Mini Review: Specificity in cytokine signal transduction: lessons learned from the IL-3:IL-5:GM-CSF receptor family

Cytokines mediate the transduction of proliferative, differentiation and survival signals in the hematopoietic system. Although the cytokine family is large and diverse, many different cytokines display broadly overlapping functions. This can be explained by the fact that cytokine receptors often share multiple subunits. Specificity in signal transduction can however be achieved through several mechanisms. This review focuses on how signal specificity can be achieved within the IL-3, IL-5 and GM-CSF receptor family. This is discussed in terms of receptor expression, recent advances in our understanding of intracellular signalling components, and analysis of null mutant knock-out mice

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

Cytokine-induced inside-out activation of FcaR (CD89) is mediated by a single serine residue (S263) in the intracellular domain of the receptor

Fc receptors play an important role in leukocyte activation and the modulation of ligand binding ("activation") is a criti-cal point of regulation. Previous studies demonstrated that the Fc receptor for IgA (FcaRI/CD89) is regulated by cytokine stimulation, switching it to a high-binding state. To investigate the mechanism by which cytokine-induced signal transduc-tion pathways result in FcaRI activation, cell lines expressing various receptor mu-tants were generated. Binding studies indicated that truncation of the C-termi- nus of the FcaRI resulted in constitutive IgA binding, removing the need for cyto-kine stimulation. Furthermore, mutagen-esis of a single C-terminal serine residue (S263) to alanine (S>A) (single-letter amino acid codes) also resulted in consti-tutive IgA binding, whereas a serine to aspartate (S>D) mutation was no longer functional. The role of S263 might be in regulating the interaction with the cy-toskeleton, because disruption of the cy-toskeleton results in reduced IgA binding to both FcaRwt and FcaR_S>A. In addi- tion, overexpression of a membrane-targeted intracellular domain of FcaR, and the introduction of cell-permeable CD89 fusion proteins blocked IgA bind-ing, implying a competition for endoge-nous proteins. The proposal is made that Fc receptors are activated by cytokines via an inside-out mechanism converging at the cytoplasmic tail of these receptors

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