MECHANISMS OF NOVEL ANTIINFLAMMATORY ACTIONS OF SLPI

分泌性白细胞蛋白酶抑制剂（SLPI）是一种有效的丝氨酸蛋白酶抑制剂，由上皮细胞分泌的中性粒细胞。 我们最近克隆了小鼠SLPI，发现这种12 kDa的蛋白质也是中性粒细胞和巨噬细胞的产物，也是人类血液的组成部分。 SLPI可以被局部以及全身诱导，以响应分别来自革兰氏阴性和革兰氏阳性细菌的微生物产物脂多糖（LPS）和脂磷壁酸（LTA）。SLPI在小鼠巨噬细胞中的表达诱导对LPS和LTA的低应答表型。 此外，我们发现，重组人SLPI抑制人内皮细胞和中性粒细胞的能力，分别通过诱导粘附分子和活性氧中间体（ROI）的分泌，以响应TNF。 因此，SLPI具有至少三种抗炎功能：（i）抑制组织降解嗜中性粒细胞丝氨酸蛋白酶;（ii）抑制LPS和LTA刺激的单核吞噬细胞产生炎症介质;和（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的新抗炎作用的机制：（ii）和（iii）上述。 工作假设是SLPI通过蛋白酶非依赖性机制调节巨噬细胞、中性粒细胞和内皮细胞的炎症反应，可能由其N-末端结构域介导。 为了验证这一假设，我们将集中在：（1）通过比较野生型SLPI，其抗蛋白酶缺陷突变体及其截短形式的抗炎活性，表征SLPI新抗炎作用的结构要求（N-末端或C-末端半SLPI），使用重组蛋白和表达这些蛋白的稳定转染细胞;（2）通过表面交联和酵母双杂交筛选SLPI影响Toll样受体信号通路的特异分子或SLPI相关的特异蛋白靶点，探讨SLPI抗炎作用的分子机制;和（3）在使用SLPI转基因小鼠的内毒素休克模型中测试外源性和内源性SLPI保护小鼠的能力，并将重组SLPI施用于脓毒症小鼠。 阐明SLPI如何干扰LPS和LTA信号传导中的特定步骤，可能有助于对脓毒性休克病理生物学的更广泛的困惑及其治疗新策略的发展提供重要的见解。

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.