ROLE OF DPPI & SERINE PROTEASES IN INFLAMMATORY DISEASES

描述（由申请人提供）：本项目的长期目标是充分表征中性粒细胞丝氨酸蛋白酶调节炎症反应的机制。我们希望从这些研究中获得的信息可以用于制定策略，以抑制这些蛋白酶在炎症性疾病中的活性，同时保持它们杀死入侵病原体的能力。在过去的几年中，我们已经了解到，中性粒细胞丝氨酸蛋白酶不仅是降解酶，还可以通过调节细胞因子和趋化因子的释放以及激活特异性受体来充当炎症的特异性调节剂。然而，这些蛋白酶发挥这些调节作用的确切机制仍然未知。为了进一步在体外和体内表征这些调控机制，我们提出了以下目标：1。我们将定义细胞表面结合的组织蛋白酶G（CG）调节中性粒细胞效应子功能的机制。我们的数据表明，细胞外CG切割尚未鉴定的分子（或分子），这种蛋白水解修饰导致细胞骨架重组，细胞扩散和效应功能。我们已经确定了两个候选蛋白作为CG的潜在底物，syndecan-4和CD 43。在这个目标中，我们将确定CG是否直接蛋白水解syndecan-4和CD 43，以及这种酶修饰是否对CG依赖的中性粒细胞效应子功能至关重要。2.我们将产生蛋白酶3（PR 3）的功能丧失突变模型，以确定其在细胞因子产生中的作用及其对体内炎症的贡献。我们的初步数据表明，在几种炎症模型中，PR 3在促炎细胞因子和趋化因子的局部产生或加工中起重要作用。为了明确研究PR 3在体内炎症中的作用，我们建议在PR 3中产生功能缺失突变。我们将充分表征PR 3缺陷小鼠，并使用这些突变小鼠进行体外试验和体内模型，以确定PR 3的生理作用。3.我们将建立抗中性粒细胞胞浆抗体（ANCA）介导的炎症的小鼠模型，并确定决定疾病发展的因素。ANCA与几种小血管炎有关，包括韦格纳肉芽肿病。在90%的韦格纳氏病中，ANCA是针对PR 3的，尽管也发现了针对其他丝氨酸蛋白酶的ANCA。我们还推测，肾脏中补体调节因子表达的降低可能是影响靶器官疾病严重程度的决定因素。

DESCRIPTION (provided by applicant): The long-term goal of this project is to fully characterize the mechanisms by which neutrophil serine proteases regulate the inflammatory response. We hope that information gained from these studies can be used to develop strategies to inhibit the activity of these proteases in inflammatory diseases while preserving their ability to kill invading pathogens. Over the past several years, we have learned that, more than being degradative enzymes, neutrophil serine proteases can act as specific regulators of inflammation by modulating the release of cytokines and chemokines as well as activating specific receptors. Yet, the exact mechanisms by which these proteases exert these regulatory effects are still unknown. To further characterize these regulatory mechanisms in vitro and in vivo, we propose the following aims: 1. We will define the mechanisms by which cell-surface-bound cathepsin G (CG) modulates neutrophil effector functions. Our data indicate that extracellular CG cleaves a yet-unidentified molecule (or molecules) and this proteolytic modification leads to cytoskeleton reorganization, cell spreading, and effector functions. We have identified two candidate proteins as potential substrates for CG, syndecan-4 and CD43. In this aim, we will determine whether CG directly proteolyses syndecan-4 and CD43 and whether this enzymatic modification is critical for CG-dependent neutrophil effector functions. 2. We will generate a loss-of-function mutation model for proteinase 3 (PR3) to define its role in cytokine production and its contribution to inflammation in vivo. Our preliminary data suggest that in several inflammatory models, PR3 plays an important role in the local production or processing of pro- inflammatory cytokines and chemokines. To definitively study the role of PR3 in inflammation in vivo, we propose to generate a loss-of-function mutation in PR3. We will fully characterize the PR3-deficient mice and use these mutant mice for in vitro assays and in vivo models to define the physiologic role of PR3. 3. We will generate a murine model of anti-neutrophil cytoplasmic antibody (ANCA)-mediated inflammation and determine the factors that dictate disease development. ANCAs are associated with several small vessel vasculitides, including Wegener's granulomatosis. In 90% of Wegener's, ANCAs are directed against PR3, although ANCAs specific for other serine proteases are also found. We propose to determine whether all ANCAs are potentially pathogenic. We also hypothesize that decreased expression of complement regulators in the kidney may be a determinant that influences disease severity in target organ.