CYTOADHESION MOLECULES IN CYTOKINE-DIRECTED EOSINOPOIESIS

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Experiments are proposed to examine the effects of adhesion molecule-regulated cell-cell and cell-extracellular matrix (ECM) interactions on cytokine-directed eosinopoiesis of human progenitor cells. Studies of the expression of the differentiation antigens, CD34 and CD33, and mRNA encoding eosinophil major basic protein (MBP) and eosinophil peroxidase (EPO) during eosinopoiesis will be performed using a newly developed preparation of cultured eosinophils derived from human umbilical cord blood. Studies will be performed utilizing multi-color flow cytometry, progenitor-ECM coculture and a newly developed endothelial cell adhesion assay to assess the expression and functionality of adhesion molecules on developing and mature cultured eosinophils. Data from fully mature, cultured cells will be compared to cells extracted from the peripheral blood from normal human volunteers. The central hypothesis of the first series of studies is that a specific CD34 + subpopulation of progenitor cells present in the mononuclear fraction of human umbilical cord blood defines a population of committed precursor cells responsive to the hematopoietic cytokines, stem cell factor (SCF), granulocyte-macrophage colony- stimulating factor (GM-CSF), interleukin (IL)-3 and IL-5. Experiments are proposed to isolate subpopulations of progenitor cells in high purity and to culture these cells in the presence of different concentrations of single cytokines and in various combinations and sequences of cytokines. Studies will reveal proliferative and differentiative responses of the various subpopulations under these conditions. We will perform parallel studies of mRNA expression and granule protein mediator and lipid mediator production by quiescent and activated cells produced in these studies. A second series of studies will characterize the surface adhesion molecule ontogeny of developing eosinophils in vitro and their interactions with ECM molecules and/or with other cells present in the cord blood mononuclear cell fraction. Using the cord blood system of eosinopoiesis, specific surface molecules, ICAM-1 LFA-1, VLA-4 and CD44 will be analyzed by single and multi- color flow cytometry during eosinophil development. These receptors which may be involved in cell-cell or ECM-cell interactions also will be studies under optimal and suboptimal conditions of cytokine stimulation. These studies will provide direct correlations between cytokine-stimulation, adhesion molecule receptor expression and eosinopoiesis. Data derived from these studies should provide insight into the bi-directional mechanisms of adhesion molecule development and regulation in eosinopoiesis and suggest unique avenues of therapeutic intervention in the eosinophilic inflammatory responses seen in asthma and allergic disease.