APOPTOSIS IN TUMOR NECROSIS FACTOR-ALPHA-DEPENDENT, MONOCYTE-MEDIATEDLEUKEMIC-CELL DEATH - A FUNCTIONAL, MORPHOLOGIC, AND FLOW-CYTOMETRIC ANALYSIS

Little is known about the precise ways in which monocytes and macropha ges recognize tumor cells and how they exert their cytolytic and/or cy tostatic effects. By a functional, morphologic, and flow-cytometric ap proach, we have studied monocyte/macrophage- and cytokine-mediated cyt otoxicity 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 presen ce of interferon (IFN)-gamma-activated monocytes. Light and electron m icroscopic examination showed the characteristic features of apoptosis of U937 cells after incubation with either monocytes or tumor necrosi s factor (TNF)-alpha. TNF-alpha-induced apoptosis (104 U/mL) as measur ed by multiparameter flow cytometry (propidium iodide [PI]) paralleled the functional decrease in cell viability (MTT assay) of 20 +/- 3% af ter 24 hours up to a maximum of 50 +/- 4% after 48 hours. Apoptosis co uld be confirmed by the detection of DNA degradation into multiples of 200-bp subunits by agarose gel electrophoresis. After prolonged incub ation times, monocyte-mediated leukemic cell death could be quantified as apoptosis by flow cytometry, whereas no decrease in net cell viabi lity of tumor cells relative to the initial cell number could be obser ved by MTT spectrophotometry. In conclusion, our data provide evidence that apoptosis is the major mode of TNF-alpha-dependent monocyte-medi ated cytotoxicity against U937 cells. Furthermore, multiparameter flow -cytometric analysis offers a sensitive method to quantify cytokine- a nd cell-induced apoptosis in leukemia.