Effects of oxygen tension and pH on the respiratory burst of human neutrophils

Abstract The respiratory burst of human neutrophils was measured under conditions of hypoxia and low pH. O2 -- production by neutrophils activated with opsonized zymosan fell slowly as the oxygen concentration declined to 1%, then dropped more sharply, reaching negligible levels at oxygen concentrations less than 0.25%. Production was half maximal at an oxygen concentration of 0.35% (equivalent to approximately 10-microM dissolved oxygen). O2- production by the cell- free O2- -forming system prepared from zymosan-activated neutrophils showed a similar dependence on oxygen concentration. A drop in pH caused decreases in both oxygen consumption and O2-- production by zymosan-treated neutrophils, values at PH 6.0 being 10%--20% of those observed at pH 7.5. Experiments with the cell-free O2-- -forming system suggested that this decline in respiratory burst activity at low pH was due to inefficient activation of the O2-- -forming enzyme under acidic conditions.

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Effects of oxygen tension and pH on the respiratory burst of human neutrophils

Blood ◽

10.1182/blood.v53.6.1133.bloodjournal5361133 ◽

1979 ◽

Vol 53 (6) ◽

pp. 1133-1139 ◽

Cited By ~ 3

Author(s):

TG Gabig ◽

SI Bearman ◽

BM Babior

Keyword(s):

Oxygen Consumption ◽

Oxygen Concentration ◽

Respiratory Burst ◽

Low Ph ◽

Human Neutrophils ◽

Respiratory Burst Activity ◽

Similar Dependence ◽

Activated Neutrophils ◽

Acidic Conditions ◽

O2 Production

The respiratory burst of human neutrophils was measured under conditions of hypoxia and low pH. O2 -- production by neutrophils activated with opsonized zymosan fell slowly as the oxygen concentration declined to 1%, then dropped more sharply, reaching negligible levels at oxygen concentrations less than 0.25%. Production was half maximal at an oxygen concentration of 0.35% (equivalent to approximately 10-microM dissolved oxygen). O2- production by the cell- free O2- -forming system prepared from zymosan-activated neutrophils showed a similar dependence on oxygen concentration. A drop in pH caused decreases in both oxygen consumption and O2-- production by zymosan-treated neutrophils, values at PH 6.0 being 10%--20% of those observed at pH 7.5. Experiments with the cell-free O2-- -forming system suggested that this decline in respiratory burst activity at low pH was due to inefficient activation of the O2-- -forming enzyme under acidic conditions.

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The Flavonols Quercetin, Myricetin, Kaempferol, and Galangin Inhibit the Net Oxygen Consumption by Immune Complex- Stimulated Human and Rabbit Neutrophils

Zeitschrift für Naturforschung C ◽

10.5560/znc.2012-0122 ◽

2014 ◽

Vol 69 (7-8) ◽

pp. 346-356 ◽

Cited By ~ 6

Author(s):

Andréa S. G. Figueiredo-Rinhel ◽

Everton O. L. Santos ◽

Luciana M. Kabeya ◽

Ana Elisa C. S. Azzolini ◽

Livia M. C. Simões-Ambrosio ◽

...

Keyword(s):

Oxygen Consumption ◽

Immune Complex ◽

Respiratory Burst ◽

Human Neutrophil ◽

Inflammatory Diseases ◽

Cell Types ◽

Oxygen Electrode ◽

Experimental Models ◽

Human Neutrophils ◽

Experimental Conditions

Stimulated human neutrophils exhibit increased net oxygen consumption (NOC) due to the conversion of O2 into the superoxide anion by the NADPH oxidase enzymatic complex during the respiratory burst. In several inflammatory diseases, overproduction of these oxidants causes tissue damage. The present study aims to: (a) optimize the experimental conditions used to measure the NOC in serum-opsonized zymosan (OZ)-and insoluble immune complex (i-IC)-stimulated human and rabbit neutrophils; and (b) compare the effect of four flavonols (quercetin, myricetin, kaempferol, and galangin) on this activity. We used a Clark-type oxygen electrode to measure the NOC of stimulated neutrophils. Eliciting the neutrophil respiratory burst with OZ and i-IC yielded similar maximum O2 uptake levels within the same species, but the human neutrophil NOC was almost four times higher than the rabbit neutrophil NOC. The optimal experimental conditions established for both cell types were 4·106 neutrophils mL-1, 2 mg mL-1 OZ, and 240 µg mL-1 i-IC. Upon stimulation with OZ or i-IC, the tested flavonols reduced the human and rabbit neutrophil NOC in the same order of potency - quercetin and galangin were the most and the least potent, respectively. These compounds were around four times more effective in inhibiting the rabbit as compared to the human neutrophil NOC, respectively. The four flavonols were not toxic to human or rabbit neutrophils. The experimental conditions used are suitable for both the determination of human and rabbit neutrophil NOC and for the assessment of the modulatory effects of natural compounds on these activities. The relationship between the level of NOC and the inhibitory potency of the flavonols suggests that rabbit neutrophils can be useful experimental models to predict the effect of drugs on immune complexstimulated human neutrophils.

