An influx of eosinophils into the interstitium of airways is characteristic of chronic inflammation associated with asthma. Eosinophils release factors that may damage airway epithelial cells, and indeed, degranulated eosinophils are seen in close association with the basement membrane in asthmatic lungs. Tissue eosinophils produce and release an abundance of 92 kDa gelatinase (92 (G'ase), a matrix metalloproteinase (MMP) with a broad substrate specificity. Neutrophils also store an abundance of 92 kDa gelatinase but do not have the capacity to produce additional enzyme. Thus, the sustained production of this MMP by eosinophils may significantly contribute to tissue damage and altered cell function associated with asthma. Preliminary data demonstrate that circulating eosinophils from asthmatics contain 92 G'ase, whereas blood eosinophils from healthy humans do not. These findings suggest that in normals 92 G'ase is induced in eosinophils upon or subsequent to extravasation, whereas enzyme expression is initiated at an earlier stage in asthma. Once in the tissue environment, upregulation and the site-specific release of 92 G'ase may be mediated by cell:cell and cell:matrix contacts. These hypotheses will be explored in this project. Eosinophil-derived 92 G'ase can degrade numerous matrix molecules, and this proteolytic activity may contribute to the pathophysiology of asthma. The following studies are proposed to assess the regulation and role of this potentially important enzyme in asthma. 1. To test the hypothesis that the proteolytic phenotype of eosinophils is altered in asthmatics and is further changed as the cells move from blood vessels into tissues, specific histochemical and biochemical assays will be used to assess the expression of MMPs in blood, tissue, and alveolar eosinophils from normals and asthmatics. Proteinase expression will be correlated with ability of eosinophils to migrate through a matrix barrier. 2. To identify factors responsible for induction of 92 (G'ase, blood eosinophils from normals will be exposed to key soluble factors, and enzyme expression will be measured with sensitive molecular and immunologic assays. Eosinophils will also be cultured on various matrix proteins and in contact with activated endothelial cells, T lymphocytes, and airway epithelial cells to assess if interactions within the tissue space influence 92 G'ase expression and release. 3. The molecular mechanisms controlling induction and upregulation of 92 G'ase in eosinophils will be characterized with an emphasis on identifying cis-regulatory elements. 4. Using ex vivo and cell culture models, the ability of activated eosinophils and purified 92 G'ase to directly affect airway epithelial integrity will be assessed. In addition, MMP inhibitors and 92 G'ase-deficient mice will be used to determine the role of 92 G'ase in the development of airway damage in an ovalbumin-challenged mouse model of asthma. All studies proposed in this project involve interactions with the other SCOR investigators and will require the assistance of the Human Subjects, Immunopathology and Mouse Transgenic Cores. These studies will demonstrate that 92 G'ase is a prominent and destructive proteinase that plays a critical role in the pathogenesis of asthma.
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