Studies are proposed to examine the role of apoptosis during eosinophilopoiesis and the role of the extracellular matrix heparans sulfate (HS), via a potential recepto, CD31 on modulating survival and development of eosinophils, cells central to pathogenesis of asthma. The central gypothesis of this proposal is tht ceptor-mediated interaction of precursor cells with HS causes the upregulation of survival mechansims in the precursor cells contributing to an increase in eosinophil production. To test this hypothesis, we propose three specific aims: (1) Determine the effects of HS and CD31 ligation on the in Vitro production of eosinophils (CDE) developing from isolated progenitor cells. The mechanism by which heparan sulfate enhances CDE production in vitro will be examined. Experiments will: a) assess Il-3 and IL-5-directed production of maturing CDE from isolated cord blood progenitor (CD34+) cells in tissue-culture plates pretreated with HS or irrelevant matrix molecules over a determined dose range; b) determine receptor specificity of the response of CDE to heparan sulfate by using epitope-specific mAb; c) assess effects of CD31 stimulatory monoclonal antibody in terms of eosinophil production and compare to HS stimulation. (2) Determine the susceptibility of developing CDE to induction of apoptosis through 'passive' cytokine withdrawal and 'active' Fas ligation and the mechanistic roles of the Bcl-2 family and the ICE family in these processes.a) Using 'naive' or cytokine-induced progenitor cells, we will measure the relative susceptibility of the cells to cytokine withdrawl and to Fasligation;b) measure the expression of Bcl-2-related and ICE mRNA and proteins in eosinophils at different maturational stages, using RT-PCR, Northern, Western and flow cytometric assays to identify specific intracellular mechanisms of regulation of apoptosis; c) using specific Bcl-2 and ICE inhibition of susceptible and resistant eosinophil precursors, assess the relative roles of these regulators during development. (3) Determine the role of Heparan sulfate and/or CD31 ligation in the suppression of apoptosis of developing eosinophils and the molecular mechanisms involved suppression of these apoptotic events. Using cord blood-derived progenitor cells and CDE, we will a) detemine the effect of culture on heparan sulfate or stimulation by anti-CD31 on the apoptotic response to cytokine withdrawal and Fas ligation in different maturational stages of eosinophils; b) assess the contributions of specific Bcl-2 and ICE family members to CD31/HS modulated response to apoptotic stimuli. We anticipate that these studies will establish a novel strategy of examining eosinophilopoiesis and will elucidate mechanisms of matrix-mediated regulation of hematopoiesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI032654-05
Application #
2003790
Study Section
Special Emphasis Panel (ZRG4-ALTX-2 (01))
Project Start
1992-04-01
Project End
2000-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Chicago
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
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Munoz, N M; Hamann, K J; Rabe, K F et al. (1999) Augmentation of eosinophil degranulation and LTC(4) secretion by integrin-mediated endothelial cell adhesion. Am J Physiol 277:L802-10
White, S R; Dorscheid, D R; Rabe, K F et al. (1999) Role of very late adhesion integrins in mediating repair of human airway epithelial cell monolayers after mechanical injury. Am J Respir Cell Mol Biol 20:787-96
Hamann, K J; Dorscheid, D R; Ko, F D et al. (1998) Expression of Fas (CD95) and FasL (CD95L) in human airway epithelium. Am J Respir Cell Mol Biol 19:537-42
Hamann, K J; Neeley, S P; Dowling, T L et al. (1996) Effect of interleukin-5 exposure during in vitro eosinophilopiesis on MAC-1 adhesion molecule expression and function on cultured human eosinophils. Blood 88:3575-82
Hamann, K J; Dowling, T L; Neeley, S P et al. (1995) Hyaluronic acid enhances cell proliferation during eosinopoiesis through the CD44 surface antigen. J Immunol 154:4073-80
Neeley, S P; Hamann, K J; Dowling, T L et al. (1994) Augmentation of stimulated eosinophil degranulation by VLA-4 (CD49d)-mediated adhesion to fibronectin. Am J Respir Cell Mol Biol 11:206-13
Neeley, S P; Hamann, K J; White, S R et al. (1993) Selective regulation of expression of surface adhesion molecules Mac-1, L-selectin, and VLA-4 on human eosinophils and neutrophils. Am J Respir Cell Mol Biol 8:633-9
Hamann, K J; Strek, M E; Baranowski, S L et al. (1993) Effects of activated eosinophils cultured from human umbilical cord blood on guinea pig trachealis. Am J Physiol 265:L301-7