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 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.

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 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 Early plasma-membrane-potential changes during stimulation of lymphocytes by concanavalin A.

1983 ◽  
Vol 210 (3) ◽  
pp. 885-891 ◽  
Author(s):  
S M Felber ◽  
M D Brand
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Concanavalin A ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
K Channel ◽  
Ionophore A23187 ◽  
Resting Cells ◽  
Plasma Membrane Potential ◽  
Stimulation Of

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.

scholarly journals Selective inhibition of human neutrophil functional responsiveness by erbstatin, an inhibitor of tyrosine protein kinase

Blood ◽  
1990 ◽  
Vol 76 (10) ◽  
pp. 2098-2104 ◽  
Author(s):  
PH Naccache ◽  
C Gilbert ◽  
AC Caon ◽  
M Gaudry ◽  
CK Huang ◽  
...  
Keyword(s):  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Functional Responses ◽  
Chemotactic Factors ◽  
Cytoplasmic Free Calcium

Abstract The role of tyrosine kinases in the responses of human neutrophils to chemotactic factors was examined using the recently described inhibitor erbstatin. Pre-incubation with erbstatin decreased the amount of tyrosine phosphorylation induced by the formylated oligopeptide formyl- methionyl-leucyl-phenylalanine (fMet-Leu-Phe) without effecting the binding of [3H]-fMet-Leu-Phe. Erbstatin also dose-dependently inhibited the production of superoxide anion induced by fMet-Leu-Phe and platelet- activating factor, but did not affect the oxidative burst induced by either the calcium ionophore A23187 or the phorbol ester phorbol 12- myristate 13-acetate. Furthermore, erbstatin diminished the cytosolic acidification elicited by fMet-Leu-Phe, platelet-activating factor, and leukotriene B4. In contrast, erbstatin was without effect on the increase in the levels of cytoplasmic free calcium and polymerized actin elicited by fMet-Leu-Phe, C5a, leukotriene B4 and platelet- activating factor, whereas the increase in cytoplasmic free calcium elicited by platelet-derived growth factor was inhibited by erbstatin. In addition, erbstatin affected neither the release of elastase stimulated by these agonists nor the release of beta-glucosaminidase, lysozyme or vitamin B12-binding protein induced by fMet-Leu-Phe. These results indicate that tyrosine protein kinases are involved in the signaling pathways employed by chemotactic factors in the stimulation of selective functional responses (and superoxide production in particular) in human neutrophils.

scholarly journals Adenosine: a physiological modulator of superoxide anion generation by human neutrophils.

1983 ◽  
Vol 158 (4) ◽  
pp. 1160-1177 ◽  
Author(s):  
B N Cronstein ◽  
S B Kramer ◽  
G Weissmann ◽  
R Hirschhorn
Keyword(s):  
Superoxide Anion ◽  
Calcium Ionophore ◽  
Neutrophil Function ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Surface Receptors ◽  
Superoxide Anion Generation ◽  
Marked Enhancement ◽  
Nucleoside Uptake

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.

Effect of the calcium ionophore A23187 on superoxide generation in phorbol ester stimulated human neutrophils

1985 ◽  
Vol 63 (12) ◽  
pp. 1543-1546 ◽  
Author(s):  
Colette F. Strnad ◽  
Kenneth Wong
Keyword(s):  
Protein Kinase C ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Superoxide Generation ◽  
Kinase C ◽  
Dose Dependent

The calcium ionophore, A23187, and the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), interacted synergistically to elicit an accelerated superoxide production response in human neutrophils. The lag period preceding PMA-induced superoxide generation was decreased in a dose-dependent manner by A23187 at a concentration range from 1.0 × 10−8 to 1.0 × 10−5 M. Superoxide production rate, however, was subject to biphasic effects. While the rate was potentiated in a dose-dependent manner at A23187 concentrations below 1.0 × 10−6 M, inhibitory influences became manifest at higher concentrations. Total superoxide production was subject to inhibitory effects, characterized by a mean inhibitory dose of 1.3 × 10−6 M. The synergistic interaction of A23187 with PMA is consistent with a role for protein kinase C in neutrophil activation. Inhibition at high A23187 concentrations appeared to result from the effects of elevated intracellular Ca2+ levels on either NADPH oxidase itself, or some step in the transduction process linking protein kinase C to the oxidase complex.

scholarly journals Human neutrophil adherence to laminin in vitro. Evidence for a distinct neutrophil integrin receptor for laminin.

1990 ◽  
Vol 171 (4) ◽  
pp. 1221-1237 ◽  
Author(s):  
J F Bohnsack ◽  
S K Akiyama ◽  
C H Damsky ◽  
W A Knape ◽  
G A Zimmerman
Keyword(s):  
Endothelial Cell ◽  
Divalent Cation ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Surface Proteins ◽  
Human Neutrophils ◽  
Ionophore A23187 ◽  
Cell Monolayers ◽  
Beta 2

We used mAbs against polymorphonuclear leukocyte (PMN) surface proteins to investigate the mechanisms by which stimulated human neutrophils (PMNs) adhere in vitro to laminin, the major glycoprotein of mammalian basement membrane. mAb IB4, which is directed against the common beta 2 chain of the CD11/CD18, only partially inhibited the adherence of PMA-stimulated PMNs to both laminin and to subendothelial matrices. In contrast, IB4 completely inhibited PMA-stimulated PMN adherence to gelatin, fibronectin, collagen IV, and endothelial cell monolayers. PMA-stimulated PMNs from a patient with severe congenital CD11/CD18 deficiency also adhered to laminin, but not to gelatin or endothelial cell monolayers. Therefore, PMA-stimulated PMNs adhere to laminin by both CD11/CD18-dependent and CD11/CD18-independent mechanisms. Expression of CD11/CD18-independent adherence to laminin was agonist dependent, occurring after stimulation with the calcium ionophore A23187 and recombinant TNF-alpha, but not with the chemotactic factors FMLP, platelet activating factor, or recombinant human C5a. Expression of CD11/CD18-independent adherence was also divalent cation dependent, occurring in the presence of Mg2+ but not Ca2+ as the sole added divalent cation. The mAbs AIIB2 and 13, which are directed against the beta 1 subunit of the VLA integrins, significantly inhibited the CD11/CD18-independent adherence of normal PMNs to laminin, and completely abolished the adherence of CD11/CD18-deficient PMNs to laminin. Both anti-beta 1 mAbs bound to PMNs, as demonstrated by flow cytometry, and immunoprecipitated a membrane molecule of Mr 130,000 daltons from 125I-labeled, detergent-solubilized PMNs. These data suggest that human PMNs possess beta 1 and beta 2 classes of integrins, and that both mediate PMN adherence.

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