TARGET CELL DAMAGE BY IMMUNE MECHANISMS

为了检验淋巴细胞的颗粒吐出模型 细胞毒性，我们检测了大鼠粘膜的细胞毒活性。 肥大细胞瘤细胞株RBL转导细胞毒基因后的研究 淋巴细胞颗粒成分。特别是，我们试图测试 被称为颗粒酶的颗粒丝氨酸蛋白酶是否在 靶DNA降解伴随细胞毒性。我们已经建造了 表达细胞溶素(Cy)的双亲和单亲RBL细胞及 颗粒丝氨酸蛋白酶颗粒酶A(GZA)。Rbl-Cy转染体显示 对红细胞靶标有较强的杀伤力，对 肿瘤靶点。然而，后者的DNA降解可以忽略不计。 而RBL-GZA转染体表达GZA的水平与克隆的水平相当 CTL并分泌它以响应Iger交联物，它们没有 可检测到细胞毒活性。Rbl-Cy-GZA转基因细胞表现良好 这两种颗粒成分的表达均显示出细胞溶解活性 在RBC和肿瘤靶点上与RBL-Cy相当。靶DNA击穿 由这些双转染细胞明显呈阳性，具有剂量效应 曲线平行于裂解，但效率稍低。琼脂糖凝胶 标记的靶DNA显示出梯状条带特征 核小体间分裂。这些结果清楚地表明，颗粒酶A 可以在靶DNA的分解中发挥作用。作为对能力的直接测试 当被引入细胞质时诱导细胞毒作用的蛋白水解酶 作为靶细胞，我们向肿瘤细胞“注入”了各种不同的蛋白酶。 用渗透裂解法裂解胞质小体。内源性蛋白水解酶-胰酶， 凝乳酶、蛋白酶K和木瓜酶都被发现可以溶解淋巴瘤 以剂量依赖的方式，如通过5ICr释放来测量；这 死亡的测量之前是DNA的释放。从微观上看，这些 显示细胞经历了明显的膜起泡和收缩， 以“细胞凋亡”为特征。

In order to test the granule exocytosis model for lymphocyte cytotoxicity, we have examined the cytotoxic activity of the rat mucosal mast cell tumor line RBL after transfection with genes for cytotoxic lymphocyte granule components. In particular we have sought to test whether the granule serine proteases known as granzymes play a role in target DNA degradation accompanying cytotoxicity. We have constructed double and single RBL transfectants expressing cytolysin (cy) and the granule serine protease granzyme A (gza). RBL-cy transfectants show potent killing on red blood cell targets, and moderate cytotoxicity on tumor targets. However, DNA degradation in the latter is negligible. While RBL-gza transfectants express gza at levels comparable to cloned CTL and secrete it in response to IgER crosslinking, they have no cytotoxic activity detectable. RBL-cy-gza transfectants showing good expression of both these granule components showed cytolytic activity comparable to RBL-cy on both RBC and tumor targets. Target DNA breakdown by these double transfectants was clearly positive, with dose response curves parallel to lysis but somewhat less efficient. Agarose gels of labelled target DNA showed the ladder pattern characteristic of internucleosomal cleavage. These results clearly show that granzyme A can play a role in target DNA breakdown. As a direct test of the ability of proteases to induce cytotoxicity when introduced into the cytoplasm of a target cell, we have "injected" various proteases into tumor cells using osmotic lysis of pinosomes. The endoproteases trypsin, chymotrypsin, proteinase K, and papain were all found to lyse lymphoma cells in a dose dependent manner, as measured by 5ICr release; this measure of death was preceded by DNA release. Microscopically, these cells were shown to undergo a marked membrane blebbing and shrinking, characteristic of "apoptosis".