REGULATION OF MACROPHAGE DEVELOPMENT

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White blood cell differentiation is controlled by hormone like factors that act to regulate complex patterns of gene expression. A number of leukemia cell lines from the granulocyte or monocyte/macrophage with agents such as retinoic acid and phorbol esters. Aspects of macrophage development will be studied by determining the effects of differentiating agents on the expression of genes in the stimulated cell lines. The 5- and 15- lipoxygenase (5-LO, 15-LO) enzymes are produced in mature macrophages and produce leukotrienes and lipoxins from arachadonic acid. The developmental regulation of the 5-LO and 15-LO genes will be investigated by looking for variable gene expression in HL-60, THP-1, and U-937 cells after treatment with differentiating agents. Levels of 5-LO and 15-LO mRNA will be measured using Northern blot hybridization and ribonuclease protection assays. Nuclear run on studies are proposed to study the rates of 5-LO and 15-LO transcription initiation. Screening strategies for cDNA libraries derived from mRNA in stimulated cells are proposed to identify a network of genes that are induced by retinoic acid and phorbol esters. The specific cDNA clones activated in differentiated cells will be initially characterized by sequence analysis and determining tissue specific expression. Antisera raised against peptides coded by these cDNAs will be used in immunofluorescence studies to determine subcellular localization. Selected clones will be further analyzed to identify their functional roles in macrophages involved in normal and pathologic conditions. The subset of clones expressed in monocyte derived foam cells isolated from atherosclerotic lesions will be assessed for the functions they might perform in the process of atherogenesis. Experimental manipulations that alter the expression of individual clones or interfere with their protein products will be tested for their effects on macrophage development, the expression of specific macrophage properties, and LDL metabolism in foam cells.