MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

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Studies are proposed for competitive continuation of investigations into mechanisms by which eosinophils cause augmented bronchomotor responsiveness in the human asthmatic state. In the prior grant period, investigations were completed that demonstrated that eosinophil secretion of leukotriene (LT) C4 caused direct contraction of guinea pig trachealis in vivo. The current proposal tests the hypothesis that molecular adhesion of eosinophils to endothelium and airway matrix primes eosinophil secretion and augments airway smooth muscle contraction. This proposal utilizes a newly developed method for measurement of cell-cell interactions causing airway contractile responses in microsections of explanted human airways (obtained through an NHLBI-sponsored collaboration with the Krankenhaus Gro/beta/hansdorf, Hamburg, Germany) in which a very small number of human eosinophils (greater than or equal to 50,000/chamber) is utilized. Studies are proposed to examine the mechanism by which adhesion of eosinophils to human umbilical vein endothelial cells (HUVEC) and to the matrix protein fibronectin (FN) causes augmented secretion of bronchoactive LTC4, which then causes augmented contraction of human bronchial explants. The mechanism by which direct binding of the beta1-integrin, VLA-4, to FN and of beta2- integrin or VLA-4 to endothelium causes augmented synthesis and secretion of LTC4 in eosinophils will be examined directly. Using the newly developed videomicrometry system, the direct effects of molecular adhesion of human eosinophils to HUVEC or FN in augmenting smooth muscle contraction from human airways will be examined. Preliminary investigations have established that adhesion molecule ligation causes augmented secretion of LTC4 from eosinophils. Further preliminary studies indicate that ligation to FN causes increased activity of cytosolic PLA2 (cPLA2) and increased sPLA2 activity with corresponding translocation of these isoforms. Utilizing specific monoclonal antibodies (mAb) to the beta2 subunit or to VLA-4 and by selective use of mAb directed against isoforms of PLA2, studies are proposed to examine the hypothesis that molecular adhesion augments stimulated constriction of human airways through augmented secretion of LTC4, which is caused by increased activity of cPLA2 after adhesion-induced translocation to nuclear membrane and sPLA2 to plasma membrane/perfusate. These investigations should establish one mechanism that accounts for the selective activation of human eosinophils and establish the direct relevance of this mechanism to augmented contraction of human airway smooth muscle in the hyperreactive state. Data derived from these studies should suggest direct mechanisms for physiologically relevant approaches to the treatment of human asthma.