scholarly journals Oxidative inactivation of myeloperoxidase released from human neutrophils

1987 ◽  
Vol 245 (3) ◽  
pp. 925-928 ◽  
Author(s):  
S W Edwards ◽  
H L Nurcombe ◽  
C A Hart
Keyword(s):  
Respiratory Burst ◽  
Cytochalasin B ◽  
The Other ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Cellular Activity ◽  
Chemotactic Peptide ◽  
Oxidative Inactivation ◽  
Stimulation Of

Within 1 min of stimulation of human neutrophils by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B, myeloperoxidase (together with other granule enzymes) was secreted and detected extracellularly. In contrast with the other granule constituents assayed (vitamin B12-binding protein and beta-glucuronidase), the activity of released myeloperoxidase rapidly decreased, so that, by 10 min after stimulation, only about 5% of the total cellular activity was detected. This inactivation was shown to be dependent on oxidant generation during the respiratory burst, since inactivation was not observed (a) after stimulation of anaerobic suspensions or (b) after release from neutrophils from a patient with chronic granulomatous disease; purified myeloperoxidase was rapidly inactivated after incubation with H2O2, presumably owing to the formation of an inactive enzyme-H2O2 complex. These results show that experiments designed to assess the role of myeloperoxidase in neutrophil functions which utilize assays based on peroxidase activity will grossly underestimate this enzyme if oxidant generation during the respiratory burst has also been activated.

scholarly journals Regulation of superoxide generation by myeloperoxidase during the respiratory burst of human neutrophils

1986 ◽  
Vol 237 (2) ◽  
pp. 601-604 ◽  
Author(s):  
S W Edwards ◽  
T F Swan
Keyword(s):  
Respiratory Burst ◽  
Cytochalasin B ◽  
Superoxide Radical ◽  
Human Neutrophils ◽  
Second Phase ◽  
Chemotactic Peptide ◽  
O2 Uptake ◽  
Superoxide Generation ◽  
Lower Magnitude

The role of myeloperoxidase in the regulation of the respiratory burst of human neutrophils activated by the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) plus cytochalasin B was determined by using anti-(human myeloperoxidase) antibody. The respiratory burst activated under these conditions consisted of an initial (1-2 min) phase with high rates of O2 uptake, luminol-dependent chemiluminescence and superoxide radical (O2-.) generation and a second, more sustained, phase of lower magnitude of chemiluminescence and O2 uptake: O2-. generation did not occur during this second phase. In cell suspensions stimulated in the presence of anti-(human myeloperoxidase) antibody, the magnitude of the initial phase of both O2 uptake and O2-. generation was unaffected, but these high rates were maintained over much longer periods than in control suspensions. It is therefore proposed that a product of myeloperoxidase normally regulates the duration of O2-. generation during the respiratory burst, possibly by inhibition of NADPH oxidase.

scholarly journals NADPH-binding component of the respiratory burst oxidase system: studies using neutrophil membranes from patients with chronic granulomatous disease lacking the beta-subunit of cytochrome b558.

1994 ◽  
Vol 179 (1) ◽  
pp. 291-297 ◽  
Author(s):  
S Tsunawaki ◽  
H Mizunari ◽  
H Namiki ◽  
T Kuratsuji
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Beta Subunit ◽  
Human Neutrophils ◽  
Granulomatous Disease ◽  
Oxidase System ◽  
Cytochrome B558 ◽  
Respiratory Burst Oxidase ◽  
Stimulation Of ◽  
Binding Component

