The importance of airway inflammation in the pathogenesis of asthma has ,considerable experimental and clinical support. In particular, the mast cell, eosinophil, T lymphocyte and alveolar macrophage are considered to play a pivotal proinflammatory role in the production of airway inflammation, bronchial hyperreactivity and acute episodes of asthma. The ability in vitro of cytokines (IL-5, TNF, GM-CSF, IL-3) derived from these inflammatory cells to activate eosinophils as well as histamine releasing factors and cytokines (IL-1, IL-3) to degranulate human basophils and/or mast cells has suggested a mechanism for cytokines and HRF's to amplify the airway inflammatory response to an inhaled antigen. Therefore, the focus of this grant proposal is to assess both the airway levels, the cellular source and the molecular regulation of cytokines (HRF-A, TNF, IL-3, GM-CSF, and IL-5) in airway cells derived from asthmatics. This study will utilize airway cells obtained from symptomatic asthmatics undergoing bronchoalveolar lavage, and cells form asthmatic airway obtained before and after local endobronchial antigen challenge. Mast cell activation will be assessed by release of both preformed (histamine, tryptase) and newly generated mediators (LTC4, PGD2) while eosinophil activation will be assessed by percoll density gradient centrifugation (% hypodense eosinophils), LTC4 generation and electronmicroscopy. Levels of IL-1 (alpha and beta) and IL-2 will be used as indices of alveolar macrophage and T cell activation respectively. Methods for the detection of cytokines in asthmatic airway will include (a) IL-1, IL-2, IL-3, and GM-CSF ELISA's, (b) TNF, IL-2 and HRF-A (histamine releasing factor-asthma) bioassays, (c) RNA-PCR, (d) in situ hybridization (utilizing cDNA probes for TNF, IL-5, IL-3, GM-CSF, IL-2 and IL-B mRNA) and (e) northern blots. Strategies for the further biochemical purification and characterization of HRF-A will include gel filtration HPLC, Accell-QMA anion exchange HPLC, SDS gel electrophoresis, and production of monoclonal antibodies. The characterization of cytokines responsible for local airway inflammation in asthmatics may allow determination of their cellular sources (airway cells will be separated by adherence, cell sorting and percoll gradients), as well as identify possible novel therapeutic strategies to interrupt the cytokine activated airway inflammation.
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