MONOCYTE/MACROPHAGE FREE RADICAL BIOLOGY ASSESSED BY EPR

吞噬细胞产生超氧化物和过氧化氢是关键 宿主防御但也会导致损害正常组织的事件。在……里面 在体外，这两种氧化剂在存在过渡的情况下发生反应 金属催化剂(如铁)形成羟基自由基(OH)。无论OH是不是 吞噬细胞的生理产物或需要外源催化剂 由于缺乏特定的羟基检测系统，目前尚不清楚。我 帮助开发了这样一个系统，它利用自旋捕获技术在 结合电子顺磁共振光谱分析。使用 这个系统我们没有发现中性粒细胞(PMN)或单核细胞 产生吞噬细胞(单核细胞或单核细胞来源的巨噬细胞[MDM]) 哦，在没有外源铁的情况下。即使补充了铁， PMN乳铁蛋白和髓过氧化物酶(MPO)释放抑制OH 一代。然而，单核巨噬细胞表现出持续的OH 在相同的条件下形成，可能是由于缺乏这些 抑制性化合物。此应用程序建议在这些基础上进行扩展 考察内外物理化学因素的观察 这可能会影响单核细胞产生羟基的潜力 吞噬细胞。已经确定了五个具体的研究目标。目标1 将决定自由基的性质和动力学 世代因以下研究而异：1)贴壁细胞与非贴壁细胞；2) 组织(肺)巨噬细胞，而不是MDM；以及3)铁超载 单核巨噬细胞。目标2将检查由一种 多种细胞外(非吞噬)靶标可能允许OH 由于单核吞噬细胞而发生的世代)2 还原。红细胞、细菌和细菌铁载体，以及 装载铁蛋白的脂质体(用作真核细胞的模型)将 使用。目标3将检查吞噬体环境是否 与可能改变OH可能性的条件有关 当靶细胞被单核细胞吞噬时形成 吞噬细胞。AIM 4利用最近合成的自旋捕捉剂 随着脂类溶解度的增加，试图增加可能的 检测在目标内的受限位置发生的OH 粒子。单核巨噬细胞摄取乳铁蛋白和MPO 氧化反应可受其他炎症产物的影响 有效细胞-弹性蛋白酶、组织蛋白酶G、肿瘤坏死因子、白介素1和2、GM- 脑脊液等。AIM 5将调查暴露于单核细胞 吞噬细胞对这些因子的作用改变了它们产生羟基的能力。 这项提案是一项综合性计划，其目标是明确 内生和外生因素对羟基磷灰石可能性的影响 由于单核巨噬细胞02减少而形成。是这样的 信息将为今后的工作奠定基础，以澄清 在杀菌活性方面与S组织损伤有关 吞噬细胞氧化剂的产生。

Phagocyte production of superoxide and hydrogen peroxide is critical to host defense but also leads to events which damage normal tissues. In vitro these two oxidant species react in the presence of a transition metal catalyst (e.g. iron) to form hydroxyl radical (OH). Whether OH is a physiologic product of phagocytes or requires an exogenous catalyst has not been clear due to the lack of a specific OH detection system. I helped develop such a system which utilizes spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry. With this system we found no evidence that neutrophils (PMNs) or mononuclear phagocytes (monocytes or monocyte-derived macrophages [MDM]) generated OH in the absence of exogenous iron. Even with iron supplementation, PMN lactoferrin and myeloperoxidase (MPO) release inhibited OH generation. Mononuclear phagocytes, however, exhibited sustained OH formation under the same conditions, presumably due to the lack of these inhibitory compounds. This application proposes to expand on these observations by examining internal and external physico-chemical factors which could influence the potential for OH generation by mononuclear phagocytes. Five specific aims have been identified for study. Aim 1 will determine whether the nature and dynamics of free radical generation varies with study of: 1) adherent vs non-adherent cells; 2) tissue (pulmonary) macrophages as opposed to MDM; and 3) iron-overloaded mononuclear phagocytes. Aim 2 will examine whether iron provided by a variety of extracellular (non-phagocytosed) targets may allow OH generation to occur as a consequence of mononuclear phagocyte )2 reduction. Erythrocytes, bacteria and bacterial siderophores, and ferritin-loaded liposomes (used as models of eukaryotic cells) will be used. Aim 3 will examine whether the phagosomal environment is associated with conditions which may alter the likelihood of OH formation occurring when targets are phagocytosed by mononuclear phagocytes. Aim 4 utilizes recently synthesized spin trapping agents with increased lipid solubility in an attempt to increase the possible detection of OH occurring at a restricted site within the target particle. Mononuclear phagocytes take up lactoferrin and MPO and their oxidative response can be influenced by other products of inflammatory effective cells - elastase, cathepsin G, TNF, Interleukin 1 and 2, GM- CSF etc. Aim 5 will investigate whether exposure of mononuclear phagocytes to these actors alters their potential for generation of OH. This proposal is a comprehensive program whose objective is to define the impact of endogenous and exogenous factors on the likelihood of OH formation as a consequence of mononuclear phagocytes 02 reduction. Such information would set the stage for future work to clarify the role of OH in the microbicidal activity s tissue damage associated with phagocyte oxidant production.