Role of free radical processes in stimulated human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes produce large quantities of superoxide when they attack and kill bacteria. However, superoxide is a weak oxidizing and reducing agent, and other more reactive oxygen species derived from reactions of superoxide are suggested to participate in the killing processes. To test the hypothesis that a reactive free radical or singlet oxygen is involved in bactericidal activity, human polymorphonuclear leukocytes were exposed to phagocytozable particles containing lipids that contain the easily autoxidized 1,4-diene moiety. After incubation the preparations were extracted and the extracts reduced with NaBH4 to convert hydroperoxides to stable alcohols. Using gas chromatography/mass spectrometry to analyze the extracts, we were unable to detect products unless iron salts were added to the medium. The products obtained by extraction are those that would be expected if both free radical chain autoxidation and 1O2 oxidation were taking place. In summary, we find that polymorphonuclear leukocytes do not cause peroxidation, implying that formation of strongly oxidizing free radicals is not an intrinsic property of the leukocyte. Added iron catalyzes peroxidation by activated leukocytes yielding an unusual distribution of hydroxylated products.

Co-localization of superoxide generation and NADP formation in plasma membrane fractions from human neutrophils.

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.

Arachidonic acid metabolism in polymorphonuclear leukocytes from patients with chronic granulomatous disease.

The effect of the calcium ionophore A23187 on the release and metabolism of [3H]arachidonic acid was examined in normal polymorphonuclear leukocytes and those obtained from patients with chronic granulomatous disease. The ionophore A23187 which stimulates oxidative metabolism in normal polymorphonuclear leukocytes was ineffective in increasing oxidative metabolism (chemiluminescence) in polymorphonuclear leukocytes from patients with chronic granulomatous disease. However, the ionophore A23187 stimulated the release of [3H]arachidonic acid from chronic granulomatous disease neutrophil phospholipids and stimulated its metabolism into hydroxyeicosatetraenoic acids and leukotrienes.

Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.

We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and 160 +/- 13 attomoles DCF per cell, respectively, in 15 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by 100 ng/ml PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by 100 ng/ml PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., 1 ng/ml PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.

Further evaluation of luminol-enhanced luminescence in the diagnosis of disorders of leukocyte oxidative metabolism: role of myeloperoxidase.

Chemiluminescence can be used to identify defects in the oxidative metabolism of granulocytes. This procedure has recently been adopted for use with microliter quantities of whole blood, appropriate for prenatal or neonatal study. Although the contribution of myeloperoxidase to the chemiluminescence assay has been noted, the possible diagnostic confusion between chronic granulomatous disease of childhood (which is rare and severe) and myeloperoxidase deficiency (which is common and of little clinical consequence) has not been stressed. We report a father and his infant daughter whose cells emitted no light in the luminol-enhanced luminescence assay; both patients are totally peroxidase deficient. These results emphasize the hereditary nature of myeloperoxidase deficiency, and the possibility for erroneous diagnosis of chronic granulomatous disease of childhood based on the luminol-enhanced luminescence test.

Mechanism of arachidonic acid release in human polymorphonuclear leukocytes.

Previous studies have demonstrated that [3H]arachidonic acid is released from prelabeled human neutrophil phospholipids when the cells are stimulated by calcium ionophore A23187 or by opsonized zymosan. Neither lysophospholipid generated by phospholipase A2 activity, diacylglycerol nor monoacylglycerol produced via phospholipase C/diacylglycerol lipase action have been identified following neutrophil challenge. The inability to detect any intermediates during the release of arachidonate is due to either rapid reacylation of lysophospholipid or conversion of diacylglycerol (monoacylglycerol) to cellular acylglycerols. The addition of exogenous [14C]fatty acid at the time of challenge was employed to determine the involvement of either phospholipase A2 or phospholipase C activities. Neutrophil stimulation with calcium ionophore A23187 resulted in an incorporation of exogenous [14C]arachidonate into phosphatidylinositol and phosphatidylcholine, those phospholipids which specifically release arachidonate. When the saturated fatty acid, [14C]stearate, replaced [14C]arachidonate, very little [14C]fatty acid was incorporated into any of the phospholipid species. Lipid phosphorus measurements revealed no significant mass change in any phospholipid class following ionophore challenge. Production of [14C]phosphatidic acid was not detected, as would be expected if diacylglycerol kinase and de novo phospholipid metabolism were significantly involved.

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.

Comparison of the calcium ionophore and phorbol myristate acetate on the initiation of the respiratory burst in human neutrophils.

