Role of DPPI & Serine Proteases in Inflammatory Diseases

炎症过程中中性粒细胞的迁移是一个高度调控的过程，对宿主防御入侵生物至关重要。然而，中性粒细胞的过度聚集和激活可能会导致破坏性的影响。中性粒细胞丝氨酸蛋白酶[中性粒细胞弹性蛋白酶(NE)、组织蛋白酶G(CG)、蛋白酶3(PR3)]在成熟的中性粒细胞中特异表达。我们最近创造了一只溶酶体半胱氨酸蛋白酶二肽基肽酶I(DPPI)零突变的小鼠。我们发现DPPI是许多丝氨酸蛋白酶的加工和激活所必需的，包括NE、CG和PR3。此外，我们的初步结果表明，DPPI缺陷和NE x CG缺陷的小鼠对被动转移单抗到II型胶原诱导的急性关节炎具有保护作用。具体地说，在蛋白酶缺陷小鼠的关节中没有中性粒细胞的积累。这些结果支持DPPI(可能通过NE和CG的作用)在特定炎症部位的中性粒细胞迁移和/或中性粒细胞募集中发挥非多余作用的假说。因此，我们提出以下具体目标：1.我们将确定DPPI和丝氨酸蛋白酶在中性粒细胞迁移中的作用(S)。中性粒细胞的迁移需要多个黏附分子的激活和相互作用。在这个特定的目标中，我们将检测黏附分子L-选择素和β2整合素在内毒素激活后的表达。我们还将在体外检测中性粒细胞黏附和迁移过程中对DPPI和丝氨酸蛋白酶的需求。2.明确DPPI和丝氨酸蛋白酶在急性关节炎模型中的体内作用(S)。我们的初步结果表明，炎症部位的中性粒细胞存在原发的移行缺陷或继发的中性粒细胞激活和募集缺陷。在这个目标中，我们将表征DPPI缺陷小鼠对关节内IL-8的直接反应。我们还将通过骨髓移植建立嵌合小鼠，以确定炎症部位野生型中性粒细胞的积累和激活是否足以招募DPPI缺陷的中性粒细胞。3.我们将探讨(S)中性粒细胞衍生丝氨酸蛋白酶在其他急性和慢性炎症模型中的作用。为此，我们将确定中性粒细胞迁移/募集的缺陷是否适用于其他器官，如皮肤。此外，我们还将研究另外两种关节炎模型，一种是感染性关节炎，另一种是慢性炎症。

Neutrophil emigration during inflammation is a highly regulated process and is critical for host defense against invading organisms. However, excessive accumulation and activation of neutrophils can lead to damaging effects. Neutrophil serine proteases [neutrophil elastase (NE), cathepsin G (CG), proteinase 3 (PR3) are expressed specifically in mature neutrophils. We have recently created a mouse with a null mutation in the lysosomal cysteine protease dipeptidyl peptidase I (DPPI). We showed that DPPI is required for the processing and activation of many serine proteases including NE, CG, and PR3. Furthermore, our preliminary results indicate that the DPPI- deficient and NE x CG-deficient mice are protected against acute arthritis induced by passive transfer of monoclonal antibodies to type II Collagen. Specifically, there is no accumulation of neutrophils in the joints of protease-deficient mice. These results support the hypothesis that DPPI (probably through the action of NE and CG) plays a non-redundant role in neutrophil emigration and/or neutrophil recruitment at specific inflammatory sites. Thus, we propose the following specific aims: 1. We will define the role(s) of DPPI and serine proteases in neutrophil migration. Neutrophil migration requires the activation and interaction of several adhesion molecules. In this specific aim, we will examine the expression of adhesion molecules L-selectin and beta2 integrins upon activation with LPS. We will also examine the requirement for DPPI and serine proteases in neutrophil adhesion and transmigration in vitro. 2. We will define the in vivo role(s) of DPPI and serine proteases in the model of acute arthritis. Our preliminary results suggest that there is either a primary defect in neutrophil emigration or a secondary defect in neutrophil activation and recruitment at sites of inflammation. In this aim, we will characterize the direct response of DPPI-deficient mice to intra-articular IL-8. We will also establish chimeric mice by bone marrow transplantation to determine whether accumulation and activation of wild type neutrophils at sites of inflammation will be sufficient to recruit DPPI-deficient neutrophils. 3. We will explore the role(s) of neutrophil-derived serine proteases in other models of acute and chronic inflammation. In this aim, we will determine whether the defect in neutrophil emigration/recruitment is generalized to other organs, such as skin. In addition, we will also study two additional arthritis models, one of septic arthritis and the other involving chronic inflammation.