HYDROXYL RADICAL AND BACTERIAL KILLING BY NEUTROPHILS

氧化代谢在吞噬细胞的杀微生物活性中至关重要。 细胞 在摄取微生物的过程中，氧气通过 细胞膜，并还原为超氧阴离子（O2-）， 快速生成过氧化氢（H2 O2）。 在铁的存在下，H2 O2 与O2-相互作用形成羟基自由基（.OH）。 .OH的生成 刺激的中性粒细胞已被测量，并显示是一种手段， 从而杀死细菌。 我们已经证明，乳铁蛋白（LF）， 在嗜中性粒细胞的特定颗粒中发现的铁结合蛋白， 增强整个嗜中性粒细胞、 中性粒细胞和化学生成系统。 通过结合铁，LF 提供铁，有效地在中性粒细胞中产生.OH。 本项目将进一步确定LF、铁和.OH在细菌中的作用。 杀人 细胞膜、嗜天青颗粒和特异性颗粒 在蔗糖密度梯度上分离， 单独或与其它化合物组合对氧自由基形成（O2-，H2 O2， .OH）和细菌杀灭进行研究。 更具体地看， 将使用铁饱和LF、中性粒细胞LF的杀菌活性 在用黄嘌呤氧化酶系统的杀菌测定中。 对照研究 将探讨LF分离的抗菌作用的可能性 从. OH的增强。 LF的铁源是一个重要的 在这些研究中考虑。 使用免疫沉淀的实验 技术和亲和层析将确定结合的铁的量 从中性粒细胞到LF。 将使用59 Fe进行研究，以探索 铁可能从细菌或其他铁结合 proteins. 氧自由基的产生，细菌的杀灭，LF含量， 还将在几种缺乏状态下测量铁状态， .OH产生、特定颗粒或LF的异常可能导致 功能异常 由于中性粒细胞和它们产生的氧自由基与 炎症的组织损伤，这些研究在 研究其炎症反应和抗菌作用 中性粒细胞 通过了解中性粒细胞的生化事件 功能，我们可能能够更好地操纵吞噬细胞， 增强杀微生物活性或使组织损伤最小化。

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.