In distinction to the more commonly recognized morphologic eosinophil phenotype with a segmented nucleus that is associated with eosinophilic disease states, we have recently observed that eosinophils of a lesser centrifugation density (hypodense eosinophils) can also be mononuclear. Previously, we observed that freshly isolated, bilobed peripheral blood eosinophils from healthy individuals (normodense eosinophils) treated with the eosinophilopoietic cytokines, interleukin (IL)-5, IL-3, or granulocyte-macrophage colony-stimulating factor (GM-CSF), are protected from apoptosis and glucocorticoid-induced cytotoxicity. These eosinophils are converted to a hypodense phenotype that is primed for ligand- initiated activation of the 5-lipoxygen-ase (5-LO) pathway and superoxide generation, and for antibody-dependent and independent cytotoxicity. The focus of Project 4 is to define the pro-inflammatory characteristics of the mononuclear eosinophil, an immature phenotype that is achieved after 28 days of culture of pleuripotent granulocyte precursors and appears comparable to mononuclear eosinophils in lesional sites by physical and morphologic criteria. The culture system for mononuclear eosinophils utilizes freshly isolated cord blood mononuclear cells cultured in vitro with IL-3 and IL-5 on a reconstituted basement membrane (Matrigel-Tm). After 14 days of culture, a hybrid mononuclear cell with both eosinophilic and basophilic granules predominates. This hybrid cell resembles a morphologic phenotype previously described in association with certain leukemic states but not further characterized. The intermediate hybrid cell phenotype, and especially the mononuclear eosinophil generated after 28 days of culture, will be characterized for susceptibility to apoptosis without and with glucocorticoid treatment, ligand-initiated and (or) calcium ionophore-stimulated superoxide generation and leukotriene production, antibody-mediated target cell cytotoxicity, type and size of cell-associated proteoglycan(s), presence of selected eosinophil-granule markers, and ultrastructural morphology. Particular attention will be paid to the development of the 5-LO pathway by monitoring steady-state transcription, immunoblot detection of protein, and individual assay of cytosolic phospholipase A2, 5-LO, 5- lipoxygenase-activating peptide, and LTC4 synthase. The recent cloning of the cDNA for LTC4 synthase as described for Project 3 now permits the characterization of the full 5-LO pathway during eosinophilopoiesis to the mononuclear phenotype and with conversion of normodense eosinophils to the hypodense phenotype with a segmented nucleus.
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