scholarly journals Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils

1986 ◽  
Vol 239 (3) ◽  
pp. 723-731 ◽  
Author(s):  
P G Heyworth ◽  
A W Segal
Keyword(s):  
Respiratory Burst ◽  
Protein A ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Receptor Signal ◽  
Latex Beads ◽  
Respiratory Burst Oxidase ◽  
Kinetics Of

Phosphorylation of a 47 kDa protein in human neutrophils is induced by phorbol 12-myristate 13-acetate (PMA), opsonized latex beads, fMet-Leu-Phe, calcium ionophore A23187 and fluoride. All of these stimuli activate the specialized microbicidal respiratory burst of neutrophils, and in each case the kinetics of activation correspond with the kinetics of phosphorylation of the 47 kDa protein. Trifluoperazine (50 microM) and chlorpromazine (100 microM), inhibitors of calmodulin and protein kinase C, abolish the increase in oxygen consumption and selectively prevent phosphorylation of the 47 kDa protein after PMA stimulation. Treatment of neutrophils with pertussis toxin totally inhibits both superoxide production and phosphorylation of this protein in response to fMet-Leu-Phe, but not in response to PMA, indicating that a GTP-binding protein modulates the fMet-Leu-Phe receptor signal. Phosphorylation of the 47 kDa protein, a phenomenon absent from the neutrophils of subjects with autosomal recessive chronic granulomatous disease, which lack the respiratory burst, appears to be the common trigger for activation of the burst in normal neutrophils.

The appearance of glycoconjugates associated with cortical granule release during mouse fertilization

Development ◽  
1988 ◽  
Vol 102 (3) ◽  
pp. 595-604 ◽  
Author(s):  
S.H. Lee ◽  
K.K. Ahuja ◽  
D.J. Gilburt ◽  
D.G. Whittingham
Keyword(s):  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Meiotic Spindle ◽  
Cortical Granule ◽  
Ionophore A23187 ◽  
Perivitelline Space ◽  
Cell Stage ◽  
Artificial Activation ◽  
First Time ◽  
Kinetics Of

For the first time we have shown with appropriately labelled lectins that fucosyl- and sialyl-rich glycoconjugates are released into the perivitelline space of the mouse oocyte after activation by the fertilizing spermatozoon or artificial activation by the calcium ionophore A23187 or ethanol. The glycoconjugates show a punctate distribution over the oocyte surface except for the microvilli-free area overlying the second meiotic spindle from which they are absent. Their appearance in the perivitelline space is associated with the release of the cortical granule suggesting that they represent part of the cortical granule exudate. Soon after the glycoconjugates appear, they begin to aggregate. The process continues until the beginning of cytokinesis at first cleavage when a single large aggregate is found within the cleavage furrow. Most of the labelled glycoconjugates disappear by the late 2-cell stage and no evidence was found for their presence during the later preimplantation period. This technique is suitable for monitoring the kinetics of the cortical reaction in mammalian oocytes and investigating the importance of the glycoconjugates in early preimplantation period.

scholarly journals Inhibition of neutrophil oxidative metabolism by lysosomotropic weak bases

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 334-342 ◽  
Author(s):  
B Styrt ◽  
MS Klempner
Keyword(s):  
Phorbol Myristate Acetate ◽  
Oxidative Metabolism ◽  
Respiratory Burst ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Enzyme Complex ◽  
Myristate Acetate ◽  
Ph Gradients ◽  
Weak Bases

