Role of extracellular signal-regulated kinase and phosphatidylinositol-3 kinase in chemoattractant and LPS delay of constitutive neutrophil apoptosis.

The present study examined the role of mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3 kinase-stimulated Akt (PI-3K/Akt) in the regulation of constitutive human neutrophil apoptosis by bacterial lipopolysaccharide (LPS) and two chemoattractants, fMLP and leukotriene B(4) (LTB(4)). LPS and LTB(4) inhibited apoptosis, while fMLP had no effect. Inhibition of extracellular signal-regulated kinase (ERK) with PD098059 significantly inhibited the anti-apoptotic effect of both LPS and LTB(4), while inhibition of p38 kinase with SB203580 had no effect. Inhibition of PI-3K with wortmannin and LY294002 significantly attenuated the anti-apoptotic effect of LTB(4), but not LPS. LPS, fMLP, and LTB(4) stimulated similar levels of ERK and Akt activation. LTB(4) and LPS inhibited neutrophil apoptosis when added simultaneously with fMLP, and LTB(4) and LPS demonstrated an additive effect. We conclude that the ERK and/or PI-3K/Akt pathways are necessary, but not sufficient, for LPS and LTB(4) to delay apoptosis, but other anti-apoptotic pathways remain to be identified.

Bacterial phagocytosis activates extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in human neutrophils.

The hypothesis that bacterial phagocytosis by human polymorphonuclear neutrophils (PMNs) stimulates MAPK cascades that regulate respiratory burst activation was tested. Extracellular response kinase (ERK) and p38 kinase, but not c-Jun NH2-terminal kinase, activities were increased within 5 min of phagocytosis of plasma-opsonized Staphylococcus aureus (S-SA), reached maximum at 20-30 min, and remained elevated through 60 min. The role of Fcy receptors was examined using gamma globulin-opsonized SA (IgG-SA), whereas CR3 receptors were activated by particulate beta-glucan. IgG-SA stimulated a maximal ERK activity at 30 min, whereas p38 activity was maximal at 5 min. Beta-glucan stimulated maximal ERK activity at 5 min and maximal p38 activity at 2 min. Non-opsonized bacteria were ingested at 10% of the level of S-SA and stimulated a minimal increase in ERK and p38 activity at 60 min. S-SA stimulation of ERK was inhibited by wortmannin, LY294002, and genistein, but not calphostin C; whereas p38 stimulation was inhibited by calphostin C and genistein, but not wortmannin and LY294002. Simultaneous measurement of phagocytosis and H2O2 production by flow cytometry was used to assess the role of ERKs and p38 kinase in phagocytosis. The MEK inhibitor PD098059 had no significant effect on phagocytosis or H2O2 production. The p38 kinase inhibitor SB203580 significantly attenuated H2O2 production, whereas phagocytosis was unaffected. In conclusion, bacterial phagocytosis stimulates ERK and p38 activation by distinct signal transduction pathways. Phagocytosis-stimulated p38 kinase activity is necessary for optimal H2O2 production.

Activation of mitogen-activated protein kinase cascades during priming of human neutrophils by TNF-alpha and GM-CSF.

The signal transduction pathways activated by tumor necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) that lead to priming of polymorphonuclear leukocytes (PMNs) are unknown. The hypotheses that these cytokines stimulate multiple mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), and p38 MAPK, and that these MAPKs participate in priming of human PMNs were examined. TNF-alpha stimulated a dose-dependent increase in ERK and p38 MAPK activities that was maximal at 10 min. JNKs were not stimulated by TNF-alpha or GM-CSF. GM-CSF stimulated ERK activity comparable to that of TNF-alpha, but GM-CSF was a less potent stimulus of p38 MAPK activity. The tyrosine kinase inhibitor, genistein, inhibited ERK and p38 MAPK stimulation by both cytokines. The phosphatidylinositol 3-kinase inhibitor, wortmannin, attenuated stimulation of ERKs and p38 MAPK by GM-CSF, but not TNF-alpha. GM-CSF, but not TNF-alpha, stimulated wortmannin-sensitive activation of Raf-1. TNF-alpha and GM-CSF priming of superoxide release stimulated by N-formyl-methionyl-leucyl-phenylalanine was significantly attenuated by the MEK inhibitor, PD098059, and the p38 MAPK inhibitor, SB203580. Incubation with both MAPK inhibitors produced an additive effect. Our data suggest that TNF-alpha and GM-CSF activate ERKs and p38 MAPK by different signal transduction pathways. Both ERK and p38 MAPK cascades contribute to the ability of TNF-alpha and GM-CSF to prime the respiratory burst response in human PMNs.

Signal transduction during Legionella pneumophila entry into human monocytes.