We investigated the ability of the calcium ionophore A23187 and phorbol myristate acetate to elicit a respiratory burst in human neutrophils. In general, the ionophore was considerably more potent than phorbol myristate acetate in generating a chemiluminescent response and slightly less active in generating superoxide anion. In contrast, the ionophore caused a much smaller stimulation of glucose oxidation via the hexose monophosphate shunt. This relative inability of the ionophore to stimulate the shunt could not be ascribed to an effect on glucose transport or to a direct inhibition of any of the enzymes involved in glucose metabolism. These data suggest that different stimuli may have markedly different effects on various activities associated with the respiratory burst and emphasize the necessity for measurement of more than one parameter to assess the oxidative metabolism of the neutrophil.

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

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.

Platelet activating factor. Stimulation of the lipoxygenase pathway in polymorphonuclear leukocytes by 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine.

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (AAGPC) triggered the release of [3H]arachidonate but not [14C]stearate from cellular phospholipids in cytochalasin B-treated rabbit polymorphonuclear leukocytes. Concentrations of AAGPC up to 20 nM caused a dose-dependent release and subsequent metabolism of the released [3H]arachidonic acid. Most of the release of the [3H]arachidonate had taken place within the first 2 min of stimulation. Phosphatidylinositol and phosphatidylcholine served as the sources of [3H]arachidonate with about 50% of the label coming from each pool. Challenge of cytochalasin B-treated polymorphonuclear leukocytes with AAPGC led to the production of [3H]hydroxyeicosatetraenoic acids and [3H]dihydroxyeicosatetraenoic acids. No significant production of [3H]prostaglandins or [3H]thromboxanes was detected. AAGPC also caused a dose-dependent degranulation of cytochalasin B-treated rabbit polymorphonuclear leukocytes as shown by the release of beta-glucuronidase and lysozyme. Both the AAGPC-stimulated production of arachidonate metabolites and the degranulation response were blocked by eicosatetraynoic acid and non-dihydroguaiaretic acid at similar inhibitor concentrations. These findings suggest the bioactions of AAGPC on polymorphonuclear leukocytes may be mediated by the release of arachidonic acid and the production of mono- and dihydroxyeicosatetraenoic acids.

Does tuftsin alter phagocytosis by human polymorphonuclear neutrophils?

The physiological significance of the putative phagocytosis-promoting peptide, tuftsin, was investigated by measurement of chemiluminescence generated during phagocytosis and by assay of the uptake of radiolabeled bacteria. We found no differences in either assay when we compared serum from splenectomized patients (which purportedly lacks tuftsin) with normal serum. Further, there was no difference when serum from splenectomized patients was employed in the presence of absence of exogenous tuftsin. Similar results were obtained under a variety of conditions, utilizing three different challenge particles with varying particle-cell ratios and serum from 20 different splenectomized patients. These results do not agree with the hypothesis that tuftsin plays a major role in promoting phagocytosis.

Chemiluminescence of human neutrophils induced by soluble stimuli: effect of divalent cations.

The effect of three soluble stimuli, phorbol myristate acetate, concanavalin A, and the calcium ionophore A23187, on the luminol-enhanced chemiluminescence of human neutrophils was investigated. All three stimuli elicited a strong burst of chemiluminescence which was dose dependent. The effect of phorbol myristate acetate was independent of the presence of divalent cations in the medium and, in fact, was greater in the presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. The effect of concanavalin A was greatly stimulated by the presence of calcium in the medium, although some reaction was observed in the absence of this cation. In contrast, chemiluminescence induced by A23187 was absolutely dependent upon the presence of extracellular calcium. These results suggest that the mobilization of calcium into the cell is a sufficient, but not necessary, stimulus for initiation of the respiratory burst. Compounds such as phorbol myristate acetate, which act independently of extracellular divalent cations, may function by altering the intracellular ratio of bound/free calcium.

Evaluation of chronic granulomatous disease by a chemiluminescence assay of microliter quantities of whole blood.

In our modification of a chemiluminescence assay to detect chronic granulomatous disease of childhood, whole blood is used and dark-adaptation is not required. This improvement was made possible by the use of luminol as an amplification device and of phorbol myristate acetate as a stimulant. Using this simplified assay, we can assess neutrophil function with 25-100 microL of whole blood, permitting simple evaluation even in very young pediatric patients.

Neutrophil responses to platelet-activating factor.

1-O-Alkyl-2-O-acetyl-sn-glycerol-3-phosphorylcholine (i.e., platelet-activating factor) was prepared and confirmed to possess potent platelet aggregating activity. It was also potent in aggregating and degranulating rabbit and human neutrophils. When injected into rabbits, the lipid induced profound neutropenia, thrombocytopenia, and anaphylactic symptoms. The lyso derivative of this lipid, 1-O-alkyl-sn-glycerol-3-phosphorylcholine, was inactive or several orders of magnitude weaker in inducing these responses. The acetylated lipid appears to be a potent stimulator of both platelets and neutrophils. Its anaphylactic-like toxicity may be related, at least in part, to its ability to aggregate or otherwise stimulate these cells.