Colotta F, Peri G, Villa A, Mantovani A
J Immunol. 1984 Feb;132(2):936-44.
Preincubation of tumor cells with actinomycin D (Act D) rendered various murine and human lines susceptible to killing by unstimulated human peripheral blood mononuclear cells (PBM) in a 6-hr 51Cr-release assay. The murine WEHI 164 sarcoma was selected for analysis of drug-dependent cellular cytotoxicity (DDCC) because high levels of killing were detected with this tumor, and it was considerably resistant to natural killer (NK) cell activity. Optimal conditions for induction of susceptibility to lysis included a 3-hr preincubation with 1 microgram/ml Act D. Effector cells of cytotoxicity against Act D-treated WEHI 164 cells were plastic adherent (greater than 85% monocytes). Cells nonadherent to plastic and nylon wool (less than or equal to 1% monocytes) had no appreciable DDCC activity. In contrast NK activity against K562 cells was mediated by nonadherent cells. When PBM were fractionated on a one step discontinuous gradient of Percoll designed to enrich for monocytes (greater than 90% pure), DDCC activity was found in the monocyte fraction, and the lymphoid cell-enriched fraction had no cytotoxicity against Act D-treated WEHI 164 cells. In contrast, NK activity against K562 was recovered with lymphoid cells, and monocytes had no NK cytotoxicity. Upon fractionation on a six step Percoll gradient designed to enrich for large granular lymphocytes (LGL), the denser lymphocytes (fraction 4-6) and the less dense LGL with NK activity (fraction 2-3) had no cytotoxicity against Act D-treated WEHI 164 sarcoma cells. DDCC activity sedimented in fraction 1 in association with monocytes. PBM were fractionated according to monoclonal antibody-defined surface markers by using a fluorescence-activated cell sorter. Effector cells of DDCC were positive for monocyte markers (Mo2, UCHM1) and were negative for NK cell (B73.1, HNK1), T cell (T11), and B cell (Leu-10) markers. Macrophages obtained by culturing blood monocytes in vitro for 5 to 10 days had DDCC activity. Similarly, peritoneal and bronchoalveolar macrophages had considerable cytotoxicity against Act D-treated target cells, whereas minimal or no NK activity was found at these anatomic sites. Cells of human or murine origin, preincubated with Act D for 3 hr, were heterogeneous in their susceptibility to monocyte killing in a 6-hr 51Cr-release assay. High levels of cytotoxicity were observed with the murine WEHI 164 sarcoma and 3T3 "fibroblast" line and with the human CEM leukemia. Monocytes were weakly (but significantly) cytotoxic against the ALAB breast carcinoma (human) and the 8387 sarcoma (human).(ABSTRACT TRUNCATED AT 400 WORDS)
在6小时的51Cr释放试验中，用放线菌素D（Act D）对肿瘤细胞进行预孵育，可使多种小鼠和人类细胞系易于被未刺激的人外周血单核细胞（PBM）杀伤。选择小鼠WEHI 164肉瘤来分析药物依赖性细胞毒性（DDCC），因为用这种肿瘤检测到高水平的杀伤，并且它对自然杀伤（NK）细胞活性具有相当的抗性。诱导易被裂解的最佳条件包括用1微克/毫升Act D预孵育3小时。对经Act D处理的WEHI 164细胞具有细胞毒性的效应细胞可黏附于塑料（大于85%为单核细胞）。不黏附于塑料和尼龙毛的细胞（小于或等于1%为单核细胞）没有明显的DDCC活性。相比之下，对K562细胞的NK活性由不黏附细胞介导。当PBM在一步不连续的Percoll梯度上进行分离以富集单核细胞（纯度大于90%）时，在单核细胞部分发现了DDCC活性，而富含淋巴细胞的部分对经Act D处理的WEHI 164细胞没有细胞毒性。相反，对K562的NK活性在淋巴细胞中恢复，单核细胞没有NK细胞毒性。在设计用于富集大颗粒淋巴细胞（LGL）的六步Percoll梯度上进行分离时，密度较大的淋巴细胞（第4 - 6部分）和具有NK活性的密度较小的LGL（第2 - 3部分）对经Act D处理的WEHI 164肉瘤细胞没有细胞毒性。DDCC活性沉淀在第1部分，与单核细胞相关。通过荧光激活细胞分选仪根据单克隆抗体定义的表面标志物对PBM进行分离。DDCC的效应细胞对单核细胞标志物（Mo2、UCHM1）呈阳性，对NK细胞（B73.1、HNK1）、T细胞（T11）和B细胞（Leu - 10）标志物呈阴性。通过体外培养血单核细胞5至10天获得的巨噬细胞具有DDCC活性。同样，腹腔和支气管肺泡巨噬细胞对经Act D处理的靶细胞具有相当的细胞毒性，而在这些解剖部位发现的NK活性最小或没有。在6小时的51Cr释放试验中，经Act D预孵育3小时的人或小鼠来源的细胞对单核细胞杀伤的敏感性各不相同。在小鼠WEHI 164肉瘤、3T3“成纤维细胞”系和人CEM白血病中观察到高水平的细胞毒性。单核细胞对ALAB乳腺癌（人）和8387肉瘤（人）具有较弱（但显著）的细胞毒性。（摘要截断于400字）
