MECHANISMS OF AIRWAY HYPERRESPONSIVENESS

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The major objective of the proposed research is to test the hypothesis that bidirectional interactions between eosinophils and sensory C-fiber nerves enhance airway inflammation and promote airway hyperresponsiveness. We postulate that each cell type recruits and/or stimulates the other to release its bioactive mediators, and that this positive feedback cycle is necessary for the full expression of allergen- or capsaicin-induced airway hyperresponsiveness. In this application, experiments are proposed to evaluate the mechanisms and functional relevance of eosinophil- sensory C-fiber interactions. To achieve this broad objective, the investigators plan studies in vitro that will first define the mechanisms by which these cell species each recruit and/or stimulate the other to release its bioactive mediators. Then, they will test the importance of these mechanisms in vivo, using two animal models of airway constrictor hyperresponsiveness, in which the proposed cyclic interaction is initiated either by: (i) primary stimulation of airway sensory C-fibers (capsaicin aerosol inhalation), or (ii) primary recruitment and activation of eosinophils (allergen aerosol inhalation after immune- sensitization). Three specific aims are put forth: (1) Identify mechanisms by which eosinophils promote sensory C-fiber neuropeptide release within the airways. Neonatal rat dorsal root ganglion cells, in primary culture, will be exposed to eosinophil- conditioned media or to individual eosinophil mediators, to test the hypotheses that eosinophil-derived substances act on sensory C- fibers to: (i) directly stimulate sensory neuropeptide release; (ii) potentiate release of sensory neuropeptides induced by non- eosinophil stimuli; and (iii) promote tachykinin synthesis. (2) Identify mechanisms by which sensory C-fibers promote eosinophil infiltration and activation. Quiescent human peripheral blood eosinophils will be exposed to dorsal root ganglion cell- conditioned medium or individual sensory neuropeptides, to test the hypotheses that C-fibers promote airway eosinophil recruitment and activation by: (i) upregulating the expression and activity of the eosinophil integrins LFA-1, VLA-4, and Mac-1 (using immunofluorescent flow cytometry and a novel eosinophil adhesion assay); (ii) increasing expression of eosinophil adhesion ligands (ICAM-1, VCAM-1, and E-selectin [ELAM]) on systemic vascular endothelium; and (iii) directly stimulating the release of bioactive mediators. (3) Delineate the roles of eosinophils and sensory C-fibers in allergen-and capsaicin-induced airway hyperresponsiveness in vivo. Guinea pigs will be used to test the hypothesis that both eosinophils and sensory C-fibers are necessary participants in both allergen- and capsaicin-induced airway hyperresponsiveness; functional inhibition of either cell type (i.e., of recruitment or activation, using monoclonal antibodies or pharmacologic interventions) reduces airway eosinophil infiltration and airway hyperresponsiveness after either allergen or capsaicin aerosol exposure. From these studies, the investigators should learn whether and how eosinophils and sensory c-fibers interact to promote airway inflammation and hyperresponsiveness in vivo. Insight gained from this work may shed light on parallel mechanisms that may operate in human asthma.