞核 损伤通过先前提出的半胱氨酸天冬氨酸酶发生。

To define the molecular pathway of CTL-mediated cytotoxicity, we previously tested the possibility that precursors of caspases (endogenous "death proteases") are the physiologically relevant granzyme substrates leading to target lysis. By using two types of caspase inhibitor, we found that caspases were required for the FasL/Fas cytotoxicity pathway and also for nuclear damage by the granule exocytosis pathway, but were not required for target lysis by the latter pathway. We have now tested whether other pre-lytic non-nuclear target damage inflicted by the granule exocytosis pathway is caspase-dependent. Using flow cytometry and a lipid probe to mark target cells, we have found that target cell phosphatidyl serine is rapidly exposed on the target cell outer membrane in response to damage by CTL or NK cells using both the granule exocytosis and FasL/Fas pathways. PS exposure via the former pathway is not affected by peptide-FMK caspase inhibitors, while PS exposure via the latter is specifically and completely blocked. We have used DiOC5 to measure mitochondrial potential psi and found that both pathways of CTL-induced damage give a rapid loss of psi. Caspase inhibitors again block loss via the FasL/Fas pathway but not the granule exocytosis pathway. We have also examined target cells microscopically for the appearance of membrane blebs, and found that CTL induce rapid blebbing by both damage pathways. Again caspase inhibitors block blebbing induced by the FasL/Fas pathway but not the granule exocytosis pathway. Thus for three different non-nuclear measures of target damage shared by the two damage pathways, caspase inhibitors block the FasL/Fas pathway but not the granule exocytosis pathway. These findings support our previously proposed model that granzymes and caspases share common substrates leading to target cell death, while granzyme-induced nucelar damage occurs via caspases as previously proposed.