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 (H2-CDCF) diacetate as starting material, we synthesized its succinimidyl
ester derivative. H2-CDCF-conjugated
IgG prepared from the succinimidyl
ester derivative was used to opsonize targets. In vitro studies have shown that H2-CDCF becomes fluorescent upon exposure to reactive
oxygen metabolites, including
hydrogen peroxide. Using video intensified epifluorescence microscopy, we observed that reactive
oxygen metabolites are deposited on
tumor cell membranes during neutrophil-mediated antibody-dependent cellular cytotoxicity (ADCC). This deposition process is
catalase sensitive. The role of reactive
oxygen metabolites produced by neutrophils in triggering the oxidation of H2-CDCF is further supported by the observation that neutrophils from
chronic granulomatous disease (CGD) patients did not affect target fluorescence. YAC
tumor cells were also labeled with
dihydrorhodamine 123 or
dihydrotetramethylrosamine. The oxidized forms of these
reagents were found within the cytoplasm of YAC cells. During ADCC normal neutrophils, but not neutrophils obtained from CGD patients, triggered the oxidation of
dihydrorhodamine 123 and
dihydrotetramethylrosamine within
tumor cells. Using two-color automated epifluorescence microscopy, we could not detect temporal intermediates with fluorescence in only one compartment, i.e., either solely on the plasma membrane or in the cytoplasm. These observations suggest that reactive
oxygen metabolites cross target membranes (< 12 sec. These studies show that reactive
oxygen metabolites are deposited both onto and into
tumor cells during ADCC, wherein both compartments could become vulnerable to
oxidant-mediated damage.