scholarly journals Tyrosine-specific protein phosphorylation during activation of human neutrophils

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2445-2452
Author(s):  
RL Berkow ◽  
RW Dodson
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Ionophore ◽  
Specific Protein ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Early Event ◽  
Ionophore A23187 ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine

The activation of human neutrophils by a variety of receptor-dependent and receptor-independent agonists induces the phosphorylation of a large number of proteins. Since we have previously shown that human neutrophils have at least two distinct tyrosine kinase activities, we examined protein tyrosine phosphorylation in human neutrophils stimulated with a variety of agonists. Using a monoclonal antibody specific for phosphotyrosine, the present study shows that the chemotactic peptides FMLP and leukotriene B4, the phorbol ester phorbol myristate acetate (PMA), and the calcium ionophore A23187 induce an increase in tyrosine phosphorylation of a number of neutrophil proteins. This increased protein tyrosine phosphorylation was dependent on the concentration of the agonist, as well as on the time of exposure to the agonist. Fractionation experiments showed that both a 150,000 g cytosolic and a particulate preparation showed increases in protein tyrosine phosphorylation with stimulation by FMLP or PMA, and showed that the pattern of protein tyrosine phosphorylation was slightly different in the FMLP- and PMA-stimulated cells. These data indicate that protein tyrosine phosphorylation is an early event in the activation of human neutrophils by a variety of receptor-dependent and receptor-independent agonists.

scholarly journals Tyrosine-specific protein phosphorylation during activation of human neutrophils

Blood ◽  
1990 ◽  
Vol 75 (12) ◽  
pp. 2445-2452 ◽  
Author(s):  
RL Berkow ◽  
RW Dodson
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Ionophore ◽  
Specific Protein ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Human Neutrophils ◽  
Early Event ◽  
Ionophore A23187 ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine

Abstract The activation of human neutrophils by a variety of receptor-dependent and receptor-independent agonists induces the phosphorylation of a large number of proteins. Since we have previously shown that human neutrophils have at least two distinct tyrosine kinase activities, we examined protein tyrosine phosphorylation in human neutrophils stimulated with a variety of agonists. Using a monoclonal antibody specific for phosphotyrosine, the present study shows that the chemotactic peptides FMLP and leukotriene B4, the phorbol ester phorbol myristate acetate (PMA), and the calcium ionophore A23187 induce an increase in tyrosine phosphorylation of a number of neutrophil proteins. This increased protein tyrosine phosphorylation was dependent on the concentration of the agonist, as well as on the time of exposure to the agonist. Fractionation experiments showed that both a 150,000 g cytosolic and a particulate preparation showed increases in protein tyrosine phosphorylation with stimulation by FMLP or PMA, and showed that the pattern of protein tyrosine phosphorylation was slightly different in the FMLP- and PMA-stimulated cells. These data indicate that protein tyrosine phosphorylation is an early event in the activation of human neutrophils by a variety of receptor-dependent and receptor-independent agonists.

scholarly journals Protein tyrosine phosphorylation in rabbit peritoneal neutrophils

1990 ◽  
Vol 269 (2) ◽  
pp. 431-436 ◽  
Author(s):  
C K Huang ◽  
V Bonak ◽  
G R Laramee ◽  
J E Casnellie
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Leukotriene B4 ◽  
Early Event ◽  
Ionophore A23187 ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Group A ◽  
Group B ◽  
Stimulation Of

