Analysis of Signal Transduction Pathways in Human Eosinophils Activated by Chemoattractants and the T-Helper 2–Derived Cytokines Interleukin-4 and Interleukin-5

Abstract

Activation and recruitment of eosinophils in allergic inflammation is in part mediated by chemoattractants and T-helper 2 (Th2)-derived cytokines. However, little is known concerning the signal transduction mechanisms by which this activation occurs. We have investigated tyrosine kinase-mediated activation of phosphatidylinositol 3-kinase (PI3K) and compared this with the activation of the p21ras-ERK signaling pathway in human eosinophils. The related cytokines interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF), all induced PI3K activity detected in antiphosphotyrosine immunoprecipitates. Furthermore, the chemoattractants platelet-activating factor (PAF), RANTES, and C5a were also able to induce phosphotyrosine-associated PI3K activity. Protein kinase B (PKB) is a downstream target of PI3K activation by growth factors. Induction of PKB phosphorylation in human eosinophils was transiently induced on activation with the cytokines IL-4 and IL-5, as well as the chemoattractants PAF, C5a, and RANTES showing a broad activation profile. Surprisingly, analysis of the activation of the mitogen-activated protein (MAP) kinases p44ERK1 and p42ERK2, showed that ERK2, but not ERK1, was transiently activated in human eosinophils after stimulation with IL-5 or PAF. Activation kinetics correlated with activation of p21ras by both cytokines and chemoattractants as measured by a novel assay for guanosine triphosphate (GTP)-loading. Finally, using specific inhibitors of both the p21ras-ERK and PI3K signaling pathways, a role was demonstrated for PI3K, but not p21ras-ERK, in activation of the serum-treated zymosan (STZ)-mediated respiratory burst in IL-5 and PAF-primed eosinophils. In summary, these data show that in human eosinophils, Th2-derived cytokines differentially activate both PI3K and MAP kinase signal transduction pathways with distinct functional consequences showing complex regulation of eosinophil effector functions.

Pathways for eosinophil lipid body induction: differing signal transduction in cells from normal and hypereosinophilic subjects

“…In normal eosinophils, PAF has previously been shown to induce the transient phosphorylation of several proteins [25]. Our attempts to identify differential expression of tyrosine phosphorylation in HES and normal eosinophils 1 h after PAF stimulation, however, were not revealing.…”

Singlet Oxygen–induced Activation of Akt/Protein Kinase B is Independent of Growth Factor Receptors¶

“…After incubation for 30 min cells were harvested in 23 SDS sample buffer, and lysates were subjected to Western blotting with antibodies to phospho-Akt and total Akt (A and C), as well as phospho-PDGFR and total PDGFR (B and D). potential mechanism for growth factor receptor-independent activation of PI3-K involves cyclic adenosine 39:59monophosphate (cAMP) -mediated signaling because agents that raise intracellular cAMP levels can activate both PI3-K and Akt (31,48). In addition, it has been reported that Akt is activated by G-protein-coupled receptors (GPCR) and that the pathway connecting G-proteins to Akt implicates signals emanating from Ga q , Ga i , and G bc dimers through a pathway that involves PI3-K activity (49,50).…”

“…Akt is generally activated through a PI3-K-mediated mechanism, although it is also activated independent of PI3-K under some circumstances (31). Other oxidants, H 2 O 2 and ONOO ÿ , induce Akt activation by a PI3-K-dependent mechanism (14,19).…”

Chemokines, chemokine receptors and hematopoiesis

“…Several lines of evidence have suggested that chemokine receptor signaling activates Akt prior to occurrence of PI3Kg gene deletion. These involve IL-8, SDF-1, MIP-1a and RANTES (333)(334)(335). However, the biological meaning of the chemokine receptor-mediated Akt activation remains unknown.…”