Phagocytic cells contain enzyme systems which allow purposeful reduction of 02. 02 reduction by phagocytic cells has attracted tremendous interest because of the role reduction products play in host defense (e.g. microbicidal and tumorcidal processes) as well as tissue damage they produce. 02- is unstable and microenvironmental conditions will determine subsequent reactions including formation of H202, OH., O21- and OH.. Oh. is the most potent of these oxidizing agents and has been implicated in complement mediated lung damage and other forms of tissue injury. Spin trapping is a tool which has allowed us to measure intraphagosomal formation of free radicals. Our results show that OH. is formed by human neutrophils in response to S aureus, but not phorbol myristate acetate or opsonized zymosan. These results contradict some earlier studies, but offer insight into the difficulties which surround measurement of this radical. OH. formation in our studies can be clearly attributed to the Haber- Weiss reaction. Our results suggest that the way in which iron is presented to the phagocte is more important than the absolute concentration of iron. We have also conducted experiments which suggest that phagocytes limit the formation of OH. when it is formed in extracellular fluids. In this proposal we outline studies to identify conditions which allow phagocytes (PMNS, differentiated HL-60 cells, PMN cytoplasts) to make OH., and mechanisms available to the cell to prevent accumulation of this radical. Equally important, we intend to study the effects of phagocyte free radical formation on autologous membrane peroxidation and membrane fluidity. These latter results will be correlated to cell aging and death. Identifying conditions which allow hydroxyl radical formation offers the potential to harness this reaction to host defenses, and to better understand the pathogenesis of these reactions in a variety of inflammatory diseases.
