Evans D L, Harris D T, Staton D L, Jaso-Friedmann L
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens.
Nat Immun Cell Growth Regul. 1990;9(6):353-65.
In the present study a rat leukemia NK cell line designated CRC- (derived from RNK-16 cells) was shown to spontaneously transform into a noncytolytic (NL) line referred to as CRC-/NL cells. CRC- and CRC-/NL cells were utilized to study pathways of NK activation by phorbol esters, calcium ionophore (A23187), and monoclonal antibody (mAb). 10(-6)-10(-7) M phorbol myristate acetate (PMA) but not phorbol didecanoate or 4-beta-phorbol activated CRC-/NL to lyse YAC-1 targets. Activated CRC-/NL cells produced 20-90% specific cytotoxicity compared to 0-5% for nonactivated cells. 10(-7) M PMA inhibited normal CRC- cytotoxicity. The optimum concentration of PMA for activation was 10(-6)-10(-7) M and 3-6 h treatment time. Augmentation of cytotoxicity by PMA occurred at different E:T ratios. The time required to reverse the PMA activation of CRC-/NL cells was approximately 9-10 h posttreatment. In an effort to attempt to differentiate pathways which initiated activation, CRC-/NL cells were treated with FAM binding mAb, or with combinations of mAb and ionophore, mAb and PMA, or PMA and A23187. mAb singly or in combination with 10(-7) M PMA increased cytotoxicity. However, A23187 either singly or when combined with PMA or mAb did not produce an augmented lysis of YAC-1 target cells. Additional experiments were conducted to determine if PMA activation was associated with FAM binding. This was accomplished by analyzing redirected killing of various FAM mAb-producing myeloma cells in the presence of 10(-7) M PMA. PMA treatment of the CRC-/NL cells caused a significant increase in the lysis of myeloma/mAb-producing cells compared to control cells. Further evidence that FAM binding was associated with cytotoxicity was presented by demonstrating specific inhibition of redirected lysis by homologous mAb. Phenotype analysis of CRC- and CRC-/NL cells demonstrated that OX-7 and OX-1 expression on CRC-/NL cells was increased by 71.8 and 86.8% respectively compared to CRC-. FAM expression (78-83% positives) by CRC- and CRC-/NL cells was not different. These experiments indicated at the functional level that rat NK cells can be activated for increased cytotoxicity by FAM-specific mAb binding and/or by treatment with the diacylglycerol analogue PMA. This implies that protein kinase C mobilization either singly or in concert with inositol-1,4,5-trisphosphate activation following FAM mAb binding may play important roles in NK cell cytotoxicity.
在本研究中，一种名为CRC-（源自RNK-16细胞）的大鼠白血病NK细胞系被证明可自发转化为一种非细胞溶解（NL）系，即CRC-/NL细胞。利用CRC-和CRC-/NL细胞研究佛波酯、钙离子载体（A23187）和单克隆抗体（mAb）激活NK细胞的途径。10(-6)-10(-7) M的十四酰佛波醇乙酯（PMA）可激活CRC-/NL细胞以裂解YAC-1靶细胞，而佛波十二烷酸酯或4-β-佛波醇则无此作用。与未激活的细胞相比，激活的CRC-/NL细胞产生20-90%的特异性细胞毒性，未激活细胞的细胞毒性为0-5%。10(-7) M的PMA可抑制正常CRC-的细胞毒性。激活的最佳PMA浓度为10(-6)-10(-7) M，处理时间为3-6小时。PMA增强细胞毒性的作用在不同的效靶比下均有发生。逆转CRC-/NL细胞PMA激活所需的时间约为处理后9-10小时。为了尝试区分启动激活的途径，用FAM结合mAb或mAb与离子载体、mAb与PMA或PMA与A23187的组合处理CRC-/NL细胞。单独的mAb或与10(-7) M PMA联合使用均可增加细胞毒性。然而，单独的A23187或与PMA或mAb联合使用时，均未增强对YAC-1靶细胞的裂解。进行了额外的实验以确定PMA激活是否与FAM结合有关。这是通过分析在10(-7) M PMA存在下各种产生FAM mAb的骨髓瘤细胞的重定向杀伤来实现的。与对照细胞相比，PMA处理CRC-/NL细胞导致骨髓瘤/mAb产生细胞的裂解显著增加。通过证明同源mAb对重定向裂解的特异性抑制，进一步证明了FAM结合与细胞毒性相关。CRC-和CRC-/NL细胞的表型分析表明，与CRC-相比，CRC-/NL细胞上OX-7和OX-1的表达分别增加了71.8%和86.8%。CRC-和CRC-/NL细胞的FAM表达（阳性率78-83%）无差异。这些实验在功能水平上表明，大鼠NK细胞可通过FAM特异性mAb结合和/或用二酰基甘油类似物PMA处理而被激活，以增加细胞毒性。这意味着在FAM mAb结合后，蛋白激酶C的动员单独或与肌醇-1,4,5-三磷酸激活协同作用可能在NK细胞的细胞毒性中起重要作用。
