Through their activation-dependent release of preformed secretory granule mediators, membrane lipid-derived mediators, and proinflammatory cytokines, mast cells are likely to contribute to the pathogenesis of bronchial asthma. In contrast to most other cells of the hematopoietic system, mast cells leave the bone marrow as unrecognized progenitors, circulate through the bloodstream, and undergo essentially all of their further development at multiple tissue sites. Studies utilizing a limited number of late-expressed mast cell phenotypic characteristics such as histochemical staining indicate that the tissue microenvironment of the mast cell is likely to dictate the final mature phenotype, but there is little knowledge about the developmental stages between the bone marrow progenitor and mature tissue mast cells in vivo. The molecular cloning of seven murine mast cell-specific proteases, including several that are expressed preferentially by immature, bone marrow culture- derived mst cells, affords the means to identify early mast cell progenitors in situ and to elucidate the tissue-defined steps of mast cell development, so as to gain an appreciation of how mast cells acquire their phenotypic diversity in vivo. The first Specific Aim will use immunocytochemical and in situ hybridization assessments of mast cell protease gene expression to define the development of mast cells in mast cell-deficient WBB6F1-W/Wv mice that have been infused with bone marrow cells from congenic normal mice to elicit the development of mast cells from the donor progenitor cells. Because of the paucity of intestinal mucosal mast cells in mice, this approach may favor the recognition of mast cells of the micro-vascular connective tissue; therefore, we will also analyze in kinetic protocols the development of mast cells in helminth-infected normal mice, whose mast cell progenitors and mucosal mast cell numbers are elevated. The second Specific Aim will determine with a combination of in situ hybridization and immunochemical approaches the spectrum of cytokines that are expressed by mast cells developing at multiple tissue sites, including tracheobronchial mast cells, in the two models studied in the first Specific Aim. The third Specific Aim of this project will seek to determine which constituent(s) of mast cells causes the mast cell-dependent pulmonary hyperresponsiveness to methacholine that occurs in three mouse models: after intravenous infusion of anti- IgE, after inhalation of ozone, and after active sensitization to ragweed allergen followed by aerosolized challenge. To achieve this aim, we will assess pulmonary hyperresponsiveness in the three models with mice generated in Project 4 that fail to express one or more mast cell proteases through targeted gene disruption. By using inhibitors of LTC4 and receptor antagonists of 5-hydroxytryptamine, we will also determine if these mast cell mediators contribute to the development of hyperresponsiveness. Overall, the studies in this project are expected to generate new insights into the in vivo developmental biology of mast cells and the contribution of their mediators to pulmonary hyperresponsiveness, a hallmark of bronchial asthma.
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