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Lipoxin A₄ Counterregulates GM-CSF Signaling in Eosinophilic Granulocytes¹

Vitaliy Starosta^*, Konrad Pazdrak^*, Istvan Boldogh, Tetyana Svider and Alexander Kurosky^2,*,

^* Department of Biochemistry and Molecular Biology, Department of Microbiology and Immunology, and the National Heart, Lung, and Blood Institute Proteomics Center, University of Texas Medical Branch, Galveston, TX 77555

Eosinophils are granulated leukocytes that are involved in many inflammation-associated pathologies including airway inflammation in asthma. Resolution of eosinophilic inflammation and return to homeostasis is in part due to endogenous chemical mediators, for example, lipoxins, resolvins, and protectins. Lipoxins are endogenous eicosanoids that demonstrate antiinflammatory activity and are synthesized locally at sites of inflammation. In view of the importance of lipoxins (LXs) in resolving inflammation, we investigated the molecular basis of LXA₄ action on eosinophilic granulocytes stimulated with GM-CSF employing the eosinophilic leukemia cell line EoL-1 as well as peripheral blood eosinophils. We report herein that LXA₄ (1–100 nM) decreased protein tyrosine phosphorylation in EoL-1 cells stimulated with GM-CSF. Additionally, the expression of a number of GM-CSF-induced cytokines was inhibited by LXA₄ in a dose-dependent manner. Furthermore, using a proteomics approach involving mass spectrometry and immunoblot analysis we identified 11 proteins that were tyrosine phosphorylated after GM-CSF stimulation and whose phosphorylation was significantly inhibited by LXA₄ pretreatment. Included among these 11 proteins were -fodrin (nonerythroid spectrin) and actin. Microscopic imaging showed that treatment of EoL-1 cells or blood eosinophils with GM-CSF resulted in the reorganization of actin and the translocation of -fodrin from the cytoplasm to the plasma membrane. Importantly, -fodrin translocation was prevented by LXA₄ but actin reorganization was not. Thus, the mechanism of LXA₄ action likely involves prevention of activation of eosinophilic granulocytes by GM-CSF through inhibition of protein tyrosine phosphorylation and modification of some cytoskeletal components.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by the National Institutes of Health National Heart, Lung, and Blood Institute’s Proteomics Initiative N01-HV-28184 (to A.K.), National Institute for Environmental Health Sciences Center Grant P30-ES006676 (to J. Halpert), National Institute of Allergy and Infectious Diseases Grant P01 AI062885 (to A. Brasier) and a James W. McLaughlin postdoctoral fellowship grant to V. Starosta.

² Address correspondence and reprint requests to Dr. Alexander Kurosky, Department of Biochemistry and Molecular Biology, 301 University Boulevard, University of Texas Medical Branch, Galveston, TX 77555. E-mail address: akurosky{at}utmb.edu

³ Abbreviations used in this paper: LX, lipoxin; 1DE, one-dimensional electrophoresis; 2DE, two-dimensional electrophoresis; GMRβ, GM-CSF β-chain receptor; IPG, immobilized pH gradient; LDS, lithium dodecyl sulfate; LXA₄, lipoxin A₄, 5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid; MS, mass spectrometry; RT, room temperature.