Experiments are proposed to examine the effects of cytokines interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the synthetic-induction of bioactive eosinophil proteins during poiesis. Studies of the expression of m-RNA encoding the core major basic protein (MBP) and matrix proteins [eosinophil cationic protein (ECP), eosinophil- derived neurotoxin (EDN) and eosinophil peroxidase (EPO)] during eosinopoiesis will be performed using a newly developed preparation of cultured eosinophils derived from human umbilical cord blood. Data from fully mature cells will be compared to cells extracted from the peripheral blood of normal human volunteers. Studies will be performed in a newly developed guinea pig """"""""living explant"""""""" preparation and an isolated-perfused rat bronchial preparation in vivo to assess the biological activity of maturing eosinophils corresponding to cytokine-induced mRNA expression of the same cells in vitro. Data from mature isolated human peripheral eosinophils will be used at each step to validate the relevance of the biological activity of cultured cells. The central hypotheses of these studies are 1) IL-5 induces synthesis of granular protein mRNA and that incremental synthesis caused by IL-3 and GM-CSF depends upon co-stimulation with Il-5. 2) The biological activity of eosinophils will be increased if cytokine-stimulation exceeds that required for maturation alone. Cells isolated immediately postpartum from neonatal cord blood will be cultured and induced to differentiate as eosinophil progenitors by stimulation with IL-3, IL-5 and GM-CSF both singly and in combination. The mRNA expression for MBP, ECP, EDN, and EPO will be assessed in cultured cells initially and during weeks 1 (< 5% explicit differentiation), 2, 3, 4, and 5 (~ 98% purity). Using newly developed in situ methods for assessing force generation in airways activated by inflammatory stimulation, biological activity of cultured cells from each stage will be assessed as change in isometric force generation (guinea pig explant preparation) and endothelially activated increase in lung resistance (rat bronchial preparation). Activation of eosinophils in these studies will be assessed in vitro by a newly developed kinetic assay for EPO and by measurement of LTC4 secretion. All essential feasibility studies have been completed for both stages of the proposal. These studies will provide a direct correlation between the effects of cytokine-stimulation and development of biologically active proteins in eosinopoiesis. Data derived from these studies should provide insight into the mechanism by which eosinophils elicit airway hyperresponsiveness in conditions such as human allergy and asthma.
