MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

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This proposal utilizes technologies developed in preliminary investigations in our laboratory and by our collaborators to define mechanisms by which eosinophils cause airway hyperresponsiveness. Studies are proposed to investigate the regulation of bronchomotor tone at 3 levels: 1) the role of intact, activated eosinophils; 2) specific effects of purified eosinophil proteins/mediators; 3) cell-cell interactions between epithelial/endothelial tissues caused by these mediators. Prior studies have been limited to in vitro investigations from excised airways and did not permit assessment of either neurocirculatory interactions nor the role of airway edemagenesis in airway hyperresponsiveness caused by eosinophil proteins. To obviate prior limitations, we have developed an in situ "living explant" preparation of guinea pig trachea, which preserves innervation and circulation to the airway. This preparation demonstrates 1) direct, characteristic contractile responses to the major purified eosinophil proteins, 2) evidence for synthesis of augmenting (rather than inhibitory) mediators during eosinophil infiltration, and 3) evidence for major contractile interactions with underlying vascular endothelium. Studies are proposed to test the hypothesis that activated eosinophils cause direct airway smooth muscle contraction and augment other contractile stimuli through these interactions. A major emphasis of this work is defining the mechanism causing the unique responsiveness of each eosinophil protein with respect to 1) site of action, 2) ion charge, 3) cytotoxic effect, and 4) RNase activity; additional studies are proposed using monoclonal antibodies to eosinophil-proteins to determine the mechanism and concentrations required to block the interactions with protein matrices for application to studies of intact eosinophils. Using the guinea pig tracheal preparation, monoclonal antibodies will be used to block the effects of degranulated eosinophilic proteins and thus to assess specifically which proteins are responsible for airway hyperresponsiveness in activated eosinophils. A final series of experiments is proposed to examine further the response of intact activated eosinophils in causing airway hyperresponsiveness. In preliminary studies, a new preparation permitting selective perfusion of the bronchial circulation of the rat has been developed. This open circuit preparation will be utilized to assess 1) the role of circulating activated eosinophils in augmenting lung resistance responses in the conducting airways of the rat; 2) the role of normo- and hypodense eosinophils in this process; 3) the specific protein mediators involved; 4) the role of edemagenesis induced by eosinophil activation in bronchial hyperresponsiveness; and 5) the role of LFA-1alpha and Mac-1 receptors in promoting eosinophil migration, activation and consequent upregulation of airway responsiveness. Preliminary studies are completed for necessary technical aspects of the proposed studies. Data derived from these studies suggest therapeutic approaches for disorders of airway hyperresponsiveness.