Lowrey D M, Hameed A, Lichtenheld M, Podack E R
Department of Microbiology and Immunology, New York Medical College, Valhalla 10595.
Cancer Res. 1988 Aug 15;48(16):4681-8.
Cytotoxic granules were isolated from human lymphokine-activated killer (LAK) cells and analyzed for their biochemical properties. Isolated granules of approximately 85-95% purity were obtained by differential centrifugation followed by discontinuous Percoll gradient centrifugation. The murine lymphocyte granule marker N-alpha-carbamazepine-L-lysine thiobenzyl ester-esterase as well as cytotoxic activity toward the human tumor cell lines K562, Raji, Daudi, and CEM were associated with LAK granule fractions. Granule-associated N-alpha-carbamazepine-L-lysine thiobenzyl ester-esterase activity increased in recombinant interleukin 2 expanded human LAK cells in parallel with cytotoxic activity for Raji tumor cell targets. Cytotoxic LAK cell granules mediated calcium-dependent killing of the tumor cell lines K562, Raji, Daudi, and CEM. However, no calcium-dependent hemolytic activity was found. Preincubation of human granules with calcium, a treatment which totally inactivates the hemolytic and cytotoxic activity of murine lymphocyte granules [perforin 1 (P1)] had no effect on human LAK granule cytotoxicity for nucleated cells. Human LAK granules appear to contain P1 detected as cross-reactive antigen detected by mouse anti-P1 and human anti-C9 in Western blot analysis. In addition, Northern blot analysis of polyadenylated RNA isolated from human LAK cells using a murine P1 complementary DNA probe showed a cross-hybridizing 2.8- to 3.0-kilobase mRNA species identical in size to murine P1 mRNA. These results demonstrate that despite similar biochemical composition, functional differences exist between human and murine cytotoxic granules. Human LAK granules were synthesized in response to recombinant interleukin 2 activation and appeared in parallel with cytotoxicity for tumor targets, suggesting an important role for LAK granules in tumor cell cytotoxicity by human LAK cells.
从人淋巴因子激活的杀伤细胞（LAK细胞）中分离出细胞毒性颗粒，并对其生化特性进行分析。通过差速离心，然后进行不连续的Percoll梯度离心，获得了纯度约为85%-95%的分离颗粒。小鼠淋巴细胞颗粒标志物N-α-卡马西平-L-赖氨酸硫代苄酯酯酶以及对人肿瘤细胞系K562、Raji、Daudi和CEM的细胞毒性活性与LAK颗粒组分相关。颗粒相关的N-α-卡马西平-L-赖氨酸硫代苄酯酯酶活性在重组白细胞介素2扩增的人LAK细胞中与对Raji肿瘤细胞靶标的细胞毒性活性平行增加。细胞毒性LAK细胞颗粒介导对肿瘤细胞系K562、Raji、Daudi和CEM的钙依赖性杀伤。然而，未发现钙依赖性溶血活性。用人颗粒与钙预孵育，这种处理可完全使小鼠淋巴细胞颗粒[穿孔素1（P1）]的溶血和细胞毒性活性失活，但对人LAK颗粒对有核细胞的细胞毒性没有影响。在蛋白质印迹分析中，人LAK颗粒似乎含有作为交叉反应抗原检测到的P1，该抗原可被小鼠抗P1和人抗C9检测到。此外，使用小鼠P1互补DNA探针从人LAK细胞中分离的聚腺苷酸化RNA的Northern印迹分析显示，有一个大小与小鼠P1 mRNA相同的2.8至3.0千碱基的交叉杂交mRNA物种。这些结果表明，尽管人源和鼠源细胞毒性颗粒的生化组成相似，但它们之间存在功能差异。人LAK颗粒在重组白细胞介素2激活后合成，并与对肿瘤靶标的细胞毒性同时出现，这表明LAK颗粒在人LAK细胞对肿瘤细胞的细胞毒性中起重要作用。
