Pulmonary Collectins, Hyaluronan and Macrophages

描述（由申请人提供）：氧化和硝化应激被认为是肺损伤和随后诱导炎症反应的关键介质。蛋白质构成超氧化物（O）和一氧化氮（NO）反应性的主要靶标。在暴露于这些反应性应激下，发生蛋白质修饰，如S-亚硝基化（SNO）、羰基化和3-硝基酪氨酸（3 NT），并且已显示通过改变蛋白质功能在生理学和病理学上起作用。肺凝集素、表面活性蛋白（SP）-A和SP-D调节肺中的免疫功能，并且SP-A的硝化消除其功能。初步数据表明，在啮齿动物中博来霉素诱导的肺损伤后，SNO和3 NT的差异定位和表达。此外，我们已经记录了在损伤的肺中灌洗液透明质酸（HA）的显著增加，并且HA结合肽治疗限制了对损伤的炎症和纤维化反应。有趣的是，活性氧和氮物质（RONS）将高分子量（HMVV）HA片段化为低分子量（LMW）形式，其促进巨噬细胞活化、细胞因子基因表达和趋化性。HA受体CD 44和RHAMM在体外和体内均与这些炎症反应有关。初步数据表明，抗RHAMM抗体阻断SP-A介导的巨噬细胞趋化性和膜脂筏所需的SP和HA信号。使用啮齿类动物肺损伤的气管内博莱霉素模型，我们将检验以下假设：由于肺损伤而发生的RONS产生GAG加合物和特异性蛋白靶的翻译后修饰，其共同调节巨噬细胞活化、炎性细胞因子基因表达和趋化性，从而促进肺部炎症。使用药理学（化学阻断剂）和转基因（SP-ND敲除）方法，目的1将确定RONS对SPND的翻译后修饰、LMW HA的形成和博来霉素损伤后的炎症的贡献。此外，使用特异性抗体和肽阻断剂，Aim 2将集中于确定HA，CD 44和RHAMM在天然和硝化SP-ND体外调节巨噬细胞功能中的作用。在目的3中，将通过确定调节SP-ND和LMW HA介导的巨噬细胞行为的脂筏相关信号传导单元的形成来进一步检查改变的巨噬细胞功能的机制。然后将测试使用脂筏特异性阻断剂的治疗干预在阻断肺损伤后炎症中的功效。本提案中描述的实验将定义肺聚集蛋白和LMW HA调节巨噬细胞功能的分子机制，并将探索治疗干预的新靶点，以限制对肺损伤的炎症反应。

DESCRIPTION (provided by applicant): Oxidative and nitrative stresses are thought to be critical mediators of lung injury and subsequent induction of inflammatory responses. Proteins constitute a major target of superoxide (O) and nitric oxide (NO) reactivity. Under exposure to these reactive stresses, protein modifications such as S-nitrosylation (SNO), carbonylation and 3-nitrotyrosine (3NT) occur and have been shown to act both physiologically and pathologically by altering protein function. The pulmonary collectins, Surfactant Proteins (SP)-A and SP-D modulate immune functions in the lung and nitration of SP-A abrogates its functions. Preliminary data indicate differential localization and expression of SNO and 3NT after bleomycin-induced lung injury in rodents. In addition, we have documented dramatic increases in lavage hyaluronic acid (HA) in injured lungs and HA-binding peptide treatment limits the inflammatory and fibrotic response to injury. Interestingly, reactive oxygen and nitrogen species (RONS) fragment high molecular weight (HMVV) HA into low molecular weight (LMW) forms that promote macrophage activation, cytokine gene expression and chemotaxis. The HA receptors CD44 and RHAMM have been implicated in these inflammatory responses both in vitro and in vivo. Preliminary data indicate that anti-RHAMM antibody blocks SP-A-mediated macrophage chemotaxis and that membrane lipid rafts are required for SP and HA signaling. Using the rodent intratracheal bleomycin model of lung injury, we will test the hypothesis that RONS, occurring as a result of lung injury, generate both GAG adducts and the post.translational modification of specific protein targets that collectively regulate macrophage activation, inflammatory cytokine gene expression and chemotaxis so as to promote pulmonary inflammation. Using both pharmacologic (chemical blockers) and transgenic (SP-ND knockouts) approaches, Aim 1 will determine the contribution of RONS to post-translational modifications of SPND, formation of LMW HA and inflammation after bleomycin injury. In addition, using specific antibody and peptide blockers, Aim 2 will focus on defining the roles of HA, CD44 and RHAMM in the regulation of macrophage functions by native and nitrated SP-ND in vitro. In Aim 3, the mechanisms of altered macrophage function will be further examined by determining the formation of a lipid raft-associated signaling unit that regulates SP-ND and LMW HA-mediated macrophage behavior. Therapeutic intervention with specific blockers of lipid rafts will then be tested for their efficacy in blocking inflammation after lung injury. The experiments described in this proposal will define the molecular mechanisms of pulmonary collectin and LMW HA regulation of macrophage function and will explore novel targets for therapeutic intervention to limit the inflammatory response to lung injury.