Abstract Maintenance of an acidic intralysosomal compartment may be relevant to multiple aspects of neutrophil function. The effect of lysosomal alkalinization on the neutrophil respiratory burst was studied by measuring cytochrome c reduction in response to soluble stimuli in the presence of lysosomotropic weak bases. The weak bases chloroquine, ammonium chloride, methylamine, and clindamycin all raised the intralysosomal pH and inhibited neutrophil oxidative metabolism at concentrations ranging from 0.1 to 100 mmol/L. Inhibition was dose dependent for each base and correlated significantly with the degree of lysosomal alkalinization. Concentrations that did not alkalinize the lysosome did not inhibit the respiratory burst. Inhibition by weak bases was seen when oxidative metabolism was stimulated by phorbol myristate acetate, calcium ionophore A23187, formyl-methionyl-leucyl- phenylalanine, opsonized zymosan, or sodium fluoride. Increasing the stimulus concentration (from 5 ng/mL to 5 micrograms/mL phorbol myristate acetate and from 0.5 to 1 mumol/L A23187) diminished or abolished inhibition by weak bases. Washing the cells after incubation with bases and before stimulation substantially reversed the inhibition. None of the bases impaired detection of superoxide in a cell-free xanthine-xanthine oxidase assay. Other indexes of oxidative metabolism, including oxygen consumption and hydrogen peroxide release, were also inhibited by weak bases. Analysis of particulate NADPH oxidase activity from neutrophils stimulated in the presence of bases suggested that these cells assemble a subnormal amount of an enzyme complex with normal kinetic characteristics. Lysosomotropic weak bases alkalinized the neutrophil lysosome and produced inhibition of oxidative metabolism that was dose related, was not stimulus specific, and was largely reversed by washing the cells before stimulation. A possible explanation would be altered assembly of the enzyme complex involved in respiratory burst activation as a consequence of impaired granule/plasma membrane fusion in the presence of diminished transmembrane pH gradients.

scholarly journals Nitric oxide and peroxynitrite trigger and enhance release of neutrophil extracellular traps

2019 ◽  
Vol 77 (15) ◽  
pp. 3059-3075 ◽  
Author(s):  
Aneta Manda-Handzlik ◽  
Weronika Bystrzycka ◽  
Adrianna Cieloch ◽  
Eliza Glodkowska-Mrowka ◽  
Ewa Jankowska-Steifer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitrosative Stress ◽  
Calcium Ionophore ◽  
Neutrophil Extracellular Traps ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Inflammatory Reactions ◽  
Extracellular Traps

Abstract Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.

scholarly journals Mechanism of Fc-mediated interaction of eosinophils with immobilized immune complexes: I. Effects of inhibitors and activators of eosinophil function

1982 ◽  
Vol 56 (1) ◽  
pp. 337-356
Author(s):  
R.C. Oliver ◽  
A.M. Glauert ◽  
K.J. Thorne
Keyword(s):  
Protein A ◽  
Fc Receptors ◽  
Calcium Ionophore ◽  
Apparent Molecular Weight ◽  
Direct Consequence ◽  
Fc Receptor ◽  
Calcium Ionophore A23187 ◽  
Cytochalasin D ◽  
Ionophore A23187 ◽  
Staphylococcal Protein A

A protein of apparent molecular weight 55000, designated protein 3, becomes newly detectable on the eosinophil surface as a specific consequence of interaction with antigen-antibody complexes immobilized in agar layers. The effect of various agents upon this interaction has been determined by monitoring the appearance of this protein by lactoperoxidase-catalysed iodination. Other parameters that have been measured include: the attachment of eosinophils to the agar layers and their subsequent degranulation, as measured by the release of granule peroxidase, and the degree of spreading of the eosinophils, as assessed by electron microscopy. Attachment of eosinophils to antibody-coated layers is inhibited by heat-aggregated immunoglobulin G (IgG), suggesting that this attachment is mediated via eosinophil Fc receptors. In addition, agents, such as the eosinophil chemotactic factor Ala-Gly-Ser-Glu, that enhance the expression of Fc receptors also enhance the appearance of protein 3, while agents, such as hydrocortisone, that inhibit the expression of Fc receptors reduce its appearance. It is concluded that the appearance of protein 3 parallels the expression of Fc receptors. Attempts to block the Fc region of the bound antibody with staphylococcal protein A were not successful. These experiments indicated that the Fc region of bound IgG has different binding sites for protein A and for the Fc receptor. The correlation between the appearance of protein 3 and subsequent degranulation of the eosinophils was confirmed by the use of agents, such as cytochalasin D and levamisole, that enhance both the appearance of protein 3 and degranulation. Conversely, hydrocortisone reduces the appearance of protein 3 and inhibits degranulation. Protein 3 does not appear when eosinophils adhere to agar layers coated with concanavalin A instead of antibody and the eosinophils do not degranulate. Addition of the calcium ionophore A23187, while causing the release of granule peroxidase, does not elicit the appearance of protein 3. These observations provided additional evidence that the appearance of protein 3 is a specific consequence of the interaction of eosinophils with antibody-coated surfaces. The fact that protein 3 appears at the eosinophil surface as a direct consequence of the interaction with antibody suggests that this protein is closely associated with the eosinophil Fc receptor. The enhancement of the appearance of protein 3 in the presence of cytochalasin D indicates that the movement and reorientation of both this protein and the Fc receptor are constrained by association with cytoplasmic microfilaments.

