Asthma is a life-long chronic inflammatory disorder. Eosinophils are major effector cells in this disorder, in great extent due to their production of the eosinophil-derived granule proteins (EDGP), major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP) and Charcot-Leyden Crystal protein (CLC). However, the mechanisms by which EDGP contribute to airways inflammation are poorly understood. In addition, although stromal cells are known to produce inflammatory cytokines that amplify and contribute to the chronicity of tissue inflammation, their role in asthma has received inadequate attention. We hypothesized that 1) Fibroblasts and other stromal cells are important immune-effector cells in asthma; 2) EDGP are important stimulators of stromal cell cytokine production and 3) EDGP stimulate stromal cell cytokines via definable molecular mechanisms. In testing this hypothesis, we have demonstrated that fibroblasts and epithelial cells are important producers of a wide variety of inflammatory cytokines including interleukin (IL)-6, IL-11, GM-CSF, leukemia inhibitory factor (LIF) and IL-8, provided evidence for dysregulated IL-6, IL-11 and LIF production in late phase asthma and demonstrated that MBP augments IL-1 and TGF-beta-induced cytokine production by fibroblasts and directly stimulates IL-6, LI-11 and GM-CSF production by epithelial cells. We also demonstrated that these effects are associated with proportionate changes in mRNA accumulation and that MBP-induced IL-6 activation is mediated by an NF-IL-6 response element between -158 and -145 in the IL-6 promoter. We propose to further characterize the effects of EDGP on stromal cell cytokine production and define the mechanism and clarify the in vivo relevance of this regulation. We will: I. Characterize the effects of EDGP on fibroblast, epithelial cell and smooth muscle cell immune-effector function. Their ability to regulate the production of IL-6, IL-11, LIF and GM-CSF, alone or in combination, with IL-1 and/or TGF-beta will be characterized. II. Characterize the mechanism by which MBP and other EDGP induce stromal cell cytokine production in vitro. We will characterize: (a) the role of molecular charge in the effects of MBP; (b) the transcriptional and post-transcriptional processes that mediate these effects; and (c) the cis- elements that mediate the effects of EDGP on IL-6 and IL-11 gene transcription. III. Characterize the relationship between MBP and other EDGP, stromal cell cytokines and altered airway physiology in vivo. We will: (a) determine if intratracheal MBP or other EDGP induce IL-6, LIF or GM-CSF production in the mouse airway; (b) determine if cytokine neutralization alters EDGP-induces alterations in airway physiology; and (c) determine if the response element(s) defined in Aim II are operative in vivo using transgenic mice containing EDGp response element-reporter gene constructs.
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