Stimulation of neutrophil oxygen-dependent metabolism by human leukocytic pyrogen (original) (raw)
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Optimization of experimental settings for the analysis of human neutrophils oxidative burst in vitro
Talanta, 2009
The evaluation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) production by neutrophils is currently a matter of extensive research, with scientific reports showing an enormous variability on the detection methods as well as the concentration of the detecting probes. Also the incubation media used to test neutrophils and the respective ionic concentration, as well as the glucose concentration, varies enormously from study to study. This variability often results in different sensibility and/or response of neutrophils to stimulating agents, which can be a focus of confounding and sometime contradictory results among reports. Thus, the main objective of the present study was to appraise and compare the effect of commonly described buffering media [phosphate buffer saline (PBS), Hank's balanced salt solution (HBSS) and Tris buffer], with or without glucose, on the activation of human neutrophils by phorbol myristate acetate (PMA), using different detection probes [luminol amplified chemiluminescence, dihydrorhodamine 123 fluorescence or cytochrome c reduction (UV/vis spectrometry)].
Superoxide anion release from blood and bone marrow neutrophils is altered by endotoxemia
Circulation Research, 1990
In vivo endotoxin infusion produces neutrophil-mediated acute lung injury and increases superoxide anion release from phorbol myristate acetate (PMA)-stimulated blood neutrophils collected 18-24 hours after the infusion. Because the turnover time of circulating blood neutrophils is only 6-8 hours, it was hypothesized that the prolonged increase in superoxide anion release from peripheral blood neutrophils is associated with increased superoxide anion release from bone marrow neutrophils. To test this hypothesis, two doses of Escherichia coli endotoxin (5.0 and 0.5 micrograms/kg) were infused into chronically instrumented awake sheep. Blood and bone marrow neutrophils were collected 24 hours after the infusion, and superoxide anion release from unstimulated and PMA-stimulated neutrophils was measured in vitro. Endotoxin infusion produced an increase in pulmonary microvascular permeability, in intravascular activation (degranulation) of blood neutrophils, and in circulating blood neut...
Oxidative Metabolism of neutrophils
Polymorphonuclear neutrophils "PMN# have an important role in the host defence response to infection[ These cells produce large amounts of reactive oxygen species "O = − 1 \H 1 O 1 and ONOO − # with microbicidal activity[ PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density "Ficoll!Hypaque gradient and dextran#\ yielding a highly homogeneous cellular population[ However\ some cellular activation due to membrane perturbation is also expected[ We studied how the production of reactive oxygen species and release of myelo! peroxidase "MPO# from blood PMN are a}ected by the use of the Ficoll!Hypaque density gradient[ PMN isolated by spontaneous sedimentation and total blood were used for comparisons[ Lucigenin! and luminol!enhanced chemi! luminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli[ The release of MPO\ estimated by the chemiluminescence of the luminol:H 1 O 1 reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B\ was also higher for PMN isolated by a density gradient[ In conclusion\ it was shown that the PMN isolation procedure a}ects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results [ Copyright Þ 1999 John Wiley + Sons\ Ltd[ KEY WORDS * oxidative metabolism^myeloperoxidase^polymorphonuclear leukocytes^Ficoll!Hypaque^spontaneous sedi! mentation^blood cells^chemiluminescence ABBREVIATIONS * C\ control^FMLP\ N!formyl!methionyl!leucyl!phenylalanine^MPO\ myeloperoxidase^PMA\ phorbol miristate acetate^PMN\ polymorphonuclear leukocytes^ZY\ opsonized zymosan\ SG\ sedimentation in gradient^SS\ spontaneous sedimentation^TB\ total blood^HRP\ horseradish peroxidase
The Journal of …, 1984
Mononuclear phagocytes are known to be long-lived cells, capable of synthesis of new cellular constituents and of major alterations in metabolic activities and morphology (1). In contrast, the neutrophil generally has been considered an end-stage cell, incapable of significant functional or metabolic modulation. However, Granelli-Piperno et ai. (2, 3) have reported that human neutrophils actively synthesize protein and RNA and secrete plasminogen activator when placed in culture, and that these activities can be stimulated by incubation of the cells with concanavalin A and inhibited by incubation with glucocorticoids. These findings, and the capacity of small amounts of bacterial endotoxin (lipopolysaccharide, LPS) ~ to prime mononuclear phagocytes for enhanced stimulated release of superoxide anion (O~) (4, 5), suggested to us that neutrophil function might be modified by exposure to LPS. We report here that incubation of human neutrophils for 30-60 min with LPS primes these cells for enhanced release of O~ or hydrogen peroxide (H~O~) upon subsequent contact with a variety of stimuli. This increase in oxidative metabolism might permit increased effectiveness of the neutrophil in host defense. These results also raise the possibility that exposure of neutrophils to bacterial LPS could enhance the capacity of these cells to damage tissue, which might occur in conditions such as endotoxic shock or infection-induced adult respiratory distress syndrome. Materials and Methods Preparation of Neutrophils. Human neutrophils were isolated from venous blood anticoagulated with 10% sodium citrate (3.8% solution in water; Fisher Scientific Co., Pittsburgh, PA). 3 vol blood were added to 1 vol 6% dextran (70,000 tool we) in 0.9% sodium chloride solution (Cutter Laboratories, Berkeley, CA
