Natalie J. Avdi

Common and distinct intracellular signaling pathways in human neutrophils utilized by platelet activating factor and FMLP.

Stimulation of human neutrophils with chemoattractants FMLP or platelet activating factor (PAF) results in different but overlapping functional responses. We questioned whether these differences might reflect patterns of intracellular signal transduction. Stimulation with either PAF or FMLP resulted in equivalent phosphorylation and activation of the mitogen-activated protein kinase (MAPk) homologue 38-kD murine MAP kinase homologous to HOG-1 (p38) MAPk. Neither FMLP nor PAF activated c-jun NH2-terminal MAPk (JNKs). Under identical conditions, FMLP but not PAF, resulted in significant p42/44 (ERK) MAPk activation. Both FMLP and PAF activated MAP kinase kinase-3 (MKK3), a known activator of p38 MAPk. Both MAP ERK kinase kinase-1 (MEKK1) and Raf are activated strongly by FMLP, but minimally by PAF. Pertussis toxin blocked FMLP-induced activation of the p42/44 (ERK) MAPk cascade, but not that of p38 MAPk. A specific p38 MAPk inhibitor (SK&F 86002) blocked superoxide anion production in response to FMLP and reduced adhesion and chemotaxis in response to PAF or FMLP. These results demonstrate distinct patterns of intracellular signaling for two chemoattractants and suggest that selective activation of intracellular signaling cascades may underlie different patterns of functional responses.

Selective activation and functional significance of p38α mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils

Activation of leukocytes by proinflammatory stimuli selectively initiates intracellular signal transduction via sequential phosphorylation of kinases. Lipopolysaccharide (LPS) stimulation of human neutrophils is known to result in activation of p38 mitogen-activated protein kinase (MAPk); however, the upstream activator(s) of p38 MAPk is unknown, and consequences of p38 MAPk activation remain largely undefined. We investigated the MAPk kinase (MKK) that activates p38 MAPk in response to LPS, the p38 MAPk isoforms that are activated as part of this pathway, and the functional responses affected by p38 MAPk activation. Although MKK3, MKK4, and MKK6 all activated p38 MAPk in experimental models, only MKK3 was found to activate recombinant p38 MAPk in LPS-treated neutrophils. Of p38 MAPk isoforms studied, only p38alpha and p38delta were detected in neutrophils. LPS stimulation selectively activated p38alpha. Specific inhibitors of p38alpha MAPk blocked LPS-induced adhesion, nuclear factor-kappa B (NF-kappaB) activation, and synthesis of tumor necrosis factor-alpha (TNF-alpha). Inhibition of p38alpha MAPk resulted in a transient decrease in TNF-alpha mRNA accumulation but persistent loss of TNF-alpha synthesis. These findings support a pathway by which LPS stimulation of neutrophils results in activation of MKK3, which in turn activates p38alpha MAPk, ultimately regulating adhesion, NF-kappaB activation, enhanced gene expression of TNF-alpha, and regulation of TNF-alpha synthesis.

Role of p38 Mitogen-Activated Protein Kinase in a Murine Model of Pulmonary Inflammation

Early inflammatory events include cytokine release, activation, and rapid accumulation of neutrophils, with subsequent recruitment of mononuclear cells. The p38 mitogen-activated protein kinase (MAPK) intracellular signaling pathway plays a central role in regulating a wide range of inflammatory responses in many different cells. A murine model of mild LPS-induced lung inflammation was developed to investigate the role of the p38 MAPK pathway in the initiation of pulmonary inflammation. A novel p38 MAPK inhibitor, M39, was used to determine the functional consequences of p38 MAPK activation. In vitro exposure to M39 inhibited p38 MAPK activity in LPS-stimulated murine and human neutrophils and macrophages, blocked TNF-α and macrophage inflammatory protein-2 (MIP-2) release, and eliminated migration of murine neutrophils toward the chemokines MIP-2 and KC. In contrast, alveolar macrophages required a 1000-fold greater concentration of M39 to block release of TNF-α and MIP-2. Systemic inhibition of p38 MAPK resulted in significant decreases in the release of TNF-α and neutrophil accumulation in the airspaces following intratracheal administration of LPS. Recovery of MIP-2 and KC from the airspaces was not affected by inhibition of p38 MAPK, and accumulation of mononuclear cells was not significantly reduced. When KC was instilled as a proinflammatory stimulus, neutrophil accumulation was significantly decreased by p38 MAPK inhibition independent of TNF-α or LPS. Together, these results demonstrate a much greater dependence on the p38 MAPK cascade in the neutrophil when compared with other leukocytes, and suggest a means of selectively studying and potentially modulating early inflammation in the lung.

Adherence of neutrophils to cultured human microvascular endothelial cells. Stimulation by chemotactic peptides and lipid mediators and dependence upon the Mac-1, LFA-1, p150,95 glycoprotein family.

