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Primed Human Blood Eosinophils of September 23, 2021 Chemoattractant-Induced Signaling via the Ras−ERK and PI3K−Akt Networks, along with Leukotriene C 4 Release, Is Dependent on the Tyrosine Kinase Lyn in IL-5− and IL-3 This information is current as −Primed Human Blood Eosinophils of September 23, 2021. Yiming Zhu and Paul J. Bertics J Immunol 2011; 186:516-526; Prepublished online 24 November 2010; doi: 10.4049/jimmunol.1000955 Downloaded from http://www.jimmunol.org/content/186/1/516 References This article cites 80 articles, 27 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/186/1/516.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 23, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology 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 Yiming Zhu* and Paul J. Bertics† Human blood eosinophils exhibit a hyperactive phenotype in response to chemotactic factors after cell “priming” with IL-5 family cytokines. Earlier work has identified ERK1/2 as molecular markers for IL-5 priming, and in this article, we show that IL-3, a member of the 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 after IL-5 priming of human blood eosinophils. Administration of a peptide antagonist that targets the Src family member Lyn before cytokine (IL-5/IL-3) priming Downloaded from 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 this 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-3Rs and GM-CSFRs, 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 http://www.jimmunol.org/ primary blood eosinophils after priming with IL-3/GM-CSF, and small interfering RNA-mediated knockdown of Lyn expression completely abolished the synergistic effects of IL-3 priming on fMLP-induced ERK1/2 phosphorylation. Altogether, our data demonstrate a central role for Lyn in the mechanisms of IL-5 family priming and suggest that Lyn contributes to the upregulation 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. The Journal of Immunology, 2011, 186: 516–526. sthma is an inflammatory airway disease that has experi- such as chemotaxis, degranulation, and the release of proinflamma- enced an increase in global incidence in recent years (1, tory mediators (7, 9, 10, 12, 14, 15)]. Furthermore, the priming of A 2). Eosinophils are a multifaceted granulocyte and prom- blood eosinophils with IL-5 family members allows for these cells by guest on September 23, 2021 inent effector cell in asthma, and the infiltration of these cells into to exhibit a similar phenotype and hyperresponsiveness as that the airways is a hallmark of asthma exacerbation (3–6). The func- observed with airway eosinophils (9, 16–19). Therefore, under- tion of human eosinophils is largely regulated by IL-5 family cyto- standing the molecular bases of priming is key for delineating the kines, including IL-5, IL-3, and GM-CSF (7–10). Recently, several function of eosinophils in asthma. lines of evidence demonstrate that IL-5 family members can en- The capacity of IL-5 family cytokines to prime the action of hance blood eosinophil responsiveness to a second stimulus, such a wide variety of chemotactic factors that signal via G protein-cou- as the chemotactic factors CCL5 and fMLP, resulting in a synergis- pled receptors (GPCRs) (9, 11, 14) suggests the presence of a fun- tic response known as priming (10–15). The capacity of IL-5 fam- damental mechanism by which IL-5 family members serve to ily cytokines to prime blood eosinophils for altered chemoattractant enhance the responses induced by GPCR ligands. In addition, responsiveness has been associated with changes in intracellular the effects of cytokine priming on chemoattractant-induced intra- signaling events [e.g., an increase in chemoattractant-induced phos- cellular signaling (ERK1/2 phosphorylation) are rapid and rela- phorylation of the MAPKs ERK1/2 (11), and biological responses, tively long lasting (11). Therefore, we hypothesized that the cross talk between IL-5 family- and GPCR-mediated pathways is a key characteristic of priming, and given the rapid nature of cytokine *Molecular and Cellular Pharmacology Program and †Department of Biomolecular priming on chemoattractant-induced ERK1/2 phosphorylation, it Chemistry, University of Wisconsin, Madison, WI 