scholarly journals Interrelationships of polymorphonuclear neutrophil membrane-bound calcium, membrane potential, and chemiluminescence: studies in single living cells

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1184-1192 ◽  
Author(s):  
GW Sullivan ◽  
GR Donowitz ◽  
JA Sullivan ◽  
GL Mandell
Keyword(s):  
Membrane Potential ◽  
Calcium Release ◽  
Calcium Ionophore ◽  
Image Intensifier ◽  
Calcium Ionophore A23187 ◽  
Polymorphonuclear Neutrophil ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Ionic Fluxes ◽  
Single Living

Abstract Stimulated neutrophils show ionic fluxes that may function as “transducers” between stimuli and effector functions. Using fluorescent probes, patterns of membrane-associated calcium (chlortetracycline, CTC) and membrane potential (3–3′-dipentyloxacarbocyanine, di-O-C5 (3)) in single living human neutrophils were observed with a fluorescence microscope fitted with an image intensifier and photometer. Fluorescence changes were related to chemiluminescence. In unstimulated neutrophils, CTC and di-O-C5 (3) fluorescence was brightest in the perinuclear region. Di-O-C5 (3) fluorescence was also seen in mitochondria. Neutrophil stimulation with zymosan, phorbol myristate acetate (PMA) or calcium ionophore (A23187) resulted in loss of di-O-C5 (3) and CTC fluorescence and chemiluminescence proportional to the strength of the stimulus. Experiments demonstrated the independence of these processes. (1) Digitonin stimulation caused chemiluminescence and di-O-C5 (3) darkening without loss of CTC fluorescence. (2) Depolarization of neutrophils did not induce CTC darkening or chemiluminescence. (3) Calcium ionophore (A23187) stimulation of neutrophils in calcium-free medium resulted in normal di-O-C5 (3) and CTC darkening, but a blunted chemiluminescence peak. (4) Calcium ionophore (A23187) stimulated the loss of di-O-C5 (3) and CTC fluorescence from chronic granulomatous disease neutrophils, but did not trigger an oxidative burst. Although neutrophil depolarization, calcium release from membranes, and oxidative activity are linked, these processes can clearly be separated.

scholarly journals Involvement of guanine nucleotide binding proteins in neutrophil activation and priming by GM-CSF

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 588-591 ◽  
Author(s):  
SR McColl ◽  
C Kreis ◽  
JF DiPersio ◽  
P Borgeat ◽  
PH Naccache
Keyword(s):  
Pertussis Toxin ◽  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Gm Csf ◽  
Surface Receptor ◽  
Pre Treatment ◽  
Guanine Nucleotide Binding

Abstract Pre-incubation of human neutrophils with pertussis toxin significantly inhibited the neutrophil-directed biologic actions of granulocyte- macrophage colony-stimulating factor (GM-CSF) in three separate assays: the induction of c-fos mRNA, the enhancement of both platelet- activating factor-induced mobilization of intracellular calcium, and stimulation of leukotriene synthesis by the calcium ionophore A23187. Cholera toxin did not have an effect on the latter two assays. Pre- treatment of human neutrophils with pertussis toxin did not affect the binding of GM-CSF to its surface receptor. These results provide the first evidence that a pertussis toxin substrate plays an important mediatory role in the mechanism of action of GM-CSF.

scholarly journals Interactions between human eosinophils and schistosomula of schistosoma mansoni. II. The mechanism of irreversible eosinophil adherence

