Linda C. McPhail

Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.

Activation of the respiratory burst in phagocytic cells, an important host defense process, is not yet well understood. We now report the development of a cell-free system for activation of NADPH oxidase, the respiratory burst enzyme, in human neutrophils. Activation was achieved by the addition of arachidonic acid to a postnuclear supernatant (500 g) from disrupted unstimulated cells (no arachidonate, 0.2; with arachidonate, 3.4 nmol superoxide anion/min per mg) and was dependent on both the concentration of arachidonate and on the amount of cellular material present. Activity stimulated by arachidonate appeared to be NADPH oxidase based on a Michaelis constant for NADPH of 32 microM and a pH optimum of 7.0-7.5. Separation of the 500-g supernatant by high speed centrifugation revealed a requirement for both soluble and particulate cofactors. Activation of NADPH oxidase by arachidonate did not occur in the high speed pellet fraction from unstimulated cells but could be restored by the addition of the high speed supernatant. In addition, priming of intact neutrophils with low concentrations of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine or the tumor promoter phorbol myristate acetate replaced the soluble factor requirement for NADPH oxidase activation by arachidonate in the high speed pellet. This cell-free system can now be used to provide further insight into the biochemical basis of priming and the terminal mechanisms involved in the activation of NADPH oxidase.

Activation of the respiratory burst enzyme in human polymorphonuclear leukocytes by chemoattractants and other soluble stimuli. Evidence that the same oxidase is activated by different transductional mechanisms.

Chemoattractant-receptor coupling triggers several biologic responses in phagocytic cells including activation of the respiratory burst. Prior evidence in intact cells implied that stimulation of the respiratory burst by chemoattractants was by a mechanism different from other soluble agents suggesting the possibility that different oxidative enzymes were responsible. We now show that the chemoattractants N-formyl-methionyl-leucyl-phenylalanine and a split fragment of the fifth component of complement (C5a) stimulate an NADPH oxidase activity, measured in the 50,000-g particulate fraction from human polymorphonuclear leukocytes (PMN). Levels of oxidase activity stimulated by the chemoattractants were both time and dose dependent and required the presence of cytochalasin B during stimulation. In contrast, activation by two nonchemotactic stimuli, the ionophore A23187 and phorbol myristate acetate (PMA), did not require cytochalasin B. Temporal patterns of oxidase activation suggested that different stimuli follow different transductional pathways. Chemoattractant-mediated activation was immediate (no lag); peaked by 45 s and declined rapidly to approximately 50% of maximal by 2 min. In contrast, activation by A23187 or PMA had a 15-30-s lag and increased more slowly. Stimulation by A23187 peaked at 5 min, then declined. Stimulation by PMA plateaued at 20 min and did not decline by 90 min. Comparison of Km values for NADPH and NADH obtained by Lineweaver-Burk analysis of the oxidase activity stimulated by N-formyl-methionyl-leucyl-phenylalanine, A23187, and PMA suggested that the same enzyme was activated by all stimuli. Thus, chemoattractants and other soluble stimuli appear to activate the same respiratory burst enzyme in PMN but they utilize different transductional mechanisms and are regulated differently.

Superoxide Dismutase Activity in Leukocytes

Superoxide dismutase activity has been identified in both human neutrophils and rabbit alveolar macrophages by two distinct assay procedures. The enzyme is insensitive to both cyanide and azide and is present in the cytosol of the cell. The identification of this enzyme in phagocytic cells is compatible with the theory that superoxide anion might be involved in the bactericidal activity of the cell. It is proposed that the enzyme functions to protect the cell against superoxide generated during the phagocytic process.

Generation of chemiluminescence by a particulate fraction isolated from human neutrophils. Analysis of molecular events.

A particulate fraction isolated from human neutrophils by homogenization, then centrifugation at 27,000 g, was demonstrated to generate chemiluminescence. This luminescence required the addition of reduced pyridine nucleotide and was very low in fractions from resting normal cells. Stimulation of neutrophils with opsonized zymosan, phorbol myristate acetate, or ionophore A23187 resulted in marked enhancement of the chemiluminescence measured in subsequently isolated particulate fractions. Stimulation did not boost the luminescence produced by fractions from cells of patients with chronic granulomatous disease. The chemiluminescence of particulate fractions from stimulated neutrophils was linear with increasing protein concentration, had a pH optimum of 7.0, and was higher with NADPH as substrate than with NADH. These results confirm previous studies suggesting that the enzyme system responsible for the respiratory burst in neutrophils is present in this fraction. The particulate fraction was used to examine the nature and origin of neutrophil luminescence by investigating the effect on this phenomenon of certain chemical and enzymatic scavengers of oxygen metabolites. Results suggest that the energy responsible for the luminescence of particulate fractions and, presumably, the intact cell, is derived from more than one oxygen species and that luminescence is a product of the interaction of these species and excitable substrates within the cell.

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.

Mechanisms of Regulating the Respiratory Burst in Leukocytes

When leukocytes encounter opsonized microorganisms or a variety of inflammatory stimuli, their utilization of oxygen is substantially enhanced. This phenomenon was first observed as increased oxygen uptake by the stimulated cells (Baldridge and Gerard, 1933; Sbarra and Karnovsky, 1959) and was correlated with the production of hydrogen peroxide (Iyer et al., 1961). Concomitant with the alterations in respiration, enhanced glucose oxidation via the hexose monophosphate shunt occurs as well (Sbarra and Karnovsky, 1959). In recent years, it has become clear that oxygen utilization in activated phagocytic cells can proceed by one electron reduction steps, and that the initial product is probably superoxide anion ( 2 - ) (Babior et al., 1973). Two molecules of 2 - can then interact in a dismutation reaction, resulting in the formation of hydrogen peroxide (H2O2). These reactions are outlined in Eqs. (1) and (2):

Phorbol myristate acetate mediates redistribution of protein kinase C in human neutrophils: potential role in the activation of the respiratory burst enzyme.

{O_2} + {e^ - } \to O_2^ -

Chemoattractant-elicited alterations of cAMP levels in human polymorphonuclear leukocytes require a Ca2+-dependent mechanism which is independent of transmembrane activation of adenylate cyclase.

(1)