MECHANISMS OF NOVEL ANTIINFLAMMATORY ACTIONS OF SLPI

SLPI 的新型抗炎作用机制

• 批准号: 6166901
• 负责人: Aihao Ding
• 金额: $ 33.9万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6166901
• 关键词: antiinflammatory agents; binding proteins; biological signal transduction; crosslink; genetically modified animals; human tissue; interleukin 1; laboratory mouse; lipopolysaccharides; macrophage; membrane proteins; mutant; neutrophil; nitric oxide; pathologic process; protease inhibitor; protein structure function; recombinant proteins; septic shock; teichoate; tissue /cell culture; transfection; tumor necrosis factor alpha; vascular endothelium; yeast two hybrid system

Secretory leukocyte protease inhibitor (SLPI) is a potent inhibitor of serine protease from neutrophils that is secreted by epithelial cells. We recently cloned mouse SLPI and discovered that this 12 kDa protein is also a product of neutrophils and macrophages and a component of human blood. SLPI can be induced locally as well as systemically in response to the microbial products lipopolysaccharide (LPS) and lipoteichoic acid (LTA) from gram-negative and gram-positive bacteria, respectively. Expression of SLPI in mouse macrophages induces a hyporesponsive phenotype to both LPS and LTA. Moreover, we found that recombinant human SLPI suppresses the ability of human endothelial cells and neutrophils to respond to TNF by induction of adhesion molecules and secretion of reactive oxygen intermediates (ROI), respectively. Thus, SLPI has at least three anti-inflammatory functions: (i) inhibition of tissue-degrading neutrophil serine proteases; (ii) suppression of the production of inflammatory mediators by LPS- and LTA-stimulated mononuclear phagocytes; and (iii) suppression of inflammatory actions of TNF on endothelial cells and leukocytes. The goal of this project is to explore the mechanisms of the novel anti-inflammatory actions of SLPI: (ii) and (iii) above. The working hypothesis is that SLPI modulates inflammatory responses of macrophages, neutrophils and endothelial cells via a protease-independent mechanism, possibly mediated by its N-terminal domain. To test this hypothesis, we will focus on: (1) Characterization of the structural requirements for SLPI's novel anti-inflammatory effects by comparing the antiinflammatory activity of wild type SLPI, its anti-protease deficient mutants and its truncated forms (N-terminal or C-terminal half SLPI) using recombinant proteins and stably transfected cells expressing these proteins; (2) Identification of the molecular mechanisms involved in SLPI's anti-inflammatory functions by searching for specific molecules in Toll like receptor-signaling pathway affected by SLPI or specific protein targets associated with SLPI using surface crosslinking and yeast two hybrid screen; and (3) Testing the ability of exogenous and endogenous SLPI to protect mice in a endotoxic shock model using SLPI transgenic mice and administration of recombinant SLPI to septic mice. Elucidating how SLPI interferes with specific steps in LPS and LTA signaling may contribute important insights to the broader puzzle of the pathobiology of septic shock and in the development of novel strategies in its therapy.

分泌性白细胞蛋白酶抑制剂（SLPI）是一种由上皮细胞分泌的中性粒细胞丝氨酸蛋白酶的有效抑制剂。我们最近克隆了小鼠SLPI，发现这个12kda的蛋白也是中性粒细胞和巨噬细胞的产物，也是人类血液的一个组成部分。在革兰氏阴性菌和革兰氏阳性菌的微生物产物脂多糖（LPS）和脂壁酸（LTA）的作用下，SLPI可以局部诱导，也可以全身诱导。SLPI在小鼠巨噬细胞中的表达诱导了对LPS和LTA的低反应表型。此外，我们发现重组人SLPI分别通过诱导粘附分子和分泌活性氧中间体（ROI）来抑制人内皮细胞和中性粒细胞对TNF的反应能力。因此，SLPI至少具有三种抗炎功能：(i)抑制组织降解中性粒细胞丝氨酸蛋白酶；（ii） LPS和lta刺激的单核吞噬细胞抑制炎症介质的产生；（iii）抑制TNF对内皮细胞和白细胞的炎症作用。本项目的目标是探索SLPI的新型抗炎作用机制：（ii）和（iii）以上。工作假设是SLPI通过蛋白酶不依赖的机制调节巨噬细胞、中性粒细胞和内皮细胞的炎症反应，可能是由其n端结构域介导的。为了验证这一假设，我们将重点关注：(1)通过使用重组蛋白和稳定转染表达这些蛋白的细胞，比较野生型SLPI、其抗蛋白酶缺陷突变体及其截断形式（n端或c端一半SLPI）的抗炎活性，来表征SLPI新型抗炎作用的结构要求；(2)利用表面交联和酵母双杂交筛选，寻找SLPI影响的Toll样受体-信号通路中的特异性分子或SLPI相关的特异性蛋白靶点，确定SLPI抗炎作用的分子机制；(3)利用SLPI转基因小鼠和重组SLPI给毒小鼠，检测外源性和内源性SLPI对内源性休克模型小鼠的保护能力。阐明SLPI如何干扰LPS和LTA信号传导的特定步骤，可能有助于对脓毒性休克的病理生物学更广泛的困惑和开发新的治疗策略提供重要的见解。