The NADPH-binding site of the respiratory burst oxidase system of neutrophils has been proposed to be either at a cytosolic component or at the beta-subunit of cytochrome b558. In this study, affinity labeling of resting and stimulated membranes, the latter having been assembled by all of the oxidase components from both membrane and cytosol, was carried out using [32P]NADPH dialdehyde (oNADPH). Stimulation of human neutrophils with PMA greatly increased O2(-)-generating activity and caused considerable translocation of the cytosolic components p47phox and p67phox. Nevertheless, PMA stimulation did not produce a labeled band which included positions at 47, 67, and approximately 32 kD. The most intense band reflected a molecular mass of 84 kD regardless of the state of activation, but a labeled band was never found near the beta-subunit (91 kD) of cytochrome b558. This 84-kD protein was further confirmed in neutrophils of 14 patients with gp91phox-deficient X-linked chronic granulomatous disease. These results indicate that the NADPH-binding component is not recruited from the cytosol, and also, that a membranous redox component besides cytochrome b558 must be involved in the NADPH oxidase system.

scholarly journals Chemotactic peptide-induced changes in neutrophil actin conformation.

1984 ◽  
Vol 99 (3) ◽  
pp. 1060-1065 ◽  
Author(s):  
P J Wallace ◽  
R P Wersto ◽  
C H Packman ◽  
M A Lichtman
Keyword(s):  
Flow Cytometry ◽  
Cytochalasin B ◽  
Fluid Phase ◽  
Repetitive Stimulation ◽  
Human Neutrophils ◽  
Chemotactic Peptide ◽  
Directed Movement ◽  
Induced Changes ◽  
Dose Dependent ◽  
Stimulation Of

The effect of the chemotatic peptide, N-formylmethionylleucylphenylalanine (FMLP), on actin conformation in human neutrophils (PMN) was studied by flow cytometry using fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD)-phallacidin to quantitate cellular F-actin content. Uptake of NBD-phallacidin by fixed PMN was saturable and inhibited by fluid phase F-actin but not G-actin. Stimulation of PMN by greater than 1 nM FMLP resulted in a dose-dependent and reversible increase in F-actin in 70-95% of PMN by 30 s. The induced increase in F-actin was blocked by 30 microM cytochalasin B or by a t-BOC peptide that competitively inhibits FMLP binding. Under fluorescence microscopy, NBD-phallacidin stained, unstimulated PMN had faint homogeneous cytoplasmic fluorescence while cells exposed to FMLP for 30 s prior to NBD-phallacidin staining had accentuated subcortical fluorescence. In the continued presence of an initial stimulatory dose of FMLP, PMN could respond with increased F-actin content to the addition of an increased concentration of FMLP. Thus, FMLP binding to PMN induces a rapid transient conversion of unpolymerized actin to subcortical F-actin and repetitive stimulation of F-actin formation can be induced by increasing chemoattractant concentration. The directed movement of PMN in response to chemoattractant gradients may require similar rapid reversible changes in actin conformation.

scholarly journals Protein phosphatase inhibitors okadaic acid and calyculin A alter cell shape and F-actin distribution and inhibit stimulus-dependent increases in cytoskeletal actin of human neutrophils

Blood ◽  
1992 ◽  
Vol 80 (11) ◽  
pp. 2911-2919 ◽  
Author(s):  
P Kreienbuhl ◽  
H Keller ◽  
V Niggli
Keyword(s):  
Okadaic Acid ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Calyculin A ◽  
Chemotactic Peptide ◽  
Phosphatase Inhibitors ◽  
Protein Phosphatase Inhibitors ◽  
Actin Localization ◽  
Shape Changes

Abstract The phosphatase inhibitors okadaic acid and calyculin A were found to elicit or to modify several neutrophil responses, suggesting that dephosphorylation plays a regulatory role. The concentrations of okadaic acid (> or = 1 mumol/L) that were effective on neutrophil functions (shape changes and marginal stimulation of pinocytosis) were shown to stimulate the incorporation of 32PO4 into many neutrophil proteins several-fold. Calyculin A was effective at 50-fold lower concentrations. In the presence of the inhibitors, the cells exhibited a nonpolar shape and the polarization response induced by chemotactic peptide was inhibited. Both phosphatase inhibitors also induced the association of F-actin with the cell membrane. A steady-state phosphatase activity is thus involved in maintaining shape and F-actin localization of resting cells. Inhibitors alone had no significant effect on the amount of cytoskeleton-associated actin. The increase in cytoskeletal actin observed at 30 minutes of stimulation with phorbol ester or 5 to 30 minutes of stimulation with chemotactic peptide, however, was abolished by okadaic acid or calyculin A, suggesting an important role of a phosphatase. In contrast, the early increase in cytoskeleton-associated actin observed at 1 minute of stimulation with peptide was not affected. This finding indicates that the increased association of actin with the cytoskeleton in the early and the later stages of neutrophil activation may be mediated by different signalling pathways.