Protein tyrosine phosphorylation in rabbit peritoneal neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. Stimulation of the neutrophils with chemotactic factor fMet-Leu-Phe (10 nM) caused rapid increases of tyrosine phosphorylation of several proteins with apparent molecular masses of (Group A) 54-58 kDa and 100-125 kDa and (Group B) 36-41 kDa. Stimulation of Group A proteins was observed by fMet-Leu-Phe (10 nM, maximum at 20 s) and A23187 (1 microM, 1 min). Stimulation of Group B proteins was observed by fMet-Leu-Phe (ED50 0.15 nM, 1 min), leukotriene B4 (ED50 0.15 nM, 1 min), phorbol 12-myristate 13-acetate (PMA) (ED50 25 ng/ml, 10 min) and partially by ionophore A23187 (1 microM, 1 min). Pretreatment of the cell with the protein kinase inhibitor H-7 (25 microM, 5 min) and PMA (0.1 microgram/ml, 3 min) partially inhibited the fMet-Leu-Phe effect. However, pretreatment of the cells with quin 2/AM (20 microM, 10 min) completely inhibited the fMet-Leu-Phe effect. The results indicate that rapid regulation of tyrosine phosphorylation is an early event occurring in stimulated neutrophils. Furthermore the effect of fMet-Leu-Phe on tyrosine phosphorylation may require Ca2+ mobilization and may partially require the activity of H-7-sensitive protein kinases.

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 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

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 Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho

1997 ◽  
Vol 327 (2) ◽  
pp. 461-472 ◽  
Author(s):  
J. Luis GARCÍA ◽  
A. Juan ROSADO ◽  
Antonio GONZÁLEZ ◽  
T. Robert JENSEN
Keyword(s):  
Phospholipase C ◽  
Tyrosine Phosphorylation ◽  
Inositol Phosphate ◽  
Calcium Ionophore ◽  
Cytosolic Calcium ◽  
Significant Inhibition ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Pancreatic Acini ◽  
Pkc Activation

Recent studies show that the effects of some oncogenes, integrins, growth factors and neuropeptides are mediated by tyrosine phosphorylation of the cytosolic kinase p125 focal adhesion kinase (p125FAK) and the cytoskeletal protein paxillin. Recently we demonstrated that cholecystokinin (CCK) C-terminal octapeptide (CCK-8) causes tyrosine phosphorylation of p125FAK and paxillin in rat pancreatic acini. The present study was aimed at examining whether protein kinase C (PKC) activation, calcium mobilization, cytoskeletal organization and small G-protein p21rho activation play a role in mediating the stimulation of tyrosine phosphorylation by CCK-8 in acini. CCK-8-stimulated phosphorylation of p125FAK and paxillin reached a maximum within 2.5 min. The CCK-8 dose response for causing changes in the cytosolic calcium concentration ([Ca2+]i) was similar to that for p125FAK and paxillin phosphorylation, and both were to the left of that for receptor occupation and inositol phosphate production. PMA increased tyrosine phosphorylation of both proteins. The calcium ionophore A23187 caused only 25% of the maximal stimulation caused by CCK-8. GF109203X, a PKC inhibitor, completely inhibited phosphorylation with PMA but had no effect on the response to CCK-8. Depletion of [Ca2+]i by thapsigargin had no effect on CCK-8-stimulated phosphorylation. Pretreatment with both GF109203X and thapsigargin decreased CCK-8-stimulated phosphorylation of both proteins by 50%. Cytochalasin D, but not colchicine, completely inhibited CCK-8- and PMA-induced p125FAK and paxillin phosphorylation. Treatment with Clostridium botulinum C3 transferase, which inactivates p21rho, caused significant inhibition of CCK-8-stimulated p125FAK and paxillin phosphorylation. These results demonstrate that, in pancreatic acini, CCK-8 causes rapid p125FAK and paxillin phosphorylation that is mediated by both phospholipase C-dependent and -independent mechanisms. For this tyrosine phosphorylation to occur, the integrity of the actin, but not the microtubule, cytoskeleton is essential as well as the activation of p21rho.

scholarly journals Platelet-activating factor both stimulates and "primes" human polymorphonuclear leukocyte actin filament assembly

Blood ◽  
1987 ◽  
Vol 70 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
M Shalit ◽  
GA Dabiri ◽  
FS Southwick
Keyword(s):  
Actin Filament ◽  
Polymorphonuclear Leukocyte ◽  
Actin Polymerization ◽  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Optimal Concentration ◽  
Ionophore A23187 ◽  
Filament Assembly

