Studies are proposed to examine mechanisms by which activated inflammatory cells convert normally responsive airways into hyperreactive tissues. Investigations are planned to examine the relationship between adhesion of peripheral blood and cultured eosinophils to intra- and extracellular matrix ligands and the effect of these reactions in upregulating secretion of bronchoactive granular proteins and eicosanoids. A central hypothesis of the first series of investigations is that adhesion of peripheral blood eosinophils to vascular endothelium causes augmented secretion upon stimulation. Using immunomagnetic separation techniques that isolate eosinophils in high purity from non-atopic humans developed in preliminary studies, experiments are proposed to determine the effect of preincubation of eosinophils with human umbilical vein endothelial cells (HUVEC) on the spontaneous and stimulated secretion of eosinophils peroxidase (EPO), leukotriene C4 (LTC4), and superoxide anion (O2-). Additional studies will l) examine the incremental effects on metabolic activation and secretion of eosinophil-adhesion to HUVEC pretreated with IL-1 or IL-4 and 2) determine the effect of blockade of eosinophil adhesion molecules LFA-1, Mac-1, VLA-4, and L-selectin on adhesion-mediated regulation of eosinophil secretory function. Additional experiments will address the role of exposure to matrix protein on upregulation of secretion of bioactive eosinophil proteins and eicasonoids. A second series of experiments will examine the functional correlates of endothelial adhesion and matrix protein interactions with eosinophils on airway responsiveness. Using preparations of cultured cord-derived eosinophils (CDB) developed in preliminary studies for this proposal, cell-cell interactions between CDE and excised human bronchi will be assessed in vitro and in a guinea pig """"""""living explant"""""""" preparation in vivo. Studies are proposed to assess the effect of exogenous activation of eosinophils with formylated tripeptide and by adhesion to matrix proteins and vascular endothelium on isometric contraction and auxotonic narrowing of human airways. Using a specially developed superfusion preparation of human bronchus, the effect of epithelial activation and adhesion will be assessed in vitro by videomicrometry. In a final series of experiments, the regulation of eosinophil secretion of bronchoactive proteins and LTC4 by the beta-adrenoceptor and by endogenous phospholipase A will be related to inhibition of airway hyperresponsiveness caused by activated eosinophils. The proposed studies are designed to l) determine mechanisms causing eosinophil activation, 2) assess indices of cellular secretion corresponding to augmented airway smooth muscle contraction, and 3) model the corresponding inflammatory induction of airway hyperresponsiveness in vitro. Data derived from these investigation should provide a mechanistic basis for therapeutic intervention in the inflammatory hyperresponsiveness of human asthma.
