A central characteristic of asthma is the recruitment of eosinophils into the lungs, followed by their activation and release of proinflammatory mediators. An improved understanding of the mechanisms involved in this response is, therefore, of great relevance to asthma pathogenesis and the development of new therapeutics. The objective of this application is to further elucidate the molecular mechanisms for preferential recruitment and activation of eosinophils and basophils in asthma. Studies will focus on how integrins, cytokines, and C-C chemokines contribute to selective cell recruitment in vitro and in vivo. Experiments in Aim 1 will test the hypothesis that the novel integrin alpha d beta2 is an important ligand for VCAM-1 on eosinophils and basophils. The investigators will delineate physiologic stimuli that alter cell surface levels and function of alpha d beta2 intergrins using adhesion assay and flow cytometry, and will localize intracellular pools of these intergrins using immunogold and electron microscopy.
In Aim 2, studies will examine the regulation of integrin-dependent adhesion and migration responses by cytokines and chemokines. They will test the specific hypothesis that cytokines and chemokines that stimulate eosinophil and basophil adhesion and migration do so by shifting integrin usage in these cells away from beta1 and beta7 integrin-dominated interactions with counterligands towards beta2 intergrin-dominated interactions (e.g., via alpha d beta2 intergrin). Therefore, granulocyte adherence and transmigration assays involving beta1, beta2 and beta7 integrin ligands employing integrin and chemokine receptor blocking monoclonal antibodies (mAbs) will be performed to characterize the concentration dependence, kinetics, and receptor specificity of the cytokine and chemokine effects on integrin function.
In Aim 3, in vivo studies will use endobronchial allergen challenge to determine whether integrin function is altered in eosinophils and basophils recruited into the airways during experimental allergic inflammatory responses in the lung.
Aim 3 will also test the hypothesis that different C-C chemokines are produced within the airways, and contribute to pathophysiology, at distinct times during allergic inflammatory reactions in vivo. The investigators will perform BAL and biopsies hours or days after segmental allergen challenge of allergic subjects, as well as on normal and asthmatic subjects will asthma of varying degrees of severity. ELISA and quantitative RT-PCR assays will then be used to measure levels of mRNA and protein for several C-C chemokines in the airways in both chronic and experimental allergen-induced airway inflammation. Sites of synthesis will be determined using immunohistochemistry and in situ hybridization, and correlations between chemokine production and relevant cellular and physiologic characteristics of allergic airway inflammation will be delineated at each time point. It is anticipated that the proposed studies will lead to an improved understanding of the pathogenesis of asthma. The investigators believe that the combined use of both in vitro and in vivo models uniquely positions them to assess the relevance of cellular and molecular processes to human allergic diseases of the airways.
