The overall aim of this proposal is to uncover new information on the mechanisms of allergic reactions and anti-inflammatory steroids in humans using both in vivo and in vitro experimental model systems. Work in the last funding period has elucidated important aspects of the role of leukocyte adhesion molecules and cytokines in allergic reactions. The present application focuses more on chemokines, especially the chemokine RANTES and a novel chemokine discovered by mass cDNA sequencing, CKbeta10. Both of these chemokines have powerful eosinophil-attracting properties; exciting preliminary studies lead us to hypothesize that airways epithelium generates these chemokines resulting in recruitment of eosinophils as well as mononuclear cells to the subepithelial region, a pattern of cellular infiltration observed in allergic diseases of the airways. RT PCR, in situ hybridization and immunohistochemical assays will be used to assess the presence of RANTES, CKbeta10 and other relevant eosinophil chemoattractants in nasal and central airway epithelium as well as skin in various patient groups and after antigen challenge. ELISA and functional assays will be used for confirmation of observed results. The influence of glucocorticoid treatment on chemokine expression will be assessed in vitro and in vivo. Preliminary results indicate that cutaneous challenge with RANTES induces a distinctly different pattern of response in normal and allergic subjects. We will assess the importance of allergic disease status and state of priming of circulating eosinophils on the RANTES-induced cellular infiltrate. Several studies are proposed to elucidate the role of nine recently-discovered members of the chemokine family in allergic diseases of the airways. RT PCR and Northern blot assays will be used to establish which of these new chemokines are important in epithelial cells, endothelial cells, mast cells and other cell types relevant to allergic inflammation. Functional studies (e.g. eosinophil chemotaxis) will be performed using purified recombinant protein for each of these chemokines. Ongoing studies designed to discover new molecules involved in airways inflammation will utilize DNA differential display technology. Thus far these studies have identified a host of candidate sequences. Induction of novel genes by cytokines and inhibition by glucocorticoids will be used as criteria to implicate them in inflammation. Results of the studies described in this proposal are highly likely to have direct relevance to allergic disease and its management with glucocorticoids.
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