Chemoattractant-Induced Signaling via the Ras–ERK and PI3K–Akt Networks, along with Leukotriene C4 Release, Is Dependent on the Tyrosine Kinase Lyn in IL-5– and IL-3–Primed Human Blood Eosinophils

Abstract

Human blood eosinophils exhibit a hyper-active phenotype in response to chemotactic factors following cell “priming” with IL-5 family cytokines. Earlier work has identified ERK1/2 as molecular markers for IL-5 priming, and herein we show that IL-3, a member of IL-5 family, also augments fMLP-stimulated ERK1/2 phosphorylation in primary eosinophils. Besides ERK1/2, we also observed an enhancement of chemotactic factor-induced Akt phosphorylation following IL-5 priming of human blood eosinophils. Administration of a peptide antagonist that targets the Src family member Lyn prior to cytokine (IL-5/IL-3) priming of blood eosinophils inhibited the synergistic increase of fMLP-induced activation of Ras, ERK1/2 and Akt, as well as the release of the proinflammatory factor leukotriene-C4. In the current study, we also examined a human eosinophil-like cell line HL-60 clone-15, and observed that these cells exhibited significant surface expression of IL-3 and GM-CSF receptors, as well as ERK1/2 phosphorylation in response to the addition of IL-5 family cytokines or the chemotactic factors fMLP, CCL5 and CCL11. Consistent with the surface profile of IL-5 family receptors, HL-60 clone-15 recapitulated the enhanced fMLP-induced ERK1/2 phosphorylation observed in primary blood eosinophils following priming with IL-3/GM-CSF, and siRNA-mediated knockdown of Lyn expression completely abolished the synergistic effects of IL-3 priming on fMLP-induced ERK1/2 phosphorylation. Altogether, our data reveal a central role for Lyn in the mechanisms of IL-5 family priming, and suggest that Lyn contributes to the up-regulation of the Ras-ERK1/2 and PI3K-Akt cascades as well as the increased leukotriene-C4 release observed in response to fMLP in “primed” eosinophils.

IL-5 Induces Suspended Eosinophils to Undergo Unique Global Reorganization Associated with Priming

“…In eosinophils treated with IL-5 for 60 minutes, a subsequent 3-minute treatment with fMLF induced ERK phosphorylation in almost all cells, with the same pERK distribution as in eosinophils treated with IL-5 for 10 minutes (compare Figures 7A and 7B). These data corroborate published Western blotting results indicating that a 60-minute incubation with IL-5 renders the eosinophils more sensitive to subsequent stimulation with fMLF (24,25), even though ERK phosphorylation has returned to basal level at this time point (26).…”

“…IL-5 family cytokines also prime eosinophils to react with increased release of granule proteins, increased production of leukotriene C4, and chemotaxis on subsequent stimulation with other activators (42)(43)(44)(45)(46). fMLF induces greater ERK phosphorylation, as analyzed by Western blotting, in IL-5-primed eosinophils than in unprimed cells (25). Polarization of eosinophils is accompanied by distribution of ERK to the granular compartment as well as to the nucleopod tip.…”

“…Western blotting has shown that eosinophils treated with IL-5 for 1 hour have more robust ERK phosphorylation in response to subsequent stimulation with fMLF when compared with naive cells treated with fMLF; this phenomenon is called priming (24,25). We found no phosphorylated ERK (pERK) staining in resting eosinophils, positive pERK staining in the nucleopod and granular compartment after 10-minute incubation with IL-5, especially at the nucleopod tip and potential leading edge, and no pERK staining after a 60-minute incubation with IL-5 ( Figure 7A).…”

Essential mechanisms of differential activation of eosinophils by IL-3 compared to GM-CSF and IL-5

“…85 Interestingly though, after 1 hour of activation, IL-3 and IL-5 have a very comparable strength to prime eosinophils in order to respond to a second activation with a ligand to a G protein-coupled receptor. 95 Unlike for a short-term activation, long-term cytokine activation of eosinophils substantially changes the potential of the three βc family cytokines on eosinophil signaling. We have shown that IL-3 activation leads to a prolonged (> 48 h) phosphorylation of the ribosomal protein S6 (RPS6), downstream of p90S6K (RSK) and ERK phosphorylations, while IL-5 and GM-CSF could not maintain this intracellular signaling for more than a few hours.…”

Tyrosine Kinase Inhibitors: Views of Selectivity, Sensitivity, and Clinical Performance

“…Basic FGF, EGF, PDGF, insulin-like growth factor-1 (IGF-1), and insulin all induce airway smooth muscle cell proliferation and seem to play a role in the hyperproliferation aspect of asthma (101). Nonreceptor tyrosine kinases like Lyn are also involved in the inflammatory component of asthma: Leukotriene C4 release depends on Lyn kinase in IL-5-and IL-3-primed human blood eosinophils, which infiltrate the airways and are key players in asthma (102). Interestingly, Pyk2 (103) and Tie2 (104) were also found to play a role in a mouse asthma model.…”