Activation of RhoA and ROCK Are Essential for Detachment of Migrating Leukocytesh

Detachment of the rear of the cell from its substratum is an important aspect of locomotion. The signaling routes involved in this adhesive release are largely unknown. One of the few candidate proteins to play a role is RhoA, because activation of RhoA in many cell types leads to contraction, a mechanism probably involved in detachment. To study the role of RhoA in detachment regulation, we analyzed several subsets of expert migratory leukocytes by video microscopy. In contrast to fast-migrating neutrophils, eosinophils do not detach the rear of the cell unless stimulated with serum. When measuring the amount of active RhoA, with the use of a GSTRhotekin pulldown assay, we found that serum is an excellent activator of RhoA in granulocytes. Inhibition of RhoA or one of Rhos target proteins, the kinase ROCK, in neutrophils leads to the phenotype seen in eosinophils: the rear of the cell is firmly attached to the substratum, whereas the cell body is highly motile. ROCK-inhibition leads to impaired migration of granulocytes in filters, on glass, and through endothelial monolayers. Also, the ROCK signaling pathway is involved in changes of integrin-mediated adhesion. Eosinophil transduction by a tat-fusion construct containing active RhoA resulted in detachment stimulation in the presence of chemoattractant. From these results we conclude that activation of the RhoA-ROCK pathway is essential for detachment of migratory leukocytes

Regulation of Proliferation, Differentiation and Survival by the IL-3/IL-5/GM-CSF Receptor Family

The receptors for the Il-3/IL-5/GM-CSF cytokine family are composed of a heterodimeric com-plex of a cytokine-specific a chain and a common ß chain (ßc). Binding of IL-3/IL-5/GM-CSF to their respective receptors rapidly induces activation of multiple intracellular signalling pathways, including the Ras-Raf-ERK, the JAK/STAT, the phosphatidylinositol 3-kinase PKB, and the JNK/SAPK and p38 signalling pathways. This re-view focuses on recent advancements in understanding how these different signalling pathways are activated by IL-3/IL-5/GM-CSF receptors, and how the individual pathways contribute to the pleiotropic effects of IL-3/IL-5/ GM-CSF on their target cells, including proliferation, differentiation, survival, and effector functions

Analysis of Signal Transduction Pathways Regulating Cytokine-Mediated Fc Receptor Activation on Human Eosinophils

Igs can be potent stimulants of eosinophil activation since interaction with IgA or IgG-coated particles can lead to eosinophil degranulation. We have investigated the comparative roles of mitogen-activated protein (MAP) kinases (MAPKs; ERK1/2 and p38) and phosphatidylinositol-3 kinase (PI3K) in the priming and regulation of Fc receptor functioning on human eosinophils utilizing a MAPK kinase (MEK) inhibitor (PD98059), a p38 inhibitor SB203580, and the widely used PI3K inhibitors wortmannin and LY294002. We demonstrate that priming of human eosinophils with Th2-derived cytokines, IL-4 and IL-5, differentially activate phosphotyrosine-associated PI3K and ERK and p38 MAP kinases. This activation can be inhibited by pre-incubation with wortmannin or LY294002, PD98059, and SB203580, respectively. Analysis of the effects of the inhibitors on rosette formation between human eosinophils and IgA- or IgG-coated beads revealed that activation of MEK was not required for IgA binding after priming with IL-4 or IL-5. However, inhibition of MEK did inhibit IL-5-primed binding of IgG-beads. The rosette formation of primed eosinophils with IgA-beads could be completely inhibited by wortmannin and LY294002 treatment, demonstrating a critical role for PI3K. Interestingly, inhibition of the p38 pathway also resulted in a complete blockade of IgA rosette formation. This work demonstrates regulatory control by inside-out signaling of Fc receptors by various cytokines on human eosinophils. Thus in vivo the local production of Th2-derived cytokines will regulate the effector functions of Fc receptors

Cytokine-mediated cPLA2 phosphorylation is regulated by multiple MAPK family members

Cytosolic phospholipase A2 (cPLA2) plays a critical role in various neutrophil functions including the generation of leukotrienes and platelet-activating factor release. Enzyme activity is regulated both by translocation to the membrane in a Ca^(2+) -dependent manner and serine phosphorylation by members of the mitogen-activated protein kinase (MAPK) family. In this report, we have investigated the role of granulocyte/macrophage colony-stimulating factor (GM-CSF)- mediated signalling pathways in the regulation of cPLA2. GM- CSF-induced cPLA2 phosphorylation was not affected by pharmacological inhibition of p38 MAPK, phosphatidylinositol 3-kinase or Src. However, inhibition of extracellular signal- regulated kinase (ERK) MAPK activation resulted in a partial inhibition of cPLA2 phosphorylation, revealed in a slower onset of phosphorylation. A cell line stably transfected with the GM- CSF receptor was used to further analyze GM-CSF-mediated cPLA2 phosphorylation. Mutation of tyrosine residues 577 and 612 resulted in a delayed cPLA2 phosphorylation similar to the pharmacological ERK inhibition. Furthermore, inhibition of p38 MAPK in cells bearing the double mutant ßc577/612 completely abrogated GM-CSF-induced cPLA2 phosphorylation. We con- clude that GM-CSF can mediate cPLA2 phosphorylation through the redundant activation of both p38 and ERK MAP kinases

Differential fMet-Leu-Phe- and Platelet-activating Factor-induced Signaling Toward Ral Activation in Primary Human Neutrophils

