This research proposal is focused on delineating the mechanisms by which the interleukin-5 (IL-5) family of cytokines can regulate the function of the human eosinophil. The importance of this goal is emphasized by the observation that IL-5 family cytokines mediate the recruitment and activation of eosinophils that likely contributes to the pathophysiologic features of asthma. However, the pathways whereby IL-5 receptor signaling is coupled to the control of eosinophilic inflammation in asthma are not well understood. In this regard, we are now in the unique position of being able to rigorously dissect the role of specific IL-5 signaling events in the modulation of eosinophil biology using both human blood and airway eosinophils. In addition, we have three key capabilities that should permit us to obtain new insights regarding eosinophil biology and asthma, including: a) routine access to highly purified human blood and airway eosinophils, including eosinophils isolated from people with asthma, b) the identification of key signaling molecules involved in mediating IL-5 and chemokine action in peripheral and airway eosinophils, including members of the Ras G-protein superfamily, the MAP kinases (ERKs 1 and 2), and several STAT transcription factors, and c) the development of methods for the analysis of cytokine/chemokine signaling in human eosinophils, including a technique for the introduction of dominant-negative molecules into primary human eosinophils. These tools will be used to test the overall hypothesis that IL-5 receptor signaling and regulation of eosinophil function involves the action of low MW G-proteins of the Ras superfamily, ERKs 1 and 2, and several STAT factors, and that the role of these pathways is not only to mediate the direct effects of IL-5 related cytokines on eosinophil function, but that they are also critical for priming blood cells to become responsive to a variety of other factors involved in the control of this cell?s inflammatory capacity. Accordingly, the following lines of investigation are proposed: 1) Test the hypothesis that activation of Ras superfamily G-proteins is essential for IL-5 to enhance the inflammatory capacity of the human blood eosinophil. 2) Evaluate the mechanism whereby IL-5 primes human blood eosinophils to respond to chemokines and ascertain how the requirement for this priming processes is bypassed in airway cells. 3) Examine the role of STAT3- and STAT5-dependent pathways in IL-5-mediated regulation of human eosinophil gene expression and biological responsiveness.
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