Oxidative metabolism is crucial in the microbicidal activity of phagocytic cells. During ingestion of the microbe, oxygen is transported across the cell membrane and reduced to superoxide anion (O2-) which dismutates rapidly to form hydrogen peroxide (H2O2). In the presence of iron, H2O2 and O2- interact to form hydroxyl radical (.OH). Generation of .OH by stimulated neutrophils has been measured and shown to be one of the means by which bacteria are killed. We have shown that lactoferrin (LF), an iron-binding protein found in the specific granules of neutrophils, enhances .OH generation in whole neutrophils, subcellular fractions of neutrophils, and a chemical generating system. By binding iron, LF presents iron efficiently for the production of .OH in neutrophils. This project will further define the role of LF, iron, and .OH in bacterial killing. Cell membrane, azurophilic granules, and specific granules will be separated on sucrose density gradients and the contribution each makes alone or in combination with others to oxygen radical formation (O2-, H2O2, .OH) and bacterial killing will be studied. To look more specifically at the bactericidal activity of iron-saturated LF, neutrophil LF will be used in bactericidal assays with the xanthine oxidase system. Control studies will explore the possibility of an antibacterial effect for LF separate from the enhancement of .OH. The source of iron for LF is an important consideration in these studies. Experiments using immunoprecipitation techniques and affinity chromatography will define the amount of iron bound to LF from neutrophils. Studies using 59Fe will be performed to explore the possible transfer of iron from bacteria or other iron-binding proteins. Oxygen radical production, bacterial killing, LF content, and iron status will also be measured in several deficiency states where abnormalities of .OH production, specific granules, or LF may result in abnormal function. Since neutrophils and oxygen radicals they generate have been implicated in the tissue damage of inflammation, these studies have importance in the study of the inflammatory response and the antimicrobial function of neutrophils. By understanding the biochemical events of neutrophils function, we may be better able to manipulate the phagocytic cell to enhance microbicidal activity or minimize tissue damage.
