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.
