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。蛋白质是超氧化物(O)和一氧化氮(NO)反应的主要靶标。在这些应激反应下，蛋白质发生修饰，如S亚硝化(SNO)、羰化和3-硝基酪氨酸(3NT)，并已被证明通过改变蛋白质功能而在生理和病理上发挥作用。肺集合素、表面活性蛋白(SP)-A和SP-D对肺的免疫功能有调节作用，而SP-A的硝化作用使其功能丧失。初步数据表明，在博莱霉素诱导的大鼠肺损伤后，SNO和3NT的定位和表达存在差异。此外，我们还记录了损伤肺中灌洗液中透明质酸(HA)的急剧增加，以及HA结合肽治疗限制了损伤后的炎症和纤维化反应。有趣的是，活性氧和氮物种(RONS)将高分子量(HMVV)HA分解成低分子量(LMW)形式，促进巨噬细胞激活、细胞因子基因表达和趋化。在体外和体内，HA受体CD44和RHAMM都与这些炎症反应有关。初步数据表明，抗rhamm抗体阻断了SP-A介导的巨噬细胞趋化作用，并且SP和HA信号传导需要膜脂筏。利用啮齿动物气管内博莱霉素肺损伤模型，我们将检验这一假说，即肺损伤导致的RONS既产生GAG加合物，又产生翻译后的特定蛋白靶标，共同调节巨噬细胞激活、炎症细胞因子基因表达和趋化作用，从而促进肺部炎症。使用药理学方法(化学阻滞剂)和转基因方法(SP-ND敲除)，目标1将确定RONS在SPND的翻译后修饰、低分子HA的形成和博莱霉素损伤后的炎症中的作用。此外，使用特定的抗体和多肽阻滞剂，AIM 2将重点确定HA、CD44和RHAMM在天然和硝化SP-ND体外调节巨噬细胞功能中的作用。在目标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单位的形成来进一步研究巨噬细胞功能改变的机制。然后将测试特定脂筏阻滞剂的治疗干预在阻止肺损伤后炎症方面的效果。本方案中描述的实验将确定肺集合素和低分子HA调节巨噬细胞功能的分子机制，并将探索新的治疗干预靶点，以限制肺损伤的炎症反应。

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.