Pamela S. Shirley

Phagocytosis-induced release of arachidonic acid from human neutrophils

Abstract The phospholipids of human neutrophils were labeled with [3H] arachidonic acid and [14C] palmitic acid. Phagocytosis of opsonized zymosan resulted in rapid release of free arachidonic acid but not of palmitic acid. Arachidonic acid was not released when the cells were exposed to unopsonized zymosan, zymosan-activated serum, or phorbol myristate acetate. These observations suggest that phagocytosis of opsonized zymosan results in the activation of a phospholipase A2.

Deficiency of NADPH oxidase activity in chronic granulomatous disease

NADPH oxidase activity was examined in paired 27,000 x g granule fractions isolated from normal polymorphonuclear leukocytes from patients with chronic granulomatous disease. At 0.17 mM NADPH, the oxidase activity was not measurable in normal resting cells but was activated by phagocytosis. This activation was absent in CGD cells. At higher levels of NADPH, activity was present in cells from patients with CGD, although it was lower than normal, and no difference in activity was found between resting and phagocytizing cells. Granule fractions from phagocytizing normal cells exhibited higher than granule fractions from resting normal cells at all levels of NADPH. These results suggest that NADPH oxidase activity is defective in chronic granulomatous disease, and further that the defect is not the absence of the enzyme but rather a failure to activate it.

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.

Enzymes of Oxidative Metabolism in the Human Eosinophil

Summary Human eosinophils exhibit reduced pyridine nucleotide oxidase activity toward both NADH and NADPH. This activity is more active toward NADPH and appears in both the 500g pellet and 27,000g pellet upon differential centrifugation; activity toward both substrates is greater in eosinophils than neutrophils, consistent with the greater respiratory burst observed in eosinophils upon phagocytosis. Other enzymes involved in detoxification of oxygen metabolites are at least as active in eosinophils as in neutrophils, and in many cases appear to be more active. These include catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. Thus, the enzyme content of the eosinophil would prepare the cell for a role which involves generation of active oxidizing agents.

Absence of the membrane marker enzyme 5′-nucleotidase in human polymorphonuclear leukocytes

Abstract As a first step in an attempt to purify the plasma membrane from human polymorphonuclear leukocytes (PMNL), we initiated a search for a marker enzyme in order to monitor the course of the purification procedure. Nucleoside-5′-monophosphatase (5′-nucleotidase) was first used as a plasma membrane marker in the isolation of this subcellular fraction from rat liver (1); it has since been described in the plasma membranes of a variety of cell types and is considered the most specific and ubiquitous enzymatic plasma membrene marker available (2). DePierre and Karnovsky have recently described 5′-nucleotidase in guinea pig peritoneal PMNL (3), and have demonstrated its existence as an ectoenzyme in a series of elegant experiments (4, 5). In contrast, the present work suggests that this enzyme is absent in the human peripheral PMNL and hydrolysis of 5′-nucleotides in this cell type is probably due to the action of nonspecific phosphatases.

Ascorbic acid levels in phagocytic cells.

Summary The concentration of ascorbic: acid was determined in guinea pig peritoneal PMNL, normal rabbit alveolar macrophages, BCG-induced rabbit alveolar macrophages, rabbit peritoneal PMNL, and human circulating PMNL. The high levels of ascorbate seen in all cell types examined are consistent with a role for this compound in the bactericidal activity of the phagocytes. No significant differences from normal were observed in leukocytes from patients with chronic granulocytic leukemia, polycythemia vera, or in one patient with chronic granulomatous disease.

Bactericidal Activity of Superoxide Anion and of Hydrogen Peroxide: Investigations Employing Dialuric Acid, a Superoxide-Generating Drug

The addition of dialuric acid (a superoxide-generating drug) to a suspension of resting human neutrophils resulted in a stimulation of cellular hexose monophosphate shunt activity. Measurement of oxygen consumption demonstrated a rapid rate of oxygen uptake by the drug alone in aqueous solution. The subsequent addition of catalase (but not of superoxide dismutase) resulted in a substantial release of oxygen, indicating that H2O2 was accumulating in the media. The generation of O2− by the drug was verified by measuring the rate of reduction of cytochrome c by the drug in the presence and absence of authentic superoxide dismutase. The growth of Escherichia coli B and Staphylococcus aureus was inhibited in vitro by addition of the drug to a suitable culture media; the degree of inhibition was dose dependent. This inhibition of bacterial growth could be completely reversed by the addition of as little as 5 μg of purified catalase, but was not affected by concentrations of superoxide dismutase that were 2,000-fold higher. These results suggest that the dialuric acid-induced stimulation of hexose monophosphate shunt activity in neutrophils is due to accumulated H2O2. Further, the contribution of superoxide to the bactericidal activity towards E. coli and S. aureus is probably minimal compared with that of H2O2.