53706 is likely that it entails the modification or reorganization, or both, Received for publication March 24, 2010. Accepted for publication October 27, of signaling molecules via preassembly of adaptor proteins and 2010. localization of downstream effectors. This work was supported by the National Institutes of Health (Grants HL0885940, HL56396, AI070503, and HL069116 to P.J.B.). With respect to the molecular mechanisms of eosinophil priming Address correspondence and reprint requests to Dr. Paul J. Bertics, Department of by IL-5 family cytokines, little is known, although it has been repor- Biomolecular Chemistry, University of Wisconsin School of Medicine and Public ted that the Src family member Lyn can physically associate with the Health, 1300 University Avenue, Madison, WI 53706. E-mail address: pbertics@ IL-5R b-chain (which is shared with the IL-3Rs and GM-CSFRs) on wisc.edu receptor ligation. This association appears critical for the subsequent Abbreviations used in this paper: dif-HC15, differentiated HL-60 clone-15; GPCR, G protein-coupled receptor; HC15, HL-60 clone-15; HSA, human serum albumin; activation of the Ras–Raf–MEK–ERK pathway (20–25). Previous LTC4, leukotriene C4; PILR, peptide inhibitor of Lyn recruitment; RBD, Ras-binding research focusing on the role of Lyn in human eosinophil function domain of Raf; si-Lyn, siRNA against Lyn; siRNA, small interfering RNA; WCL, has also revealed that this tyrosine kinase is important for IL-5–in- whole-cell lysate. duced ERK1/2 phosphorylation, as well as cell survival and differ- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 entiation (20–27). In addition, Src family members, including Lyn, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000955 The Journal of Immunology 517 have been suggested to initiate events leading to ERK1/2 activa- and HC15, respectively. Subsequently, the cells were stimulated with IL-5 tion in numerous GPCR systems (28–31). Accordingly, in this study, family cytokines and/or the chemotactic factors fMLP and CCL5 for the we tested the idea that Lyn is central to IL-5 family cytokine- indicated time periods. In the priming experiments, cells were treated for 1 h with IL-5 family cytokines, followed by stimulation with the specified mediated eosinophil priming, and we show that Lyn is not only chemotactic factors for 2 min. When indicated, the Lyn peptide PILR and important for IL-5–induced signaling and cellular responses, but it the control peptide were added 30 min before IL-5 family cytokine serves as a key component in the cross talk between IL-5 family treatment. Cell lysates were prepared using RIPA buffer (0.1% Triton X- cytokines and the chemotactic factor fMLP.We demonstrate that the 100, 0.25% deoxycholate, 0.1% SDS) and were subjected to SDS-PAGE and immunoblotting as described previously (11). To ensure that equal synergistic increase of fMLP-induced activation of the Ras–ERK1/2 protein levels were being evaluated, we also blotted loading controls and PI3K–Akt cascades after IL-5/IL-3 priming is dependent on (Grb2, actin, b-tubulin) on the same membranes. Chemiluminescence was Lyn activity in human primary blood eosinophils and the human captured using an EpiChemi II Darkroom (Ultraviolet Products, Upland, eosinophil-like cell line HL-60 clone-15 (HC15). Furthermore, we CA) equipped with a 12-bit cooled charge-coupled device camera and observed that the enhanced release of the proinflammatory mediator quantified using Image J (National Institutes of Health, Bethesda, MD). Band intensities of the proteins of interest were normalized to the corre- leukotriene C4 (LTC4) in response to fMLP treatment of IL-5– or IL- sponding loading controls. 3–primed human blood eosinophils is also dependent on Lyn, sup- porting the concept that Lyn is involved in the enhanced inflamma- Flow cytometry tory capacity of “primed” eosinophils. These results advanced our Cells (105) were suspended in 150 ml FACS buffer and incubated on ice understanding of the molecular basis of priming in human blood with 5 ml Abs of PE–IL-5Ra, PE–IL-3Ra, FITC–GM-CSFRa, or corre- eosinophils and provide a potential therapeutic target for suppress- sponding mouse IgG control for 30 min in the dark.
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