van de Loosdrecht A A, Ossenkoppele G J, Beelen R H, Broekhoven M G, Dräger A M, Langenhuijsen M M
Department of Hematology, Academisch Ziekenhuis Vrije Universiteit, Amsterdam, The Netherlands.
Exp Hematol. 1993 Dec;21(13):1628-39.
Little is known about the precise ways in which monocytes and macrophages recognize tumor cells and how they exert their cytolytic and/or cytostatic effects. By a functional, morphologic, and flow-cytometric approach, we have studied monocyte/macrophage- and cytokine-mediated cytotoxicity against U937 cells, a human histiocytic lymphoma cell line. A rapid decrease in cell viability of U937 cells (MTT assay) could be observed at an effector-to-target cell (E:T) ratio of 10 in the presence of interferon (IFN)-gamma-activated monocytes. Light and electron microscopic examination showed the characteristic features of apoptosis of U937 cells after incubation with either monocytes or tumor necrosis factor (TNF)-alpha. TNF-alpha-induced apoptosis (10(4) U/mL) as measured by multiparameter flow cytometry (propidium iodide [PI]) paralleled the functional decrease in cell viability (MTT assay) of 20 +/- 3% after 24 hours up to a maximum of 50 +/- 4% after 48 hours. Apoptosis could be confirmed by the detection of DNA degradation into multiples of 200-bp subunits by agarose gel electrophoresis. After prolonged incubation times, monocyte-mediated leukemic cell death could be quantified as apoptosis by flow cytometry, whereas no decrease in net cell viability of tumor cells relative to the initial cell number could be observed by MTT spectrophotometry. In conclusion, our data provide evidence that apoptosis is the major mode of TNF-alpha-dependent monocyte-mediated cytotoxicity against U937 cells. Furthermore, multiparameter flow-cytometric analysis offers a sensitive method to quantify cytokine- and cell-induced apoptosis in leukemia.
关于单核细胞和巨噬细胞识别肿瘤细胞的精确方式以及它们如何发挥细胞溶解和/或细胞抑制作用，目前所知甚少。通过功能、形态学和流式细胞术方法，我们研究了单核细胞/巨噬细胞和细胞因子介导的对U937细胞（一种人类组织细胞淋巴瘤细胞系）的细胞毒性。在存在干扰素（IFN）-γ激活的单核细胞的情况下，效应细胞与靶细胞（E:T）比例为10时，可观察到U937细胞的细胞活力迅速下降（MTT法）。光镜和电镜检查显示，U937细胞与单核细胞或肿瘤坏死因子（TNF）-α孵育后具有凋亡的特征性表现。通过多参数流式细胞术（碘化丙啶[PI]）测量，TNF-α诱导的凋亡（10⁴U/mL）在24小时后与细胞活力的功能下降（MTT法）平行，下降了20±3%，48小时后最多下降50±4%。通过琼脂糖凝胶电泳检测到DNA降解为200-bp亚基的倍数，可证实凋亡。延长孵育时间后，单核细胞介导的白血病细胞死亡可通过流式细胞术定量为凋亡，而MTT分光光度法未观察到肿瘤细胞相对于初始细胞数的净细胞活力下降。总之，我们的数据提供了证据，表明凋亡是TNF-α依赖的单核细胞介导的对U937细胞细胞毒性的主要模式。此外，多参数流式细胞术分析提供了一种敏感的方法来定量白血病中细胞因子和细胞诱导的凋亡。
