DEPOSITION OF REACTIVE OXYGEN METABOLITES ONTO AND WITHIN LIVING TUMOR-CELLS DURING NEUTROPHIL-MEDIATED ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY

In this study we test the hypothesis that reactive oxygen metabolites are delivered from neutrophils to simultaneously both the cell surface and cytosol of opsonized YAC erythroleukemic target cells. Using 5' ( or 6') carboxyl-2',7'-dichlorodihydrofluorescein (H-2-CDCF) diacetate as starting material, we synthesized its succinimidyl ester derivative . H-2-CDCF-conjugated IgG prepared from the succinimidyl ester derivat ive was used to opsonize targets. In vitro studies have shown that H-2 -CDCF becomes fluorescent upon exposure to reactive oxygen metabolites , including hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive oxygen metabolites are deposite d on tumor cell membranes during neutrophil-mediated antibody-dependen t cellular cytotoxicity (ADCC). This deposition process is catalase se nsitive. The role of reactive oxygen metabolites produced by neutrophi ls in triggering the oxidation of H-2-CDCF is further supported by the observation that neutrophils from chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC tumor cells were als o labeled with dihydrorhodamine 123 or dihydrotetramethylrosamine. The oxidized forms of these reagents were found within the cytoplasm of Y AC cells. During ADCC normal neutrophils, but not heutrophils obtained from CGD patients, triggered the oxidation of dihydrorhodamine 123 an d dihydrotetramethylrosamine within tumor cells. Using two-color autom ated epifluorescence microscopy, we could not detect temporal intermed iates with fluorescence in only one compartment, i.e., either solely o n the plasma membrane or in the cytoplasm. These observations suggest that reactive oxygen metabolites cross target membranes (< 12 sec. The se studies show that reactive oxygen metabolites are deposited both on to and into tumor cells during ADCC, wherein both compartments could b ecome vulnerable to oxidant-mediated damage. (C) 1993 Wiley-Liss, Inc.