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Activation mechanisms of adherent human neutrophils

Blood ◽

10.1182/blood.v76.6.1233.1233 ◽

1990 ◽

Vol 76 (6) ◽

pp. 1233-1239 ◽

Cited By ~ 45

Author(s):

I Ginis ◽

AI Tauber

Keyword(s):

Respiratory Burst ◽

Human Neutrophils ◽

Initiation Phase ◽

Independent Form ◽

Plastic Surface ◽

Activation Mechanisms ◽

Coated Surfaces ◽

Cell Effector Function ◽

O2 Production ◽

Cell Effector

Abstract The mechanism by which unstimulated human neutrophils initiate a respiratory burst on adherence to a surface has been examined. When neutrophils adhere to a plastic surface, they immediately generate a sustained burst of superoxide (O2-). However, this respiratory burst is not initiated by adherence alone, since neutrophils attached to fibronectin fail to mount a response. Adhesion to plastic is calcium (Ca2+) independent, but O2- production requires Ca2(+)-containing buffer in the initiation phase, that is, during adhesion and the early phase of O2- production. The Ca2(+)-dependent step was shown to involve protein kinase C (PK-C) in that the O2- production, but not adherence, was blocked with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and PK-C was found to translocate from the cytosol to the membrane on adhesion. Furthermore, it may be inferred that this translocation results in the generation of a Ca2+ independent form of PK-C, PK-M, since leupeptin, which inhibits the generation of PK-M, also blocked O2- production. This finding was corroborated by showing that after 5 minutes in a Ca2(+)-containing buffer, enough time to initiate O2- production and PK-C translocation, Ca2+ is no longer required for sustained O2- release. These results, in aggregate, demonstrate that neutrophils are activated by adhesion to plastic to generate O2-, a PK- C-dependent process that appears to involve a Ca2(+)-independent form of the kinase, PK-M. Why adherent neutrophils generate a respiratory burst on plastic and not fibronectin surfaces probably reflects activation of distinct receptors, whose nature must still be defined. Another issue to address is the priming effect of adhesion, since cells adherent to plastic- or fibronectin-coated surfaces have an enhanced O2- response to formylmethionyl-leucine-phenylalanine (FMLP) compared with neutrophils stimulated in suspension. This may relate to increased Ca2+ mobilization, an important mediator of priming for FMLP responses. Thus, adhesion as a priming event does not necessarily initiate cell effector function, and the further elucidation of the plastic and fibronectin models suggests a means of characterizing the crucial event that control neutrophil activation.

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Neutrophil p29 Peroxiredoxin VI (Prdx VI) Enhances Bactericidal Activity and Chemotaxis in Myeloid Cells.

Blood ◽

10.1182/blood.v114.22.1354.1354 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1354-1354 ◽

Cited By ~ 1

Author(s):

Michael Ellison ◽

Gail Thurman ◽

Daniel R. Ambruso

Keyword(s):

Nadph Oxidase ◽

Bactericidal Activity ◽

Respiratory Burst ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Human Neutrophils ◽