The process of neutrophil adhesion to and migration through the microvascular endothelium, an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractants. Although both chemotactic peptides and lipid mediators enhance neutrophil adherence in vitro and in vivo, the mechanism(s) involved in the interaction between circulating neutrophils and microvascular endothelial cells is still not completely understood. In a microtiter well adherence assay, the chemotactic peptides, FMLP and C5a, and the lipid mediators, leukotriene B4 (LTB4) and platelet activating factor (PAF), enhanced human neutrophil adherence to cultured human microvascular endothelial cells as well as to human umbilical vein endothelial cells in a dose-dependent manner with a rapid time course. This stimulated adhesive interaction between neutrophils and cultured human endothelial cells was dependent on the expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface since neutrophils from patients with leukocyte adhesion deficiency, lacking surface expression of the adhesive glycoproteins, exhibited markedly diminished adherence to human endothelial cells in response to stimulation with chemotactic factors compared to normal control neutrophils. All four mediators enhanced expression of the glycoprotein family on the surface of normal neutrophils as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the glycoprotein common beta subunit. In addition, TS1/18 inhibited up to 100% the adherence of normal neutrophils to endothelial cells stimulated by maximal concentrations of FMLP, C5a, LTB4, or PAF. Moreover, HL-60 cells, human promyelocytic leukemia cells, neither increased glycoprotein surface expression nor adherence in response to stimulation. Thus, peptide and lipid mediators of the acute inflammatory response appear to enhance adherence of circulating neutrophils to the microvascular endothelium by a mechanism dependent on expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface.

Selective Suppression of Neutrophil Accumulation in Ongoing Pulmonary Inflammation by Systemic Inhibition of p38 Mitogen-Activated Protein Kinase

The p38 mitogen-activated protein kinase (MAPK) signaling pathway regulates a wide range of inflammatory responses in many different cells. Inhibition of p38 MAPK before exposing a cell to stress stimuli has profound anti-inflammatory effects, but little is known about the effects of p38 MAPK inhibition on ongoing inflammatory responses. LPS-induced activation of p38 MAPK in human neutrophils was inhibited by poststimulation exposure to a p38 MAPK inhibitor (M39). Release of TNF-α, macrophage-inflammatory protein (MIP)-2 (MIP-1β), and IL-8 by LPS-stimulated neutrophils was also reduced by poststimulation p38 MAPK inhibition. In contrast, release of monocyte chemoattractant protein-1 was found to be p38 MAPK independent. Ongoing chemotaxis toward IL-8 was eliminated by p38 MAPK inhibition, although the rate of nondirectional movement was not reduced. A murine model of acute LPS-induced lung inflammation was used to study the effect of p38 MAPK inhibition in ongoing pulmonary inflammation. Initial pulmonary cell responses occur within 4 h of stimulation in this model, so M39 was administered 4 h or 12 h after exposure of the animals to aerosolized LPS to avoid inhibition of cytokine release. Quantities of TNF-α, MIP-2, KC, or monocyte chemoattractant protein-1 recovered from bronchial alveolar lavage or serum were not changed. Recruitment of neutrophils, but not other leukocytes, to the airspaces was significantly reduced. Together, these data demonstrate the selective reduction of LPS-induced neutrophil recruitment to the airspaces, independent of suppression of other inflammatory responses. These findings support the feasibility of p38 MAPK inhibition as a selective intervention to reduce neutrophilic inflammation.

ACTIVATION OF MEKK BY FORMYL-METHIONYL-LEUCYL-PHENYLALANINE IN HUMAN NEUTROPHILS : MAPPING PATHWAYS FOR MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION

Mechanisms of neutrophil activation in response to chemoattractants remain incompletely understood. We have recently reported a Ras-mediated c-Raf pathway leading to the activation of mitogen-activated protein (MAP) kinase in human neutrophils stimulated with the chemoattractant formyl-Met-Leu-Phe (FMLP). However, concern that Raf activation may not fully account for the early FMLP-mediated human neutrophil responses prompted us to investigate the activation of MAP kinase/ERK kinase (MEK) by MEK kinase (MEKK). In cell lysates we identified protein species at 180, 160, 110, 72, and 54 kDa with a monoclonal antibody to MEKK. Activation of MEKK was determined on immunoprecipitates from FMLP-stimulated neutrophils by in vitro kinase assay, which utilized both MEK1 and MEK2 as substrates. It was rapid, detectable at 30 s and reaching a plateau at 5 min, and it was inhibited in a dose-dependent fashion by a specific phosphatidylinositol 3-kinase inhibitor, wortmannin. Partial inhibition by pertussis toxin was observed. We were unable to show inhibition of the MEKK response by GF 109203X, a protein kinase C-specific inhibitor. These data indicate that in neutrophils activation of MEKK in addition to Raf may underlie stimulation of MAP kinase and other MAP kinase homologues by FMLP.