We have measured the activation of the small GTPase Ral in human neutrophils after stimulation with fMet- Leu-Phe (fMLP), platelet activating factor (PAF), and granulocyte macrophage-colony stimulating factor and compared it with the activation of two other small GTPases, Ras and Rap1. We found that fMLP and PAF, but not granulocyte macrophage-colony stimulating factor, induce Ral activation. All three stimuli induce the activation of both Ras and Rap1. Utilizing specific inhibitors we demonstrate that fMLP-induced Ral activation is mediated by pertussis toxin-sensitive G-proteins and partially by Src-like kinases, whereas fMLP-induced Ras activation is independent of Src-like kinases. PAFinduced Ral activation is mediated by pertussis toxininsensitive proteins, Src-like kinases and phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase is not involved in PAF-induced Ras activation. The calcium ionophore ionomycin activates Ral, but calcium depletion partially inhibits fMLP- and PAF-induced Ral activation, whereas Ras activation was not affected. In addition, 12-O-tetradecanoylphorbol-13-acetate-induced activation of Ral is completely abolished by inhibitors of protein kinase C, whereas 12-O-tetradecanoylphorbol- 13-acetate-induced Ras activation is largely insensitive. We conclude that in neutrophils Ral activation is mediated by multiple pathways, and that fMLP and PAF induce Ral activation differently

Platelet and Fibrin Deposition at the Damaged Vessel Wall: Cooperative Substrates for Neutrophil Adhesion Under Flow Conditions

At sites of vessel wall damage, the primary hemostatic reac- tion involves platelet and fibrin deposition. At these sites, circulating leukocytes marginate and become activated. Ad- hered platelets can support leukocyte localization; however, the role of fibrin in this respect is not known. We studied the adhesion of human neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial extracellular matrix (ECM)- bound fibrin and platelets under flow conditions. ECM alone did not show PMN adhesion. ECM-coated cover slips were perfused with plasma to form a surface-bound fibrin network, and/or with whole blood to allow platelet adhesion. Unstimulated PMNs adhered to fibrin at moderate shear stress (20 to 200 mPa). ECM-bound platelets induced rolling adhesion and allowed more PMNs to adhere at higher shear (320 mPa). ECM coated with both platelets and fibrin induced more static and shear-resistant PMN adhesion. PMN adhesion to fibrin alone but not to platelet/fibrin surfaces was inhibited by soluble fibrinogen. Adhesion to fibrin alone was inhibited by CD11b and CD18 blocking antibodies. Furthermore, fibrin formed under flow conditions showed up to threefold higher PMN adhesion compared with fibrin formed under static conditions, due to structural differences. These results indicate that circulating PMNs adhere to fibrin in an integrin-dependent manner at moderate shear stresses. However, at higher shear rates (Û200 mPa), additional mechanisms (ie, activated platelets) are necessary for an interac- tion of PMNs with a fibrin network

The personality characteristics of emergency nurses

Background: There are ever increasing demands on the emergency nursing workforce so it is necessary to consider how to enhance the recruitment and retention of emergency nurses. Personality is known to influence occupational choice, yet there is a lack of research exploring how personality may influence the workforce decisions of emergency nurses. Aims: To establish the personality profile of a sample of emergency nurses, and to explore whether any relationship exists between their personality characteristics and time spent working within emergency nursing. Methods: A standardised personality assessment instrument, the NEO™-PI-3, was used along with a demographic survey. Data were collected from 72 emergency nurses in an Australian Emergency Department between July and October 2012. Descriptive statistics were used to report demographics and the personality assessment results were compared against general population norms in each of the five personality domains and their 30 associated facets using a one-sample t-test. A two-sided alpha level of .01 was determined to indicate statistical significance. Results: Emergency nurse participants scored higher than the population norms in the domains of extraversion, openness to experience and agreeableness, and in twelve facets, including excitement seeking, vulnerability and competence. Conclusion: The personality profile of this sample of emergency nurses is different from the established population norms. Further research is required to establish whether these study results are applicable to the wider emergency nurse workforce and to establish any link with personality and other nursing specialty choice, which may assist in improving nursing workforce retention and recruitment

Comparison of the roles of mitogen-activated protein kinase kinase and phosphatidylinositol 3-kinase signal transduction in neutrophil effector function

Although it is known that many stimuli can activate mitogen- activated protein kinases (MAPKs) and phosphatidylinositol 3- kinases (PI3K) in human neutrophils, little is known concerning either the mechanisms or function of this activation. We have utilized a selective inhibitor of MAPKkinase (MEK), PD098059, and two inhibitors of PI3K, wortmannin and LY294002, to investigate the roles of these kinases in the regulation of neutrophil effector functions. Granulocyte/macrophage colony- stimulating factor, platelet-activating factor (PAF) and N-for- mylmethionyl-leucyl-phenylalanine are capable of activating both p44^(ERK1) and p42^(ERK2) MAPKs and phosphotyrosine-asso- ciated PI3K in human neutrophils. The activation of extracellular signal-related protein kinases (ERKs) is correlated with the activation of p21^(ras) by both tyrosine kinase and G-protein- coupled receptors as measured by a novel assay for GTP loading. Wortmannin and LY294002 inhibit, to various degrees, super- oxide generation, neutrophil migration and PAF release. In- cubation with PD098059, however, inhibits only the PAF release stimulated by serum-treated zymosan. This demonstrates that, while neither MEK nor ERK kinases are involved in the acti- vation of respiratory burst or neutrophil migration, inhibition of PAF release suggests a potential role in the activation of cytosolic phospholipase A2 . PI3K isoforms, however, seem to have a much wider role in regulating neutrophil functioning