Pyridine nucleotide-dependent generation of hydrogen peroxide by a particulate fraction from human neutrophils.

NAD(P)H oxidase activity was determined in particulate fractions from human neutrophils by measuring the production of hydrogen peroxide. Activity was measured over a wide range of substrate concentrations from 0.0 to 4.0 mM. The activity with NADPH was consistently greater than with NADH. Activity towards both substrates was higher in a particulate fraction derived from cells which had phagocytized opsonized zymosan than in a corresponding fraction from resting cells. This increased activity was apparently due to a decreasedKm of the enzyme, although no evidence of allosteric kinetics was obtained. The activity was markedly reduced in the presence of superoxide dismutase, indicating the involvement of a superoxide-mediated chain reaction. Particulate fractions derived from cells of a patient with chronic granulomatous disease exhibited decreased activity towards both substrates and an apparent defect in the activation of the enzyme by phagocytosis.NAD(P)H oxidase activity was determined in particulate fractions from human neutrophils by measuring the production of hydrogen peroxide. Activity was measured over a wide range of substrate concentrations from 0.0 to 4.0 mM. The activity with NADPH was consistently greater than with NADH. Activity towards both substrates was higher in a particulate fraction derived from cells which had phagocytized opsonized zymosan than in a corresponding fraction from resting cells. This increased activity was apparently due to a decreased Km of the enzyme, although no evidence of allosteric kinetics was obtained. The activity was markedly reduced in the presence of superoxide dismutase, indicating the involvement of a superoxide-mediated chain reaction. Particular fractions derived from cells of a patient with chronic granulomatous disease exhibited decreased activity towards both substrates and an apparent defect in the activation of the enzyme by phagocytosis.

Oxidative Metabolism of Leukocytes and Its Relationship to Bactericidal Activity

During the course of phagocytosis, normal polymorphonuclear leukocytes undergo remarkable alterations in oxidative metabolism which are insensitive to cyanide or azide. These events, collectively referred to as the respiratory burst, are listed in Table 1. Although neutrophils are equipped with a variety of bactericidal weapons, including non-oxidative processes such as hydrolytic enzymes and cationic proteins, several lines of evidence suggest that the respiratory burst plays a major role in the killing of Table 1 The Respiratory Burst I. Increased oxygen consumption II. Increased glucose oxidation via the hexose monophosphate shunt III. Generation of hydrogen peroxide IV. Generation of superoxide anion V. Generation of chemiluminescence VI. Reduction of tetrazolium dyes many bacteria. Bacteria are ingested well under anaerobic conditions but most are not killed efficiently in this situation (1). Further, cells obtained from patients with chronic granulomatous disease fail to elicit a normal respiratory burst and are unable to adequately kill many types of microorganisms (2). Patients with this disease are highly susceptible to severe pyogenic bacterial infections which are frequently life-threatening.

A comparison between the NADPH oxidase activity of human polymorphonuclear leukocytes and the oxidase activity of several purified peroxidases

Abstract NADPH oxidase activity in granules isolated from PMNL was found to share many features in common with the NADPH oxidative activity of several purified peroxidases [horseradish peroxidase (HRP), lactoperoxidase (LPO), and human myeloperoxidase (MPO)]. Both granular oxidase activity and the oxidase activity of HRP, LPO, and MPO were stimulated by certain phenols, KCN and Mn2+, while they were inhibited by superoxide dismutase (SOD) and ascorbate. A differential effect was observed with NADH in that the reduced pyridine nucleotide was a potent inhibitor of NADPH oxidase activity in isolated granules, but it had little effect on the oxidative activity of the purified peroxidases. These results indicate that NADPH oxidase activity in granules isolated from PMNL may be a peroxidase-like enzyme, but it is likely not myeloperoxidase.