MECHANISMS OF NOVEL ANTIINFLAMMATORY ACTIONS OF SLPI

分泌性白细胞蛋白水解酶抑制物(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物。我们最近克隆了小鼠SLPI，发现这个12 kDa的蛋白也是中性粒细胞和巨噬细胞的产物，也是人类血液的一种成分。革兰氏阴性菌和革兰氏阳性菌的微生物产物脂多糖(LPS)和脂磷壁酸(LTA)分别可局部和系统地诱导SLPI。SLPI在小鼠巨噬细胞中的表达诱导了对脂多糖和LTA的低反应表型。此外，我们还发现，重组人SLPI分别通过诱导黏附分子和分泌活性氧中间产物(ROI)来抑制人内皮细胞和中性粒细胞对肿瘤坏死因子的反应能力。因此，SLPI至少有三个抗炎作用：(1)抑制组织降解性中性粒细胞丝氨酸蛋白酶；(2)抑制内毒素和LTA刺激的单核巨噬细胞产生炎症介质；(3)抑制肿瘤坏死因子对内皮细胞和白细胞的炎症作用。本项目的目标是探索SLPI新的抗炎作用的机制：(Ii)和(Iii)。工作假说是SLPI通过一种不依赖于蛋白酶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-末端结构域介导的。为了验证这一假说，我们将集中在：(1)利用重组蛋白和稳定表达这些蛋白的细胞，通过比较野生型SLPI、其抗蛋白酶缺陷突变体及其截短形式(N端或C端半SLPI)的抗炎活性，表征SLPI新的抗炎作用的结构要求；(2)通过表面交联和酵母双杂交筛选，通过寻找SLPI影响的Toll样受体信号通路中的特定分子或与SLPI相关的特定蛋白靶点，鉴定SLPI的抗炎作用的分子机制；(3)用SLPI转基因小鼠检测外源性和内源性SLPI对内毒素休克小鼠的保护作用，并给脓毒症小鼠注射重组SLPI。阐明SLPI如何干扰内毒素和LTA信号的特定步骤，可能有助于对感染性休克的病理生物学更广泛的谜团以及在其治疗中开发新的策略。

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.