TARGET CELL DAMAGE BY IMMUNE MECHANISMS

为了确定CTL细胞毒性的分子途径，我们测试了 ICE家族蛋白酶（半胱天冬酶）的前体是 生理学相关的目标颗粒酶底物。 其他实验室有 从生物化学上表明，颗粒酶B激活半胱天冬酶， 核损害。 因此，我们测试了两种类型的半胱天冬酶抑制剂， 它们阻断颗粒胞吐途径和Fas受体的能力， CTL细胞毒性途径。 我们发现，虽然细胞死亡通过Fas 配体/Fas通路与颗粒诱导的凋亡性核损伤 胞吐途径需要半胱天冬酶，通过颗粒靶向裂解 胞吐途径则不。 使用的第一类半胱天冬酶抑制剂 是肽氟甲基酮-ZVAD-FMK（ICE抑制剂）， BD-FMK（CPP 32而非ICE抑制剂）和ZFA-FMK（对照 组织蛋白酶B抑制剂）。 这些都不能抑制 丝氨酸蛋白酶颗粒酶B。 ZVAD-FMK和BD-FMK都完全 阻断靶细胞溶解（51 Cr释放）和凋亡核 通过携带Fas配体的CTL杂交瘤诱导的形态学，而ZFA-FMK 没有，证实了半胱天冬酶在这一死亡途径中的作用。 当 使用不含Fas的细胞检测颗粒胞吐途径。 目标和常规克隆CTL，这些含asp的FMK 抑制剂完全阻断了凋亡的核形态，但没有 对靶细胞裂解有显著影响。这些结果已得到证实 使用第二种半胱天冬酶抑制剂，杆状病毒蛋白p35， 其阻断该家族的所有已知成员，但不阻断粒酶B。 在 在这些实验中，我们通过瞬时表达方式在靶细胞中表达p35 转染并测量CTL释放的能力 共转染半乳糖苷酶。 尽管p35有效地阻断了细胞溶解， 通过Fas途径，通过颗粒胞吐途径的溶解不 显着抑制。 因此，我们得出结论，虽然半胱天冬酶是部分 诱导细胞凋亡核损伤的途径 颗粒胞吐途径使用另一种更快速的细胞 死亡途径，不依赖于半胱天冬酶。

To define the molecular pathway of CTL cytotoxicity, we have tested the possibility that precursors of ICE-family proteases (caspases) are the physiologically relevant target granzyme substrates. Other labs have shown biochemically that granzyme B activates caspases which cause nuclear damage. We thus tested two types of caspase inhibitor for their ability to block both the granule exocytosis pathway and the Fas pathway of CTL cytotoxicity. We find that while cell death via the Fas ligand/Fas pathway and apoptotic nuclear damage induced by the granule exocytosis pathway requires caspases, target lysis via the granule exocytosis pathway does not. The first class of caspase inhibitor used was peptide fluoromethyl ketones-- ZVAD- FMK (an ICE inhibitor), BD-FMK (a CPP32 but not ICE inhibitor), and ZFA-FMK (a control cathepsin B inhibitor). None of these inhibits the asp-selective serine protease granzyme B. Both ZVAD-FMK and BD-FMK completely blocked target cell lysis (51Cr release) and apoptotic nuclear morphology induced by a Fas ligand bearing CTL hybridoma while ZFA-FMK did not, confirming a role for caspases in this death pathway. When the granule exocytosis pathway was examined using non-Fas bearing targets and conventional cloned CTL, these asp-containing FMK inhibitors completely blocked apoptotic nuclear morphology but had no significant effect on target lysis.These results have been confirmed with second type of caspase inhibitor, the Baculovirus protein p35, which blocks all known members of this family but not granzyme B. In these experiments we expressed p35 in target cells by transient transfection and measured the ability of CTL to release cotransfected-galactosidase. Although p35 effectively blocked lysis via the Fas pathway, lysis via the granule exocytosis pathway was not significantly inhibited. Thus we conclude that while caspases are part of the pathway inducing apoptotic nuclear damage for both CTL effector pathways, the granule exocytosis pathway uses another, more rapid, cell death pathway which is independent of caspases.