TARGET CELL DAMAGE BY IMMUNE MECHANISMS

为了确定CTL介导的细胞毒性的分子途径，我们 先前测试过半胱天冬酶的前体 （内源性“死亡蛋白酶”）是生理学相关的颗粒酶 底物导致靶裂解。通过使用两种半胱天冬酶 抑制剂，我们发现半胱天冬酶是必需的FasL/Fas 细胞毒性途径以及颗粒引起的核损伤 胞吐途径，但不需要由后者的目标裂解 通路我们现在已经测试了是否其他预裂解非核目标 颗粒胞吐途径造成的损伤是半胱天冬酶依赖性的。 使用流式细胞术和脂质探针标记靶细胞，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细胞损伤的应答 使用颗粒胞吐和FasL/Fas途径。PS曝光通过 前一种途径不受肽-FMK半胱天冬酶抑制剂的影响， 而通过后者的PS暴露被特异性地和完全地阻断。 我们已经使用DiOC 5测量线粒体电位psi，并发现， CTL诱导的损伤的两种途径都导致psi的快速损失。Caspase 抑制剂再次通过FasL/Fas途径阻断损失，但不阻断颗粒 胞吐途径我们还用显微镜检查了靶细胞 膜泡的出现，并发现CTL诱导快速 两种损伤途径都会产生水泡。半胱天冬酶抑制剂再次阻断 由FasL/Fas途径而非颗粒胞吐诱导的水泡形成 通路因此，对于三种不同的非核目标损害措施， caspase抑制剂阻断了FasL/Fas的表达， 而不是颗粒胞吐途径。这些发现支持 我们先前提出的模型，即颗粒酶和半胱天冬酶有共同的 导致靶细胞死亡的底物，而颗粒酶诱导的核形成 如前所述，损伤通过半胱天冬酶发生。

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.