scholarly journals Detection of carriers of the autosomal form of chronic granulomatous disease

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 505-507 ◽  
Author(s):  
AJ Verhoeven ◽  
ML van Schaik ◽  
D Roos ◽  
RS Weening
Keyword(s):  
Chronic Granulomatous Disease ◽  
Respiratory Burst ◽  
Granulomatous Disease ◽  
Mosaic Pattern ◽  
Simple Method ◽  
Nbt Test ◽  
Activated Neutrophils ◽  
Slide Test ◽  
Microscopic Slide ◽  
Stimulation Of

The NADPH:O2 oxidoreductase catalyzing the respiratory burst in activated phagocytes from healthy individuals is not operative in phagocytes from patients with chronic granulomatous disease (CGD). In a microscopic slide test using the dye nitroblue tetrazolium (NBT), carriers of X-linked CGD can be recognized by a mosaic pattern of NBT- positive and NBT-negative cells, governed by the expression of an unaffected or an affected X chromosome, respectively. Until now, it has not been possible to detect carriers of the autosomal form of CGD (other than by family studies) because all cells of these carriers stain positive in the NBT test. We have investigated whether neutrophils from carriers of autosomal CGD can be recognized by measurement of the rate of oxygen uptake upon stimulation of the cells. It was found that with the phorbol ester PMA as a stimulus, the respiratory burst is significantly lower in autosomal CGD carriers. With serum-treated zymosan as a stimulus, no difference between controls and carriers was observed. The addition of f-Met-Leu-Phe (1 microM) to PMA-activated neutrophils of control donors caused a transient increase in oxygen consumption of about 40%. Under these conditions, an increase of more than 100% was observed in neutrophils from carriers of autosomal CGD. These findings provide a simple method for the detection of carriers of the autosomal form of CGD.

scholarly journals Cytosolic free calcium changes induced by chemotactic peptide in neutrophils from patients with chronic granulomatous disease

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 231-233 ◽  
Author(s):  
PD Lew ◽  
C Wollheim ◽  
RA Seger ◽  
T Pozzan
Keyword(s):  
Chronic Granulomatous Disease ◽  
Calcium Concentration ◽  
Cytosolic Calcium ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Granulomatous Disease ◽  
Chemotactic Peptide ◽  
Intact Cells ◽  
Calcium Indicator ◽  
Free Calcium Concentration

Abstract Cytoplasmic free calcium concentration (Ca2+)i was measured in neutrophils from patients with the classical X-linked form of chronic granulomatous disease (CGD) by trapping the fluorescent calcium indicator Quin 2 in intact cells. CGD neutrophils do not produce superoxide and are only slightly depolarized upon stimulation by the chemotactic peptide. N-formyl-methionyl-leucyl-phenylalanine (FMLP). The resting levels, as well as (Ca2+)i changes induced by FMLP in CGD cells, were quantitatively and kinetically similar to those observed in normal cells. We conclude that the defect in CGD cells is distal to, or independent of, the changes in (Ca2+)i induced by FMLP stimulation and that normal membrane depolarization does not seem to be necessary for receptor-mediated rise in free cytosolic calcium in human neutrophils.

