A B S T R A C T The capacity of human phagocytes to generate superoxide anion (02-), a free radical of oxygen, and a possible role for this radical or its derivatives in the killing of phagocytized bacteria were explored using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Superoxide dismutase, which removes 02-, consistently inhibited phagocytosis-associated nitroblue tetrazolium (NBT) reduction indicating the involvement of O2 in this process. Similarly, superoxide dismutase inhibited the luminescence that occurs with phagocytosis, implicating 0-in this phenomenon, perhaps through its spontaneous dismutation into singlet oxygen. Subcellular fractions from homogenates of both normal and CGD leukocytes generated 02-effectively in the presence of NADH as substrate. However strating the interrelationship between NBT reduction and 02 generation in phagocytizing cells. Activity of superoxide dismutase, the enzyme responsible for protecting the cell from the damaging effects of 02-, was approximately equal in homogenates of normal and CGD granulocytes. Polyacrylamide electrophoresis separated this activity into a minor band that appeared to be the manganese-containing superoxide dismutase associated with mitochondria and a more concentrated, cyanide-sensitive, cytosol form of the enzyme with electrophoretic mobility that corresponded to that of erythrocyte cuprozinc superoxide dismutase.Superoxide dismutase inhibited the phagocytic killing of Escherichia coli, Staphylococcus aureus, and Streptococcus viridans. A similar inhibitory effect was noted with catalase which removes hydrogen peroxide. Neither enzyme inhibited the ingestion of bacteria. Peroxide and 02-are believed to interact to generate the potent oxidant, hydroxyl radical (-OH). A requirement for -OH in the phagocytic bactericidal event might explain the apparent requirement for both 02-and H202 for such activity. In agreement with this possibility, benzoate and mannitol, scavengers of -OH, inhibited phagocytic bactericidal activity. Generation of singlet oxygen from 02-and -OH also might explain these findings.

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Polymorphonuclear neutrophils and monocytes emigrate from the bloodstream to areas of inflammation and play an essential role there in a variety of physiological and pathological events, including defense against infection. Although the biochemical basis for these cells' microbicidal activity is not completely understood, studies primarily with neutrophils indicate that the killing of most organisms depends upon phagocytosis-associated oxidative metabolism. During phagocytosis neutrophils remove oxygen from the surrounding medium and convert it, probably first, to superoxide anion (O2") (1, 2) and then to hydrogen peroxide (H202) (3). H202 and O~. may interact to form the potent oxidant, hydroxyl radical (-OH) (2). Oxidation of glucose through the hexose monophosphate (HMP) shunt is stimulated, perhaps as a result of increased H202 generation (4). The spontaneous dismutation of O~. is believed to result in singlet oxygen formation (reviewed in 2), and the occurrence of this reaction in neutrophils appears to be the most likely explanation for the luminescence that occurs with phagocytosis (2, 5).Like neutrophils, monocytes undergo increased HMP shunt activation (6-8), oxygen consumption (7, 8), and H202 generation (7) during ingestion. Macrophages from mice and guinea pigs generate O~. (9). We report here that human monocytes elaborate O~. and generate chemiluminescence during phagocytosis, on stimulation by a surface-active chemical agent, and on contact with fLxed aggregated IgG. Materials and MethodsPreparation of Cells. Human neutrophils were separated from other leukocytes in 96-99% purity by centrifugation of defibrinated or heparinized venous blood through a Ficoll-Hypaque mixture (2) at 310 g for 35 min at room temperature. Erythrocytes were removed by dextran sedimentation and hypotonic lysis (2), and neutrophils were washed and suspended in KrebsRinger phosphate buffer, pH 7.35, containing 0.2% glucose and 0.2% bovine serum albumin. Monocyte-lymphocyte layers from the same preparations were diluted with 4 vol of buffer and centrifuged at 370 g, 4°C for 10 min. Erythrocytes were removed by hypotenic lysis, and the * Supported by U. S.

The in vitro influence of dopamine on the in vitro incorporation of leucine-4,5-³H into rat pituitary gland prolactin and growth hormone was studied. This catecholamine primarily inhibited the release of newly synthesized prolactin from the pituitary gland, but had no effect on growth hormone production. Reserpine and perphenazine, agents that deplete catecholamine stores in the brain, greatly increased the synthesis and release of prolactin but not of growth hormone. Guanethidine, however, produced a slight inhibition in prolactin synthesis. The injection of iproniazid and pargyline, monoamine oxidase inhibitors, had only minimal effects on the in vitro incorporation of radioactive leucine into prolactin and growth hormone. The pituitary glands from rats bearing the prolactin- and growth hormone-secreting tumor MtTW5 synthesized significantly less prolactin than controls. Although no difference in catecholamine concentration was found in the hypothalamus of control and tumor-bearing rats, the injection of the latter group with reserpine or perphenazine restored prolactin to supernormal levels. These data suggest that catecholamines may have a physiological function to regulate prolactin synthesis and release in the rat pituitary gland.

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