Peroxidase-Based Toxicity In Hypereosinophilic States.'

描述(申请人提供)：嗜酸性粒细胞(EO)吞噬细胞杀死寄生虫，但也可以破坏宿主组织。虽然过敏是EO介导的病理中最常见的例子，但高嗜酸性粒细胞综合征(HES)是最引人注目的。HES是一种以多器官系统受累为特征的系统性血液系统疾病，是心脏病的一种特征，通常是致命的形式。嗜酸性心脏病(EHD)在各种高嗜酸性粒细胞状态下发生，无论其原因如何。心内膜和心肌中EO特异性颗粒蛋白的沉积被认为是介导EHD心脏毒性的机制之一，其中以嗜酸细胞过氧化物酶(EPO)最为丰富。尽管有大量的EPO和EO呼吸爆发的活力，但关于EPO介导的氧化损伤在嗜酸性炎症状态的病理中所起的作用还知之甚少。我们发现三种不寻常的底物--溴化物(BR-)、亚硝酸盐(NO2-)和硫氰酸盐(SCN-)--在H202存在的生理液体中竞争EPO的氧化，分别产生HOBr、NO2和HOSCN。这些氧化剂对人体细胞的相对毒性为HOBR&GT；NO2&GT；&GT；HOSCN；而EPO优先氧化SCN&GT；NO2-&GT；BR-。我们假设，SCN“缓冲”了EPO产生的更具细胞毒性的N02和BR类氧化剂，并因此通过饮食测定的血清SCN水平可能调节了EPO的毒性。我们拟议工作的总体目标是检验这样一种假设，即EPO产生的氧化剂以底物决定的方式对哺乳动物细胞和组织造成损害，从而导致EHD的发病机制，我们建议将其作为发生在嗜酸性粒细胞炎症状态下的任何器官损害的范例。第一个特定的目的是验证这样的假设，即HOBR和N02通过与膜成分反应破坏膜完整性而导致坏死性细胞死亡，而HOSCN施加硫基靶向的细胞内氧化应激，可以影响组织因子基因的表达并诱导细胞凋亡。第二个具体目的是验证这样的假设，即白介素5(IL.5)转基因小鼠品系将发展为进行性的功能性、解剖学和组织学表现，并同时积累EPO特异性蛋白氧化损伤，正如新开发的敏感的、底物特异的氨基酸“生物标记物”所评估的那样。第三个具体目的是在IL.5转基因小鼠身上验证EPO参与EHD发病机制的假设。我们将比较IL-5转基因品系和IL-S转基因品系与EPO基因敲除品系杂交后的EHD严重程度、寿命和EPO生物标志物水平。第四个具体目的是在IL-5转基因小鼠中验证增加血清SCN-抑制和减少SCN-促进、EPO介导的蛋白质氧化损伤和EHD严重程度的假说。如果成功，这些研究将建立HES和EHD的小鼠模型，证明EPO介导的氧化剂损伤在EHD的发病机制中起到摩尔作用，并提出一种简单的治疗策略(即，在饮食中补充廉价的SCN-)，即使在贫困社区也可以应用于更常见的过敏性疾病，如哮喘。

DESCRIPTION (provided by applicant): Eosinophi1 (EO) phagocytes destroy parasites but can also damage host tissue. Although allergies are the most common example of EO-mediated pathology, the hypereosinophilic Syndrome (HES) is the most striking. HES is a systemic hematologic disorder characterized by multi-organ system involvement, preeminently a characteristic, usually lethal form of heart disease. Eosinophilic heart disease (EHD occurs in a variety of hypereosinophilic states, irrespective of their cause. Endocardial and myocardial deposition of EO specific granule proteins, of which eosinophil peroxidase (EPO) is the most abundant, is proposed to mediate cardiotoxicity in EHD. Despite the abundance of EPO and the vigor of the EO respiratory burst, little is known about the contribution of EPO-mediated oxidative damage to the pathology of eosinophilic inflammatory states. We find that three unusual substrates - bromide (Br-), nitrite (NO2-), and thiocyanate (SCN-) - compete for oxidation by EPO in physiologic fluids in the presence of H202, yielding, respectively, HOBr, NO2., and HOSCN. The relative toxicity of these oxidants for human cells is HOBr> NO2>> HOSCN; yet EPO preferentially oxidizes SCN > NO2 - > Br-. We hypothesize that SCN "buffers" against generation by EPO of the more cytotoxic N02- and Br- based oxidants and consequently serum SCN- levels, which are dietarily determined, may modulate EPO toxicity. The overall goal of our proposed work is to examine the hypothesis that that EPO-generated oxidants impose damage in a substrate-determined manner to mammalian cells and tissue that contributes to the pathogenesis of EHD, which we propose as a paradigm for any organ damage occurring in eosinophil inflammatory states. The first specific aim is to test the hypothesis that whereas HOBr and N02 cause necrotic cell death by reacting with membrane components to destroy membrane integrity, HOSCN imposes sulfhydryl-targeted intracellular oxidative stress that can influence tissue factor gene expression and induce apoptosis. The second specific Aim is to test the hypothesis that an interleukin-5 (IL.5) transgenic mouse line will develop progressive functional, anatomic, and histologic manifestations of EHD with a parallel accumulation of EPO-specific protein oxidative damage as assessed by newly developed sensitive, substrate-specific amino acid "biomarkers." The third specific aim is to test the hypothesis that EPO contributes to the pathogenesis of EHD in the IL.5 transgenic mouse line. We will compare EHD severity, longevity, and EPO biomarker levels in the IL-5 transgenic line with that of an IL-S transgenics crossbred with an EPO 'knockout" line. The fourth specific aim is test the hypothesis that increasing serum SCN- inhibits, and decreasing SCN- promotes, EPO-mediated protein oxidant damage and the severity of EHD in IL-5 transgenic mice. These studies, if successful, will establish a murine model for HES and EHD, prove a mole for EPO-mediated oxidant damage in the pathogenesis of EHD, and suggest a simple strategy fur its treatment, (i.e., dietary supplementation with inexpensive SCN-), that could be applied even in impoverished communities as well as against more common allergic diseases, such as asthma.