Abstract The phospholipid inflammatory mediator, platelet-activating factor (PAF), can stimulate polymorphonuclear leukocyte (PMN) chemotaxis. Conversion of cytoplasmic actin from monomers to filaments is associated with PMN motile functions. Using the fluorescent actin filament stain nitrobenzodiaxole phallicidin, we have investigated PAF's effects on human PMN actin polymerization. Concentrations of PAF between 1 x 10(-11) to 1 x 10(-6) mol/L induced actin filament (F- actin) assembly. An optimal concentration of PAF (1–5 x 10(-8) mol/L) induced a significantly lower rise in relative F-actin content (1.72 +/- 0.07 SEM) than an optimal concentration (5 x 10(-7) mol/L) of the chemotactic peptide FMLP (2.21 +/- 0.06). Unlike FMLP (F-actin content: 1.25 +/- 0.04 at five seconds), PAF stimulation was associated with a delay of more than five seconds (1.04 +/- 0.01 at five seconds) before an increase in F-actin could be detected. F-actin concentration reached maximum levels by 30 to 60 seconds. Prolonged stimulation (20 minutes) with PAF was associated with two phases of polymerization and depolymerization. Like FMLP, the initiation of actin filament assembly by PAF required receptor occupancy, this reaction being totally blocked by the PAF receptor inhibitor, SKI 63–441. As evidenced by the lack of inhibition by nordihydroguaiaretic acid (5 to 20 mumol/L), the production of leukotriene B4 was not required for the PAF-induced changes in F-actin. Like FMLP, PAF's ability to stimulate PMN actin polymerization was inhibited by pertussis toxin (.05 to 2.5 micrograms/mL) but not impaired by the addition of EGTA and/or the calcium ionophore A23187. Preincubation with 1 x 10(-11) to 1 x 10(-8) mol/L PAF for 2 to 60 minutes enhanced the rise in F-actin content induced by low concentrations of FMLP (5 x 10(-12) to 1 x 10(-10) mol/L) indicating that this phospholipid was capable of “priming” the PMN actin polymerization response.

Dibutyryl Cytidine and Adenosine 3':5'-Cyclic Monophosphates Inhibit A23187-Stimulated Rat Peritoneal Macrophage Leukotriene B4 Synthesis

1988 ◽  
Vol 1 (2) ◽  
pp. 109-114
Author(s):  
G. R. Elliott ◽  
A. P. M. Lauwen ◽  
I. L. Bonta
Keyword(s):  
Peritoneal Macrophage ◽  
Calcium Ionophore ◽  
Peritoneal Macrophages ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Intracellular Camp ◽  
Ionophore A23187 ◽  
Eicosanoid Release ◽  
Stable Product

Dibutyryl cytidine and adenosine 3':5'-cyclic monophosphates (db-cCMP and db-cAMP respectively) inhibited the synthesis of thromboxane (TX) B2, the stable product of TXA2, and leukotriene (LT) B4 by 4-day carrageenin-elicited rat peritoneal macrophages stimulated by the calcium ionophore A23187. Incubation of macrophages with dbcAMP, at concentrations inhibiting eicosanoid release, was associated with an increase in intracellular cAMP concentrations. No such increase was seen when db-cCMP was used.

scholarly journals Tulathromycin Exerts Proresolving Effects in Bovine Neutrophils by Inhibiting Phospholipases and Altering Leukotriene B4, Prostaglandin E2, and Lipoxin A4Production

2014 ◽  
Vol 58 (8) ◽  
pp. 4298-4307 ◽  
Author(s):  
Carrie D. Fischer ◽  
Stephanie C. Duquette ◽  
Bernard S. Renaux ◽  
Troy D. Feener ◽  
Douglas W. Morck ◽  
...  
Keyword(s):  
Calcium Ionophore ◽  
Leukotriene B4 ◽  
Calcium Ionophore A23187 ◽  
Lipid Signaling ◽  
Prostaglandin E ◽  
Ionophore A23187 ◽  
Proinflammatory Mediators ◽  
Bovine Neutrophils

ABSTRACTThe accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4(LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4[LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytesin vitroand inMannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammationin vivoand characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4and prostaglandin E2(PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2(PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

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.

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