We have reported previously that human granulocytes have an irreversible fall in their endogenous reduced soluble sulfhydryls following zymosan stimulation. In the present study, we demonstrate that stimulated granulocytes release one or more reactive oxygen species (ROS) with the capacity to oxidize reduced glutathione (GSH). One or more of these compounds is stable enough to be detected in the supernatant. The formation of these stable oxidants appears to require H2O2 and heme or a heme-containing enzyme. However, once formed, the compound reacts with GSH without these factors. The ROS is not superoxide or hydroxyl radical, since neither superoxide dismutase nor the hydroxyl scavengers, mannitol and benzoic acid, change the rate of the reaction. Methionine has recently been demonstrated to be oxidized to a sulfoxide by a reactive oxygen species that is dependent on H2O2 and heme for its production. We found that methionine could directly react with the same ROS that degrades GSH. The ROS also has the capacity to oxidize iodide and fix halogen to proteins. Our data indicate that stimulated granulocytes release a ROS with the capacity to oxidize GSH, react with methionine, and oxidize and fix I- to protein. The compound, therefore, appears dependent on H2O2 and the myeloperoxidase system for its production, and is either hypochlorous acid (HOCI) or a compound derived from HOCI, such as a chloramine. The capacity of GSH to react with this ROS suggests an additional role for this tripeptide in cellular protection against oxidant injury.
The role of reduced glutathione in relation to hexose monophosphate shunt activity and peroxide detoxification has been well established in human erythrocytes. Less is known about the content of reduced glutathione in phagocytic leukocytes and the changes that occur during functional activity. We have measured the reduced sulfhydryl content of normal resting human granulocytes and of cells isolated from a patient with chronic granulomatous disease. Normal cells and those from the patient with chronic granulomatous disease contained similar concentrations of reduced sulfhydryls. Stimulation of a phagocytic response by incubation with opsonized zymosan particles resulted in prompt and nearly complete depletion of intracellular glutathione from normal granulocytes. This fall in reduced glutathione concentration was dependent on the phagocytic load. Exposure of chronic granulomatous disease granulocytes to a similar phagocytic load resulted in a slower and less complete fall in reduced glutathione. In normal cells, those from the chronic granulomatous disease patient, and those from an obligate carrier of the disease, the decrement in reduced glutathione during phagocytosis was correlated with oxidation of 14C-1-glucose and 14C-formate, nitroblue tetrazolium reduction, and the chemiluminescence phenomenon.
Recent studies suggest that the generation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) or superoxide (O2-) may play a role in monocyte antibody-dependent cytotoxicity (ADCC). We studied ADCC by normal human monocytes, and monocytes from chronic granulomatous disease (CGD) patients, cells unable to generate ROS, toward anti-D sensitized human red cells (RBC) and an antibody sensitized lymphoblastoid cell line (CEM) by 51Cr release. The effects of hypoxia, scavengers of ROS, and the activity of the hexose monophosphate shunt pathway (HMPS) were examined. We found that monocyte HMPS activity increased two to threefold during ADCC toward RBC but was unchanged during ADCC toward CEM cells. Hypoxia decreased lysis of RBC targets by 80% but did not affect lysis of CEM cells even though hypoxia markedly decreased monocyte HMPS activity. Monocytes from CGD patients had impaired lysis of RBC but lysed CEM cells normally. We could not, however, demonstrate protection by scavengers of ROS. We conclude that monocyte ADCC involves two independent mechanisms: a nonoxidative mechanism active in the lysis of CEM cells, and an oxidative mechanism that may involve an unidentified ROS activated during ADCC toward RBC. The activation and possible interaction of these two mechanisms is determined by the nature of the target cell and sensitizing antibody.
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