Cytokine-specific activation of distinct MAP kinase subtype cascades in human neutrophils stimulated by cytokines and the role of MAP kinases in neutrophil activation

Many receptor-linked agents that prime or activate the NADPH oxidase in polymorphonuclear neutrophils (PMNs) elicit changes in cytosolic Ca2+concentration and activate mitogen-activated protein (MAP) kinases. To investigate the role of Ca2+in the activation of p38 and p42/44 MAP kinases, we examined the effects of the Ca2+-selective ionophore ionomycin on priming and activation of the PMN oxidase. Ionomycin caused a rapid rise in cytosolic Ca2+that was due to both a release of cytosolic Ca2+stores and Ca2+influx. Ionomycin also activated (2 μM) and primed (20–200 nM) the PMN oxidase. Dual phosphorylation of p38 MAP kinase and phosphorylation of its substrate activating transcription factor-2 were detected at ionomycin concentrations that prime or activate the PMN oxidase, while dual phosphorylation of p42/44 MAP kinase and phosphorylation of its substrate Elk-1 were elicited at 0.2–2 μM. SB-203580, a p38 MAP kinase antagonist, inhibited ionomycin-induced activation of the oxidase (68 ± 8%, P < 0.05) and tyrosine phosphorylation of 105- and 72-kDa proteins; conversely, PD-98059, an inhibitor of MAP/extracellular signal-related kinase 1, had no effect. Treatment of PMNs with thapsigargin resulted in priming of the oxidase and activation of p38 MAP kinase. Chelation of cytosolic but not extracellular Ca2+completely inhibited ionomycin activation of p38 MAP kinase, whereas chelation of extracellular Ca2+abrogated activation of p42/44 MAP kinase. These results demonstrate the importance of changes in cytosolic Ca2+for MAP kinase activation in PMNs.

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Adenosine Triphosphate Neutralizes Pneumolysin-Induced Neutrophil Activation

The Journal of Infectious Diseases ◽

10.1093/infdis/jiaa277 ◽

2020 ◽

Vol 222 (10) ◽

pp. 1702-1712 ◽

Cited By ~ 1

Author(s):

Fabian Cuypers ◽

Björn Klabunde ◽

Manuela Gesell Salazar ◽

Surabhi Surabhi ◽

Sebastian B Skorka ◽

...

Keyword(s):

Adenosine Triphosphate ◽

Extracellular Space ◽

Cytolytic Activity ◽

Neutrophil Activation ◽

Human Neutrophils ◽

Pneumococcal Infections ◽

Pneumococcal Strain ◽

Neutrophil Degranulation ◽

Neutrophil Response

Abstract Background In tissue infections, adenosine triphosphate (ATP) is released into extracellular space and contributes to purinergic chemotaxis. Neutrophils are important players in bacterial clearance and are recruited to the site of tissue infections. Pneumococcal infections can lead to uncontrolled hyperinflammation of the tissue along with substantial tissue damage through excessive neutrophil activation and uncontrolled granule release. We aimed to investigate the role of ATP in neutrophil response to pneumococcal infections. Methods Primary human neutrophils were exposed to the pneumococcal strain TIGR4 and its pneumolysin-deficient mutant or directly to different concentrations of recombinant pneumolysin. Neutrophil activation was assessed by measurement of secreted azurophilic granule protein resistin and profiling of the secretome, using mass spectrometry. Results Pneumococci are potent inducers of neutrophil degranulation. Pneumolysin was identified as a major trigger of neutrophil activation. This process is partially lysis independent and inhibited by ATP. Pneumolysin and ATP interact with each other in the extracellular space leading to reduced neutrophil activation. Proteome analyses of the neutrophil secretome confirmed that ATP inhibits pneumolysin-dependent neutrophil activation. Conclusions Our findings suggest that despite its cytolytic activity, pneumolysin serves as a potent neutrophil activating factor. Extracellular ATP mitigates pneumolysin-induced neutrophil activation.

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Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

Blood ◽

10.1182/blood.v87.12.5287.bloodjournal87125287 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5287-5296 ◽

Cited By ~ 45

Author(s):

YL Zu ◽

Y Ai ◽

A Gilchrist ◽

ME Labadia ◽

RI Sha'afi ◽

...

