Proteomics of eosinophil activation

摘要 这项研究的目的是获得新的见解，嗜酸性粒细胞如何影响哮喘和其他嗜酸性粒细胞- 相关疾病和过程，通过描述非激活的蛋白质组和急性和慢性 活化的人嗜酸性粒细胞嗜酸性粒细胞在许多方面是例外的：（1）颗粒含量，（2） 受体和其他控制激活和运输的分子的补体，（3） 介体生成酶，和（4）在被细胞因子激活后极化成颗粒隔室 和核足类，一种特殊的尾足类，由嗜酸性粒细胞的独特的双叶核占据，并被一个 信号体深入的蛋白质组学分析将导致对细胞范围内过程的理解增加，例如 极化和颗粒释放，产生关于疾病中改变的途径的假设，并告知 有针对性的研究。这种研究是可行的。未活化的嗜酸性粒细胞可以从血液中可重复地纯化， 并用激动剂如白细胞介素-5（IL-5）急性激活。从支气管肺泡中纯化的嗜酸性粒细胞 在节段性抗原攻击后获得灌洗液提供了表征 已经进入并持续存在于组织环境中的慢性活化细胞，所述组织环境富含多种介体， 嗜酸性炎症我们将鉴定和定量非活化的和急性的蛋白质， 慢性激活的嗜酸性粒细胞，精确定位磷酸化位点，并进行显微镜免疫检测， 定位和其他相关研究，以验证蛋白质组学结果，并寻求建议的线索 by the data数据.在初步结果的基础上提出了一些倡议， IL-5激活的嗜酸性粒细胞中形成的信号体与研究充分的 通过B细胞受体活化的B细胞;表征嗜酸性粒细胞Nance-Horan综合征样蛋白2（NHSL2）， 其在初步研究中是急性IL 5刺激后最严重磷酸化的蛋白质之一; 并表征和定位嗜酸性粒细胞IL 18，其以出乎意料的高丰度被发现。 了解蛋白质的动力学和翻译后修饰在嗜酸性粒细胞活化是没有 这是一个系统地了解人类嗜酸性粒细胞如何在细胞内停滞，浸润， 并扰乱哮喘和其他疾病的组织。这项工作将导致识别细胞靶点， 从而调节嗜酸性粒细胞的激活，控制炎症并导致愈合。展望未来， 蛋白质组学技术，我们将建立以实现特定的目标，可以用来研究 正常人与嗜酸性粒细胞相关性贫血患者嗜酸性粒细胞蛋白质含量的差异 疾病和嗜酸性粒细胞对激活的激动剂和拮抗剂的反应性。

ABSTRACT The goal of this research is to gain new insights in how eosinophils impact asthma and other eosinophil- associated diseases and processes by describing the proteomes of non-activated and acutely and chronically activated human eosinophils. Eosinophils are exceptional in a number of ways: (1) granule content, (2) complement of receptors and other molecules that control activation and trafficking, (3) complement of mediator-generating enzymes, and (4) polarization upon activation by cytokines into a granular compartment and nucleopod, a specialized uropod occupied by the eosinophil's distinctive bilobed nucleus and capped by a signalosome. Deep proteomic analysis will lead to increased understanding of cell-wide processes such as polarization and granule release, generate hypotheses about pathways that are altered in disease, and inform targeted studies. Such studies are feasible. Non-activated eosinophils can be purified reproducibly from blood, and activated acutely with agonists such as interleukin-5 (IL5). Eosinophils purified from broncho-alveolar lavage fluid obtained after segmental antigen challenge provide a unique opportunity to characterize chronically activated cells that have entered and persisted in a tissue milieu enriched in multiple mediators of eosinophilic inflammation. We will identify and quantify the proteins of non-activated and acutely and chronically activated eosinophils, pinpoint sites of phosphorylation, and perform microscopic immuno- localization and other correlative studies to validate the proteomic results and pursue leads that are suggested by the data. Initiatives are proposed based on preliminary results to extend the observation that the signalosome forming in eosinophils activated with IL5 shares components with the well-studied signalosome of B-cells activated via the B-cell receptor; characterize eosinophil Nance-Horan syndrome-like protein 2 (NHSL2), which in preliminary studies was among the proteins most heavily phosphorylated after acute IL5 stimulation; and characterize and localize eosinophil IL18, which was found in unexpectedly high abundance. Understanding dynamics of proteins and post-translational modifications during eosinophil activation is without question necessary to developing a systematic understanding of how human eosinophils arrest in, infiltrate, and perturb tissues in asthma and other diseases. This work will lead to identification of cellular targets with which to modulate eosinophil activation in ways that control inflammation and lead to healing. Going forward, the proteomic techniques that we will establish to accomplish the specific aims can be used to study differences in protein content of eosinophils between normal and individuals with eosinophil-associated diseases and responsiveness of eosinophils to agonists and antagonists of activation.