Towards a Molecular Signature of Neutrophil Priming

描述(由申请人提供)：中性粒细胞启动是调节良好的炎症反应的一个重要方面，也是控制微生物感染和修复损伤的第一步。在这个模型中，最初的损伤会“激发”中性粒细胞，这样的二次伤害本来会在未启动的细胞中缓和，但现在会导致夸大的炎症反应。细胞因子和有毒氧自由基的过度和无节制释放会对邻近组织造成损害，导致组织功能障碍、器官衰竭和死亡。启动人类中性粒细胞启动的确切分子途径还知之甚少。本提案侧重于应用定量蛋白组学技术来了解与低剂量N-甲酰-甲基-亮氨酸-苯丙氨酸、脂多糖或血小板激活因子启动的人中性粒细胞相关的独特的蛋白质组范围的酪氨酸磷酸化事件。利用目前可用的定量磷酸蛋白质组学技术和我们实验室中的专业知识，我们将在不同的时间点对启动的中性粒细胞进行采样，并从细胞总多肽的细胞裂解产物中量化大规模酪氨酸磷酸化的相对变化。该项目将提供的信息包括：中性粒细胞启动后随时间发生的酪氨酸磷酸化事件的全球识别；启动的中性粒细胞中激活的信号转导通路网络的可视化；不同促炎介质启动的中性粒细胞随时间发生的激活事件的比较。这种对中性粒细胞启动过程中全球酪氨酸磷酸化位点的定量分析，结合传统的生化分析，将使构建中性粒细胞启动的信号通路网络模型成为可能。总而言之，这些发现将检验这样的假设，即中性粒细胞的启动状态包括在促炎介质中常见的信号事件，并可作为这一基本细胞反应的分子标志。鉴于有反应的中性粒细胞必须解释炎症部位的许多信号，这些研究的结果可能有助于开发新的诊断或治疗试剂，旨在限制患者因脓毒症和创伤而遭受的自体炎症组织损害。公共卫生相关性：中性粒细胞启动是调控良好的炎症反应的一个重要方面，也是控制微生物感染和修复损伤的第一步。启动人类中性粒细胞启动的确切分子途径还知之甚少。在这项提案中，我们引入了定量质谱学的现代方法，通过提供正常和启动的细胞的磷酸化状态的全局视图，有助于表征由各种试剂启动的中性粒细胞启动的信号通路。

DESCRIPTION (provided by applicant): Neutrophil priming is an integral aspect of a well-regulated inflammatory response and is the first step in controlling microbial infection and repair of injury. In this model, an initial injury "primes" neutrophils such that a secondary insult, which would otherwise be tempered in an unprimed cell, now results in an exaggerated inflammatory response. The excessive and unregulated release of cytokines and toxic oxygen radicals causes damage to adjacent tissues leading to tissue dysfunction, organ failure and death. The precise molecular pathways initiated upon priming of human neutrophils are poorly understood. The present proposal focuses on the application of quantitative phosphoproteomic technology to understand the unique proteome-wide tyrosine phosphorylation events associated with human neutrophil priming by exposure to low dose N-formyl-met-leu-phe, lipopolysaccharide or platelet activating factor. Using currently available quantitative phosphoproteomic technology and expertise with primary human neutrophils in our laboratories, we will sample primed neutrophils at various time points and quantify relative changes in wide-scale tyrosine phosphorylation from total cell lysates of cellular peptides. Information to be derived from this project include: global identification of tyrosine phosphorylation events that occur over time after neutrophil priming; visualization of the network of signal transduction pathways that are activated in the primed neutrophils; comparison of activation events that take place over time from neutrophils primed with different proinflammatory mediators. This type of quantitative analysis of global tyrosine phosphorylation sites during a time course of neutrophil priming along with traditional biochemical analysis will permit the construction of a signaling pathway network model for neutrophil priming. Together these findings will test the hypothesis that the primed state of the neutrophil includes signaling events that are common among proinflammatory mediators and can be presented as a molecular signature of this fundamental cellular response. Given that responding neutrophils must interpret a number of signals at a site of inflammation, the results from these studies may assist in the development of novel diagnostic or therapeutic reagents aimed at limiting the auto-inflammatory tissue damage patients suffer as a result of sepsis and trauma. PUBLIC HEALTH RELEVANCE: Neutrophil priming is an integral aspect of a well-regulated inflammatory response and is the first step in controlling microbial infection and repair of injury. The precise molecular pathways initiated upon priming of human neutrophils are poorly understood. In this proposal we introduce modern methods in quantitative mass spectrometry to facilitate the characterization of the cellular signaling pathways initiated upon neutrophil priming by a variety of agents by providing a global view of the phosphorylation state of normal and primed cells.