Neutrophil apoptosis, phagocytosis and oxidative metabolism in septic patients
Critical Care
We evaluated neutrophil activation by measuring its phagocytic ability and oxidative burst activity in 16 patients with sepsis and 16 healthy volunteers. We also focused on neutrophil apoptosis as a regulatory mechanism of the inflammatory response. Neutrophil phagocytosis was evaluated by the detection of propidium iodide (PI)-labeled Staphylococcus aureus added to whole blood. Reactive oxygen species (ROS) formation was quantified by measuring the oxidation of 2Ј,7Ј dichlorofluorescein diacetate (DCFH-DA) at baseline and after cell stimulation with phorbol myristate acetate (PMA), and bacterial cells (killed S. aureus) or products (lipopolysaccharide [LPS] and N-formyl-methionyl-leucylphenylalanine [FMLP]). Apoptosis was assessed in neutrophils stained with annexin V and PI. Neutrophil phagocytic ability was increased in patients with sepsis compared with healthy controls (median geometric mean fluorescence intensity [GMFI] was 101.9 and 54.7, respectively; P = 0.05). ROS formation was enhanced in patients with sepsis compared with healthy volunteers at baseline (median GMFI 275.6 and 52.1, respectively; P < 0.001), and after stimulation with S. aureus (median GMFI 2395.8 and 454.9, respectively; P < 0.001), PMA (median GMFI 1120.6 and 307.5, respectively; P = 0.003), FMLP (median GMFI 792.4 and 123.2, respectively; P < 0.001), and LPS (median GMFI 624.8 and 144.8, respectively; P < 0.001). Early neutrophil apoptosis was increased in patients with sepsis compared with healthy volunteers (median 11.3% and 9.1%, respectively; P = 0.03). These data demonstrate that neutrophil function is enhanced in patients with sepsis. Additionally, circulating neutrophils from patients with sepsis presented with increased early apoptosis, which may be consequence of a regulatory mechanism of the inflammatory response.
Nitric oxide reduces hydrogen peroxide production from human polymorphonuclear neutrophils
European Journal of Clinical Investigation, 1995
Nitric oxide has been reported to affect both adhesion and respiratory burst of neutrophils. This indicates a possible role of nitric oxide in regulation of acute inflammatory responses. Release of oxygen metabolites from neutrophils can be measured using luminol-enhanced chemiluminescence and this method can detect both extracellularly and intracellularly released oxygen metabolites. Neutrophils treated with nitroprusside and activated with FMLP, type I collagen or PMA decreased their extracellular release of oxygen metabolites, while their intracellular release was almost unaffected. The effect of nitroprusside was mediated by nitric oxide since treatment with cyanide had the opposite effect. N-ethylmaleimide treatment decreased both extra-and intracellular release of oxygen metabolites. This indicates that nitric oxide affects membrane-bound NADPH-oxidase either indirectly or directly, and not a cytosol factor of the oxidase as earlier shown for N-ethylmaleimide. In conclusion, extracellular nitric oxide attenuates extracellularly released oxygen metabolites from activated neutrophils in an inflammatory response.
FEBS Letters, 1994
Nitric oxide ('NO) release, oxygen uptake and hydrogen peroxide (H202) production elicited by increasing phorbol12-myristate 13-acetate (PMA) concentrations were measured in human neutrophils. Half-maximal activities were sequentially elicited at about 0.0001-0.001 pg PMA/ml CNO) and O.OOl~.Ol pg PMA/ml (H,OJ. At saturated PMA concentrations, 'NO production, oxygen uptake and H,O, release were 0.56 ? 0.04, 3.32 f 0.52 and 1.19 f 0.17 nmol min-' 1 O6 cells-'. 'NO production accounts for about 30% of the total oxygen uptake. Luminol-dependent chemiluminescence, reported to detect NO reactions in other inflammatory cells, was also half-maximally activated at about 0.00-0.01 pg PMA/ml. Preincubation with A'"-monomethyl-L-arginine (L-NMMA) decreased 0, uptake and 'NO release but increased H,Oz production, while superoxide dismutase (SOD) increased 'NO detection by 30%. Chemiluminescence was also reduced by preincubation with L-NMMA and/or SOD. The results indicate that 'NO release is part of the integrated response of stimulated human neutrophils and that, in these cells, kinetics of-NO and O;-release favour the formation of other oxidants like peroxynitrite.
On the Pharmacology of Oxidative Burst of Human Neutrophils
Physiological Research, 2015
The effect of three therapeutically used drugs and five polyphenolic compounds on the mechanism of oxidative burst was compared in whole blood and isolated neutrophils at cellular and molecular level. In 10 μM concentration, the compounds investigated decreased the oxidative burst of whole blood in the rank order of potency: N-feruloylserotonin (N-f-5HT) > curcumin (CUR) > quercetin (QUER) > arbutin (ARB) > resveratrol (RES) > dithiaden (DIT) > carvedilol (CARV) > brompheniramine (BPA). The ratio between the percentage inhibition of extracellular versus intracellular chemiluminescence (CL) followed the rank order QUER > N-f-5HT > RES > CUR > DIT and is indicative of the positive effect of the compounds tested against oxidative burst of neutrophils, demonstrating suppression of reactive oxygen species extracellularly with minimal alteration of intracellular reactive oxygen species (ROS). Activation of protein kinase C was significantly decreased by DI...