Keyword(s):

Protein Kinase ◽

Enzymatic Activity ◽

Map Kinase ◽

Phorbol Ester ◽

Map Kinases ◽

Phosphorylation Site ◽

Human Neutrophils ◽

Inhibitory Peptide

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

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Role of Cdc42-Cla4 Interaction in the Pheromone Response of Saccharomyces cerevisiae

Eukaryotic Cell ◽

10.1128/ec.00102-06 ◽

2006 ◽

Vol 6 (2) ◽

pp. 317-327 ◽

Cited By ~ 17

Author(s):

Melanie Heinrich ◽

Tim Köhler ◽

Hans-Ulrich Mösch

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Map Kinase ◽

Cell Cycle Arrest ◽

Map Kinases ◽

Negative Regulator ◽

Cycle Arrest ◽

Mitogen Activated Protein ◽

Novel Alleles

ABSTRACT In Saccharomyces cerevisiae, the highly conserved Rho-type GTPase Cdc42 is essential for cell division and controls cellular development during mating and invasive growth. The role of Cdc42 in mating has been controversial, but a number of previous studies suggest that the GTPase controls the mitogen-activated protein (MAP) kinase cascade by activating the p21-activated protein kinase (PAK) Ste20. To further explore the role of Cdc42 in pheromone-stimulated signaling, we isolated novel alleles of CDC42 that confer resistance to pheromone. We find that in CDC42(V36A) and CDC42(V36A, I182T) mutant strains, the inability to undergo pheromone-induced cell cycle arrest correlates with reduced phosphorylation of the mating MAP kinases Fus3 and Kss1 and with a decrease in mating efficiency. Furthermore, Cdc42(V36A) and Cdc42(V36A, I182T) proteins show reduced interaction with the PAK Cla4 but not with Ste20. We also show that deletion of CLA4 in a CDC42(V36A, I182T) mutant strain suppresses pheromone resistance and that overexpression of CLA4 interferes with pheromone-induced cell cycle arrest and MAP kinase phosphorylation in CDC42 wild-type strains. Our data indicate that Cla4 has the potential to act as a negative regulator of the mating pathway and that this function of the PAK might be under control of Cdc42. In conclusion, our study suggests that control of pheromone signaling by Cdc42 not only depends on Ste20 but also involves interaction of the GTPase with Cla4.

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Stimulation of the Mineralocorticoid Receptor by Aldosterone Leads to Activation of HL-60 Neutrophils,

Blood ◽

10.1182/blood.v118.21.3206.3206 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3206-3206

Author(s):

Carlos E Vazquez ◽

Gregory N Prado ◽

Enrique R Maldonado ◽

Gabriela Saca ◽

Iren M Ortiz ◽

...

Keyword(s):

Western Blot ◽

Respiratory Burst ◽

Mineralocorticoid Receptor ◽

Conflicts Of Interest ◽

Critical Role ◽

Neutrophil Activation ◽

Human Neutrophils ◽

Dead Cell ◽

Cardiovascular Morbidity And Mortality

Abstract Abstract 3206 Blockade of the mineralocorticoid receptor (MR), the receptor for aldosterone (ALDO), improves cardiovascular morbidity and mortality. There is growing evidence for a critical role of ALDO in inflammation in addition to its well-described effects on sodium homeostasis. However, the role of ALDO on neutrophil activation is not entirely clear. We studied the role of ALDO on HL-60, a human promyelocytic cell line, induced to differentiate into neutrophil-like cells by incubation for 3 days with 1.3% DMSO. We detected the presence of the mineralocorticoid receptor (MR), the receptor for ALDO, by western blot analyses and MR transcript by quantitative RT-PCR using TaqMan detection probes in these cells. Cells incubated with ALDO (10−8-10−7 M) showed a dose-dependent rise in cytosolic Ca2+ that peaked within 3 min using FURA-2AM fluorescence; an event not observed when cells were incubated with 10−8 M dexamethasone (DEXA). Consistent with these results, incubation with 10−8 M ALDO led to increases in the oxidative-respiratory burst [superoxide production] (P<0.01, n=3); an event not observed when cells were incubated with either 10−8 or 10−7 M dexamethasone. The oxidative responses to ALDO were blunted by pre-incubation of cells with 1 uM canrenoic acid (CA), a well-described MR antagonist (P<0.03, n=3). We then studied the effect of ALDO on HL-60 transmigration and observed that 2 hr incubation at 37C with 10−8 M ALDO led to augmented migration (P<0.03, n=2) when compared to vehicle as estimated by CyQuant cell migration assays. We then isolated untouched circulating human neutrophils by immunomagnetic isolation following density gradient sedimentation with PolymorphPrep from otherwise healthy subjects. Flow cytometric analyses showed greater than 97% neutrophils as these cells were positive for CD45, CD16 and CD66b. Live/dead cell automated analyses shows greater than 90% cell viability by acridine orange and propidium iodide fluorescence. These cells likewise express MR as determined by western blot analyses for MR as reported in kidney and endothelial cells. Cells incubated with ALDO (10−8 M) showed a rise in cytosolic Ca2+ and an increase in the oxidative-respiratory burst (P<0.01, n=3); a response that was sensitive to 1 uM CA. We also observed that 4 hr 10−9M ALDO incubation led to augmented neutrophil transmigration (P<0.03, n=2). Thus our results suggest that activation of MR by ALDO leads to neutrophil activation that may contribute to the inflammatory responses associated with MR activation in vivo. Disclosures: No relevant conflicts of interest to declare.