Legionella pneumophila causes Legionnaires' disease by replication in alveolar macrophages and monocytes. The bacteria are internalized most efficiently by opsonin-dependent, CR3-mediated phagocytosis. This investigation focused on determining the role of actin polymerization and phosphorylation signals in this uptake mechanism. Uptake inhibition assays and confocal microscopic analysis indicated that entry of L. pneumophila activated tyrosine kinase (TK) and protein kinase C (PKC) and induced actin polymerization at the site of bacterial entry. Upon L. pneumophila entry, six major cellular proteins (75, 71, 59, 56, 53, and 52 kDa) were TK phosphorylated in soluble fractions of monocytes, and three of these proteins (52, 53, and 56 kDa) were consistently found in insoluble (i.e., cytoskeletal) fractions of monocytes as well. Tyrosine phosphorylation was suppressed when cells were pretreated with the kinase inhibitor genistein, tyrphostin, or staurosporine. A similar tyrosine-phosphorylated protein pattern was observed with CR3-mediated entry of avirulent L. pneumophila, Escherichia coli, or zymosan into monocytes. This study has shown that PKC and TK signals which activate actin polymerization during the process of phagocytosis are induced upon L. pneumophila entry. In addition, CR3 receptor-mediated phagocytosis into monocytes may involve tyrosine phosphorylation of similar proteins, regardless of the particle being phagocytosed. Therefore, the tyrosine-induced phosphorylation observed during opsonized L. pneumophila entry is not a virulence-associated event.

Differential mitogen-activated protein kinase stimulation by Fc gamma receptor IIa and Fc gamma receptor IIIb determines the activation phenotype of human neutrophils.

Fc gamma Rs mediate immune complex-induced tissue injury. The hypothesis that Fc gamma RIIa and Fc gamma RIIIb control neutrophil responses by activating mitogen-activated protein kinases was examined. Homotypic and heterotypic cross-linking of Fc gamma RIIa and/or Fc gamma RIIIb resulted in a rapid, transient increase in ERK and p38 activity, with maximal stimulation between 1 and 3 min. Fc gamma RIIa and Fc gamma RIIIb stimulated distinct patterns of ERK and p38 activity, and heterotypic cross-linking failed to stimulate synergistic activation of either ERK or p38 activity. Both Fc gamma RIIa and Fc gamma RIIIb required activation of a nonreceptor tyrosine kinase and phosphatidylinositol 3-kinase for stimulation of ERK and p38. Inhibition of ERK activation with PD98059 enhanced H2O2 production stimulated by homotypic and heterotypic Fc gamma R cross-linking. Inhibition of p38 with SB203580 attenuated H2O2 production stimulated by Fc gamma RIIIb or heterotypic cross-linking, but had no effect on Fc gamma RIIa-stimulated H2O2 production. On the other hand, PD98059 inhibited actin polymerization stimulated by Fc gamma R cross-linking, while SB203580 had no effect. Inhibition of actin polymerization with cytochalasin D enhanced p38 activity stimulated by either Fc gamma RIIa or Fc gamma RIIIb, but cytochalasin D only enhanced H2O2 production stimulated by Fc gamma RIIIb. Our data indicate that Fc gamma RIIa and Fc gamma RIIIb independently activate ERK and p38. The two receptors demonstrate different efficacies for ERK and p38 activation, and they do not act cooperatively. ERK and p38 provide stimulatory and inhibitory signals for neutrophil responses to immune complexes. In addition, these data indicate that actin reorganization may play a role in mediating p38-dependent activation of respiratory burst upon stimulation of Fc gamma RIIIb in neutrophils.

Granulocyte-macrophage colony-stimulating factor delays neutrophil constitutive apoptosis through phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways.

Activated neutrophils play an important role in the pathogenesis of sepsis, glomerulonephritis, acute renal failure, and other inflammatory processes. The resolution of neutrophil-induced inflammation relies, in large part, on removal of apoptotic neutrophils. Neutrophils are constitutively committed to apoptosis, but inflammatory mediators, such as GM-CSF, slow neutrophil apoptosis by incompletely understood mechanisms. We addressed the hypothesis that GM-CSF delays neutrophil apoptosis by activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI 3-kinase) pathways. GM-CSF (20 ng/ml) significantly inhibited neutrophil apoptosis (GM-CSF, 32 vs 65% of cells p < 0. 0001). GM-CSF activated the PI 3-kinase/Akt pathway as determined by phosphorylation of Akt and BAD. GM-CSF-dependent Akt and BAD phosphorylation was blocked by the PI 3-kinase inhibitor LY294002. A role for the PI 3-kinase/Akt pathway in GM-CSF-stimulated delay of apoptosis was indicated by the ability of LY294002 to attenuate apoptosis delay. GM-CSF-dependent inhibition of apoptosis was significantly attenuated by PD98059, an ERK pathway inhibitor. LY294002 and PD98059 did not produce additive inhibition of apoptosis delay. To determine whether PI 3-kinase and ERK are used by other ligands that delay neutrophil apoptosis, we examined the role of these pathways in IL-8-induced apoptosis delay. LY294002 blocked IL-8-dependent Akt phosphorylation. PD98059 and LY294002 significantly attenuated IL-8 delay of apoptosis. These results indicate IL-8 and GM-CSF act, in part, to delay neutrophil apoptosis by stimulating PI 3-kinase and ERK-dependent pathways.

p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils.

Akt activation requires phosphorylation of Thr(308) and Ser(473) by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser(473) phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.