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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
1R29AI029974-01A1
Application #
3455579
Study Section
Immunological Sciences Study Section (IMS)
Project Start
1991-03-01
Project End
1996-02-29
Budget Start
1991-03-01
Budget End
1992-02-29
Support Year
1
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Sriramarao, P; DiScipio, R G; Cobb, R R et al. (2000) VCAM-1 is more effective than MAdCAM-1 in supporting eosinophil rolling under conditions of shear flow. Blood 95:592-601
Weinberger, M S; Davidson, T M; Broide, D H (1996) Differential expression of vascular cell adhesion molecule mRNA and protein in nasal mucosa in response to IL-1 or tumor necrosis factor. J Allergy Clin Immunol 97:662-71
Sullivan, S; Broide, D H (1996) Compartmentalization of eosinophil granulocyte-macrophage colony-stimulating factor expression in patients with asthma. J Allergy Clin Immunol 97:966-76
Sriramarao, P; Anderson, W; Wolitzky, B A et al. (1996) Mouse bone marrow-derived mast cells roll on P-selectin under conditions of flow in vivo. Lab Invest 74:634-43
Anderson, W H; Davidson, T M; Broide, D H (1996) Mast cell TNF mRNA expression in nasal mucosa demonstrated by in situ hybridization: a comparison of mast cell detection methods. J Immunol Methods 189:145-55
Ferguson, P; Broide, D H (1995) Environmental and bronchoalveolar lavage Dermatophagoides pteronyssinus antigen levels in atopic asthmatics. Am J Respir Crit Care Med 151:71-4
Sriramarao, P; von Andrian, U H; Butcher, E C et al. (1994) L-selectin and very late antigen-4 integrin promote eosinophil rolling at physiological shear rates in vivo. J Immunol 153:4238-46
Sullivan, S; Salapow, M A; Breen, R et al. (1994) Eosinophil peroxidase differs from neutrophil myeloperoxidase in its ability to bind antineutrophil cytoplasmic antibodies reactive with myeloperoxidase. Int Arch Allergy Immunol 105:150-4
Broide, D H; Paine, M M; Firestein, G S (1992) Eosinophils express interleukin 5 and granulocyte macrophage-colony-stimulating factor mRNA at sites of allergic inflammation in asthmatics. J Clin Invest 90:1414-24
Broide, D H; Lotz, M; Cuomo, A J et al. (1992) Cytokines in symptomatic asthma airways. J Allergy Clin Immunol 89:958-67

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