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Mechanical strain regulates syndecan-4 expression and shedding in smooth muscle cells through differential activation of MAP kinase signaling pathways

AJP Cell Physiology ◽

10.1152/ajpcell.00093.2006 ◽

2007 ◽

Vol 292 (1) ◽

pp. C517-C525 ◽

Cited By ~ 14

Author(s):

Matheau A. Julien ◽

Peiyi Wang ◽

Carolyn A. Haller ◽

Jing Wen ◽

Elliot L. Chaikof

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Map Kinase ◽

Signaling Pathways ◽

Map Kinases ◽

Muscle Cells ◽

Kinase Signaling ◽

Fold Reduction ◽

Map Kinase Signaling

Syndecan-4 (S4) belongs to a family of transmembrane proteoglycans, acts as a coreceptor for growth factor binding as well as cell-matrix and cell-cell interactions, and is induced in neointimal smooth muscle cells (SMCs) after balloon catheter injury. We investigated S4 expression in SMCs in response to several force profiles and the role of MAP kinase signaling pathways in regulating these responses. S4 mRNA expression increased in response to 5% and 10% cyclic strain (4 h: 200 ± 34% and 182 ± 17%, respectively; P < 0.05) before returning to basal levels by 24 h. Notably, the SMC mechanosensor mechanism was reset after an initial 24-h “preconditioning” period, as evident by an increase in S4 gene expression following a change in cyclic stress from 10% to 20% (28 h: 181 ± 1%; P < 0.05). Mechanical stress induced a late decrease in cell-associated S4 protein levels (24 h: 70 ± 6%; P < 0.05), with an associated increase in S4 shedding (24 h: 537 ± 109%; P < 0.05). To examine the role of MAP kinases, cells were treated with U-0126 (ERK1/2 inhibitor), SB-203580 (p38 inhibitor), or JNKI I (JNK/SAPK inhibitor). Late reduction in cell-associated S4 levels was attributed to ERK1/2 and p38 signaling. In contrast, accelerated S4 shedding required both ERK1/2 (5-fold reduction in accelerated shedding; P < 0.05) and JNK/SAPK (4-fold reduction; P < 0.05) signaling. Given the varied functions of S4, stress-induced effects on SMC S4 expression and shedding may represent an additional component of the proinflammatory, growth-stimulating pathways that are activated in response to changes in the mechanical microenvironment of the vascular wall.

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Role of calcium in the expression of MAP kinase kinases (MKKs) and MAP kinases in tomato flower abscission

AFRICAN JOURNAL OF BIOTECHNOLOGY ◽

10.5897/ajb11.2120 ◽

2012 ◽

Vol 11 (46) ◽

Author(s):

Jinhong Li

Keyword(s):

Map Kinase ◽

Map Kinases ◽

Flower Abscission

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Inhibition of p42 and p44 MAP kinase does not alter smooth muscle contraction in swine carotid artery

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.1.h131 ◽

1998 ◽

Vol 275 (1) ◽

pp. H131-H138 ◽

Cited By ~ 26

Author(s):

Isabelle Gorenne ◽

Xiaoling Su ◽

Robert S. Moreland

Keyword(s):