Boyden Chamber ◽

And Control ◽

O2 Production ◽

In Cells

Abstract Abstract 1354 Poster Board I-377 Introduction We have identified a 29 kDa protein from human neutrophils which binds to the NADPH oxidase component p67phox and enhances superoxide anion (O2−) production in a cell-free, reconstituted, NADPH oxidase system. The protein was identified as peroxiredoxin VI by sequence and the recombinant molecule was found to have both peroxiredoxin activity and calcium-independent PLA2 activity that is optimum at low pH (aiPLA2). Although p29 Prdx VI is found in many tissues, its role in myeloid cells is not well established. To explore other roles of p29, in addition to its effect on the respiratory burst, a PLB-985 cell line with shRNA mediated knockdown of p29 Prdx VI was established. Chemotaxis, as well as ingestion and killing S. aureus were determined in knockdown and control cells. Methods PLB-985 cells were transfected with a plasmid encoding a p29 Prdx VI targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx VI was confirmed by Western blot with no changes in actin or other oxidase components. After maturation of the knockdown and control cells by DMSO for 4 days, each was combined with serum opsonized Staph. aureus in a 2 to1 human cell to bacterial cell ratio, bacterial cells remaining at various times were measured by plating aliquots of the cell mixtures and counting bacterial colonies which grew overnight. To evaluate ingestion, aliquots of the cell mixtures were transferred to slides by cytospin, stained, and examined under a light microscope to determine what proportion of PLB-985 cells had internalized bacteria. To evaluate chemotaxis, distances of migration toward chemo-attractant (opsonized zymosan) in a Boyden chamber were measured for differentiated p29 Prdx VI knockdown and control cells. Results Using stable expression of shRNA p29 protein was reduced to 31+/-18% (SD) of that in non-knockdown control cells. In two separate assays of bactericidal activity, cells without knockdown of p29 Prdx VI had 17 and 13% of initial bacteria surviving at 30 min; cells with p29 Prdx VI knockdown had 30 and 56% of bacteria surviving. This defect in bactericidal activity since ingestion was no different between the two types of cells at 0, 5, 10, and 15 min after addition of the bacteria. In response to zymosan activated serum, stimulated directed migration (distance of leading front in response to zymosan activated serum minus distance of leading front in response to buffer) was greater in cells without knockdown (23.9 ± 3.0 microns, mean ± SEM, n = 4 separate experiments) than movement by cells with knockdown of p29 Prdx VI (18.3 ± 5.3 microns). The difference was significant, p<0.05 by paired t test. Conclusion Optimal O2− production during the respiratory burst in intact myeloid cells is dependent on p29. Deficient bactericidal activity was demonstrated at 30 min; this decrease could not be associated with a difference in ingestion. In addition, directed cell migration was also decreased in cells with decreased amounts of p29 Prdx VI. These results indicate that in addition to its effect on the respiratory burst, p29 Prdx supports multiple functions in neutrophils. Disclosures No relevant conflicts of interest to declare.

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Pancreatic Phospholipase A2 - Mediated Enhancement of the Respiratory Burst Response of Human Neutrophils

Zeitschrift für Naturforschung C ◽

10.1515/znc-2001-11-1237 ◽

2001 ◽

Vol 56 (11-12) ◽

pp. 1150-1156 ◽

Cited By ~ 2

Author(s):

Julia Müller ◽

Marijana Petković ◽

Jürgen Schiller ◽

Jürgen Arnhold

Keyword(s):

Respiratory Burst ◽

Laser Desorption ◽

Human Neutrophils ◽

Oxygen Species ◽

Respiratory Burst Activity ◽

Mediated Effects ◽

Flight Mass Spectrometry ◽

Pancreatic Phospholipase ◽

Pancreatic Phospholipase A2 ◽

Matrix Assisted Laser

Abstract The aim of this study was to investigate the effects of exogenously added pancreatic phos-pholipase A2 (pPLA2) on the production of reactive oxygen species by human polymorpho nuclear leukocytes (PMNs). Pancreatic PLA2 was used because PMNs do not possess a re ceptor for that enzyme and, therefore, the receptor-mediated effects could be excluded. Respiratory burst activity of PMNs was monitored by luminol-amplified chemiluminescence and the lipid composition of neutrophils after treatment with pPLA2 was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that the products of the pPLA2 digestion of the PMN membrane -lysophospholipids and the corresponding free fatty acids -significantly enhanced the respiratory burst response of human neutrophils.

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Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses

Biochemical Journal ◽

10.1042/bj2960297 ◽

1993 ◽

Vol 296 (2) ◽

pp. 297-301 ◽

Cited By ~ 769

Author(s):

A Arcaro ◽

M P Wymann

Keyword(s):

Respiratory Burst ◽

Actin Polymerization ◽

Kinase Inhibitor ◽

Inhibitory Effect ◽

Burst Activity ◽

Regulatory Subunit ◽

Human Neutrophils ◽

Respiratory Burst Activity ◽

Intact Cells ◽

Cytoskeletal Rearrangements

Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.

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