MONOCYTE/MACROPHAGE FREE RADICAL BIOLOGY ASSESSED BY EPR

吞噬细胞产生的超氧化物和过氧化氢是至关重要的 宿主防御，但也导致损害正常组织的事件。 在 在体外，这两种氧化剂在过渡存在下反应， 金属催化剂（例如铁）以形成羟基自由基（OH）。 如果OH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OH检测系统，目前还不清楚。 我 帮助开发了这样一个系统，利用自旋捕获技术， 结合电子顺磁共振光谱法。 与 在这个系统中，我们没有发现中性粒细胞（PMNs）或单核细胞 产生的吞噬细胞（单核细胞或单核细胞衍生的巨噬细胞[MDM]） OH在没有外源性铁的情况下。 即使补充铁， PMN乳铁蛋白和髓过氧化物酶（MPO）释放抑制OH 一代 然而，单核细胞吞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 抑制化合物。 本应用程序建议扩展这些 通过检查内部和外部理化因素进行观察 这可能会影响单核细胞产生OH的潜力， 吞噬细胞 确定了五个具体的研究目标。 要求1 将决定自由基的性质和动力学 世代随以下研究而变化：1）粘附细胞与非粘附细胞; 2） 与MDM相反的组织（肺）巨噬细胞;和3）铁过载 单核吞噬细胞 目标2将研究是否铁提供了一个 各种细胞外（非吞噬）目标可能允许OH 单核吞噬细胞的结果）2 还原 红细胞、细菌和细菌铁载体，以及 铁蛋白负载的脂质体（用作真核细胞的模型）将被 采用 目标3将检查吞噬体环境是否 与可能改变OH可能性的条件相关 当目标被单核细胞吞噬时发生的形成 吞噬细胞 目标4利用最近合成的自旋捕获剂 增加了脂溶性，试图增加可能的 检测目标内限制性位点处出现的OH 粒子 单核巨噬细胞摄取乳铁蛋白和MPO， 氧化反应可能受到其他炎症产物的影响， 有效细胞-弹性蛋白酶，组织蛋白酶G，TNF，白细胞介素1和2，GM- CSF等。目标5将研究单核细胞暴露是否 这些作用者的吞噬细胞改变了它们产生OH的潜力。 该提案是一项全面的计划，其目标是确定 内源性和外源性因素对OH可能性的影响 由于单核吞噬细胞O2减少而形成。 等 信息将为今后的工作奠定基础， OH在杀微生物活性的组织损伤与 吞噬细胞产生氧化剂。

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.