Smooth Muscle ◽

Muscle Contraction ◽

Map Kinase ◽

Map Kinases ◽

Kinase Activity ◽

Smooth Muscle Contraction ◽

Protein Map ◽

Mitogen Activated Protein ◽

Kinase Phosphorylation

Caldesmon inhibits myosin ATPase activity; phosphorylation of caldesmon reverses the inhibition. The caldesmon kinase is believed to be mitogen-activated protein (MAP) kinase. MAP kinases are activated during vascular stimulation, but a cause-and-effect relationship between kinase activity and contraction has not been established. We examined the role of MAP kinase in contraction using PD-098059, an inhibitor of MAP kinase kinase (MEK). MAP kinase activity was assessed using an anti-active MAP kinase antibody and direct measurement of MAP kinase catalyzed phosphorylation of myelin basic protein, MBP-(95—98). MAP kinase phosphorylation, stimulated by histamine (50 μM) or phorbol 12,13-dibutyrate (PDBu, 0.1 μM), was inhibited by PD-098059 (100 μM). PD-098059 did not alter the sensitivity or the maximal level of force in smooth muscle stimulated by histamine or PDBu, nor did PD-098059 affect contraction of β-escin-permeabilized tissue. Our data suggest that p44 and p42 MAP kinases are not involved in regulation of vascular smooth muscle contraction. These results do not, however, preclude a role for other isoforms of the MAP kinase family.

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Neutrophil activation by anti-proteinase 3 antibodies in Wegener's granulomatosis: role of exogenous arachidonic acid and leukotriene B4 generation.

Journal of Experimental Medicine ◽

10.1084/jem.184.4.1567 ◽

1996 ◽

Vol 184 (4) ◽

pp. 1567-1572 ◽

Cited By ~ 50

Author(s):

F Grimminger ◽

K Hattar ◽

C Papavassilis ◽

B Temmesfeld ◽

E Csernok ◽

...

Keyword(s):

Arachidonic Acid ◽

Wegener’S Granulomatosis ◽

Wegener's Granulomatosis ◽

Neutrophil Activation ◽

Tnf Alpha ◽

Lipid Mediator ◽

Human Neutrophils ◽

Proteinase 3 ◽

Lipoxygenase Pathway

Among the anti-neutrophil cytoplasmic antibodies (ANCA), those targeting proteinase 3 (PR3) have a high specificity for Wegener's granulomatosis (WG). It is known that a preceding priming of neutrophils with cytokines is a prerequisite for membrane surface expression of PR3, which is then accessible to autoantibody binding. Employing a monoclonal antibody directed against human PR3 and ANCA-positive serum from WG patients with specificity for PR3, we now investigated the role of free arachidonic acid (AA) in autoantibody-related human neutrophil activation. Priming of neutrophils with tumor necrosis factor (TNF-alpha) for 15 min or exposure to anti-PR3 antibodies or incubation with free AA (10 microM) as sole events did not provoke superoxide generation, elastase secretion or generation of 5-lipoxygenase products of AA. Similarly, the combination of TNF-alpha-priming and AA incubation was ineffective. When TNF-alpha-primed neutrophils were stimulated by anti-PR3 antibodies, superoxide and elastase secretion was provoked in the absence of lipid mediator generation. However, when free AA was additionally provided, a strong activation of the 5-lipoxygenase pathway was demasked, with the appearance of excessive quantities of leukotriene (LT)B4, LTA4, and 5-hydroxyeicosatetraenoic acid. Moreover, superoxide and elastase secretion were markedly amplified, and studies with 5-lipoxygenase inhibitors and a LTB4-antagonist demonstrated this was due to an LTB4-related autocrine loop of cell activation. In contrast, the increased synthesis of platelet-activating factor in response to TNF-alpha-priming and anti-PR3 stimulation did not contribute to the amplification loop of neutrophil activation under the given conditions. We conclude that anti-PR3 antibodies are potent inductors of the 5-lipoxygenase pathway in primed human neutrophils, and extracellular free AA, as provided at an inflammatory focus, synergizes with the autoantibodies to evoke full-blown lipid mediator generation, granule secretion and respiratory burst. Such events may be enrolled in the pathogenesis of focal necrotizing vascular injury in Wegener's granulomatosis.

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