The long-term goal of this project will be to investigate the regulation of the superoxide-generating NADPH oxidase of phagocytic leukocytes. Oxygen species generated by this enzyme system play a major role in host defense against infection and as well as in tissue damage during inflammation and reperfusion injury. Our work will focus on the cytochrome b component of the NADPH oxidase, in particular the gene encoding gp91-phox, the 91 kilodalton glycoprotein of the phagocyte cytochrome b heterodimer. Expression of this and closely related genes is developmentally regulated and virtually lineage-specific to myelomonocytic cells. Glucocorticoids and non-steroidal anti-inflammatory drugs (NSAID) modulate the expression of various genes and cell functions. The proposed studies will test the central hypothesis that glucocorticoids and NSAID inhibit phagocytic NADH oxydase activity by down-regulating expression of genes encoding components of the NADPH oxidase system. We will: 1. Develop a system for the differentiation of the human monocytic cell line THP-1 by cytokines that influence phagocyte function: We will examine the NADPH oxidase activity, cytochrome b content, and expression of the genes encoding gp91-phox and the other NADPH oxidase components. 2. Study the effects of glucocorticoids and NSAID on the NADPH oxidase system of cytokine-differentiated THP-1 cells: We will examine the effect of glucocorticoids and NSAID on the NADPH oxidase activity, cytochrome b content, and expression of the genes encoding gp91-phox and the other NADPH oxidase components. 3. Investigate the effects of cytokines, glucocorticoids or NSAID on the regulation of the NADPH oxidase system in human peripheral blood monocytes/macrophages: We will investigate the effects of glucocorticoids or NSAID on the NADPH oxidase activity, cytochrome b content, and expression of the genes encoding gp91-phox and the other NADPH oxidase components in human peripheral blood monocytes/macrophages under cytokine stimulation. The proposed studies should advance our fundamental knowledge of transcriptional control mechanisms in the myelomonocytic lineage and as well determine new mechanisms by which anti-inflammatory agents exert their anti-inflammatory effects, in this case by down-regulating the expression of genes encoding components of the phagocytic NADPH oxidase system. Better understanding of the pharmacological regulation of the NADPH oxidase system will provide information to help modulate its expression during inflammation and oxidant-mediated tissue damage.