1979 ◽  
Vol 150 (6) ◽  
pp. 1456-1471 ◽  
Author(s):  
AE Butterworth ◽  
MA Vadas ◽  
DL Wassom ◽  
A Dessein ◽  
M Hogan ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Concanavalin A ◽  
Protein A ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Con A ◽  
Normal Human ◽  
Culture Supernate ◽  
Eosinophil Major Basic Protein

Previous work (1)(1) has shown that normal human eosinophils show a preferential capacity, in comparison with neutrophils, to bind to antibody- coated schistosomula of Schistosoma mansoni. This effect is attributable to a temperature-dependent function of the eosinophil which renders its binding stable and irreversible by aggregated gamma globulin or Staphylococcus aureus protein A. In contrast, the binding of neutrophils is readily reversible by these agents. It has now been shown that the differences observed between eosinophils and neutrophils is a property of their interaction with living schistosomula. When dead or artificially damaged schistosomula were tested, neutrophils showed a markedly enhanced capacity to adhere, in both the presence and absence of anti-chistosomular serum. Subsequent experiments were designed to test the hypothesis that the strong, stable binding of eosinophils was attributable to degranulation, with release of granule contents which would then serve as ligands to bind the cell to the organism. First, an enhanced adherence both of eosinophils and of neutrophils could be demonstrated in the presence of eosinophil major basic protein (MBP) or of protamine, a high molecular weight cation. Second, the binding of eosinophils induced by concanavalin A (Con A) was found to differ markedly from that induced by antischistosomular serum. Con A-mediated binding of eosinophils was fully reversible by alpha-methyl-mannoside, was not associated with damage to the organism, and did not lead to degranulation of the cell, as estimated by measuring the release of MBP into the culture supernate. However, induction of degranulation of concanavalin A-bound eosinophils, but not of neutrophils, with the calcium ionophore A23187 converted the reaction into one which was no longer reversible by alpha- methylmannoside and in which damage to the organism now did occur. These findings support the hypothesis that the stable binding of eosinophils is associated with degranulation, a process which may contribute to the preferential capacity of this cell to mediate antibody-dependent damage to schistosomula.

scholarly journals Interrelationships of polymorphonuclear neutrophil membrane-bound calcium, membrane potential, and chemiluminescence: studies in single living cells

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1184-1192
Author(s):  
GW Sullivan ◽  
GR Donowitz ◽  
JA Sullivan ◽  
GL Mandell
Keyword(s):  
Membrane Potential ◽  
Calcium Release ◽  
Calcium Ionophore ◽  
Image Intensifier ◽  
Calcium Ionophore A23187 ◽  
Polymorphonuclear Neutrophil ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Ionic Fluxes ◽  
Single Living

Stimulated neutrophils show ionic fluxes that may function as “transducers” between stimuli and effector functions. Using fluorescent probes, patterns of membrane-associated calcium (chlortetracycline, CTC) and membrane potential (3–3′-dipentyloxacarbocyanine, di-O-C5 (3)) in single living human neutrophils were observed with a fluorescence microscope fitted with an image intensifier and photometer. Fluorescence changes were related to chemiluminescence. In unstimulated neutrophils, CTC and di-O-C5 (3) fluorescence was brightest in the perinuclear region. Di-O-C5 (3) fluorescence was also seen in mitochondria. Neutrophil stimulation with zymosan, phorbol myristate acetate (PMA) or calcium ionophore (A23187) resulted in loss of di-O-C5 (3) and CTC fluorescence and chemiluminescence proportional to the strength of the stimulus. Experiments demonstrated the independence of these processes. (1) Digitonin stimulation caused chemiluminescence and di-O-C5 (3) darkening without loss of CTC fluorescence. (2) Depolarization of neutrophils did not induce CTC darkening or chemiluminescence. (3) Calcium ionophore (A23187) stimulation of neutrophils in calcium-free medium resulted in normal di-O-C5 (3) and CTC darkening, but a blunted chemiluminescence peak. (4) Calcium ionophore (A23187) stimulated the loss of di-O-C5 (3) and CTC fluorescence from chronic granulomatous disease neutrophils, but did not trigger an oxidative burst. Although neutrophil depolarization, calcium release from membranes, and oxidative activity are linked, these processes can clearly be separated.

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