Suppressive action of cobalt on exocytosis and respiratory burst in neutrophils

1989 ◽  
Vol 257 (5) ◽  
pp. C859-C864 ◽  
Author(s):  
J. G. Elferink ◽  
M. Deierkauf
Keyword(s):  
Plasma Membrane ◽  
Phorbol Myristate Acetate ◽  
Respiratory Burst ◽  
Cytochalasin B ◽  
Superoxide Production ◽  
Co2 Activation ◽  
Chemotactic Peptide ◽  
Myristate Acetate ◽  
Intracellular Target ◽  
Suppressive Action

Activation of exocytosis and respiratory burst in rabbit neutrophils by the chemotactic peptide fMet-Leu-Phe is inhibited by Co2+. Inhibition is antagonized by extracellular Ca2+ and is dependent on the time of preincubation of cells with Co2+ before addition of activator. Co2+ inhibits the enhancement of 45Ca association that occurs during activation with fMet-Leu-Phe. Interference with Ca2+ translocation across the plasma membrane by Co2+ is probably not the cause of inhibition of neutrophil activation, because activation in the absence of extracellular Ca2+ is inhibited by Co2+. Activation of neutrophils by phorbol myristate acetate is inhibited at higher Co2+ concentrations than activation by fMet-Leu-Phe. Inhibition of the superoxide production by Co2+ occurs both in the presence or in the absence of cytochalasin B. Fluorescence of neutrophils loaded with quin2 is diminished by Co2+, indicating that Co2+ had entered into the cytoplasm. The results are compatible with the view that Co2+ inhibits exocytosis and respiratory burst in neutrophils by an interaction with a Ca2+-dependent intracellular target.

Source and role of diacylglycerol formed during phagocytosis of opsonized yeast particles and associated respiratory burst in human neutrophils

1991 ◽  
Vol 177 (3) ◽  
pp. 948-955 ◽  
Author(s):  
Vittorina Della Bianca ◽  
Miroslawa Grzeskowiak ◽  
Daniele Lissandrini ◽  
Filippo Rossi
Keyword(s):  
Respiratory Burst ◽  
Human Neutrophils

scholarly journals G Protein-Coupled Estrogen Receptor 1 Regulates Human Neutrophil Functions

2017 ◽  
Vol 2 (1) ◽  
pp. 1-13 ◽  
Author(s):  
M. Carmen Rodenas ◽  
Nicola Tamassia ◽  
Isabel Cabas ◽  
Federica Calzetti ◽  
José Meseguer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Estrogen Receptor ◽  
Protein Kinase ◽  
G Protein ◽  
Respiratory Burst ◽  
Human Neutrophils ◽  
G Protein Coupled ◽  
Estrogen Receptor 1

Background: The role of estrogens in immune functioning is relatively well known under both physiological and pathological conditions. Neutrophils are the most abundant circulating leukocytes in humans, and their abundance and function are regulated by estrogens, since they express estrogen receptors (ERs). Traditionally, estrogens were thought to act via classical nuclear ERs, namely ERα and ERβ. However, it was observed that some estrogens induced biological effects only minutes after their application. This rapid, “nongenomic” effect of estrogens is mediated by a membrane-anchored receptor called G protein-coupled estrogen receptor 1 (GPER1). Nevertheless, the expression and role of GPER1 in the immune system has not been exhaustively studied, and its relevance in neutrophil functions remains unknown. Methods: Human neutrophils were incubated in vitro with 10-100 µM of the GPER1-specific agonist G1 alone or in combination with lipopolysaccharide. GPER1 expression and subcellular localization, respiratory burst, life span, gene expression profile, and cell signaling pathways involved were then analyzed in stimulated neutrophils. Results: Human neutrophils express a functional GPER1 which regulates their functions through cAMP/protein kinase A/cAMP response element-binding protein, p38 mitogen-activated protein kinase, and extracellular regulated MAPK signaling pathways. Thus, GPER1 activation in vitro increases the respiratory burst of neutrophils, extends their life span, and drastically alters their gene expression profile. Conclusions: Our results demonstrate that GPER1 activation promotes the polarization of human neutrophils towards a proinflammatory phenotype and point to GPER1 as a potential therapeutic target in immune diseases where neutrophils play a key role.

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