Neutrophil Trafficking to Normal and Inflamed Lung

中性粒细胞转运至正常肺和发炎肺

• 批准号: 7232630
• 负责人: BRIAN R DULING
• 金额: $ 18.03万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7232630
• 关键词: CD antigens; bioterrorism /chemical warfare; cell migration; diagnostic respiratory lavage; electron microscopy; flow cytometry; genetically modified animals; immunocytochemistry; in situ hybridization; inflammation; integrins; interstitial; laboratory mouse; leukocyte adhesion molecules; lung; lung injury; molecular pathology; myocardium disorder; neutrophil; pathologic process; pneumonia; respiratory circulation; selectins; septicemia

Neutrophil trafficking to normal and inflamed lung. Neutrophil recruitment to the lung is of critical importance for various pathologies including adult respiratory distress syndrome (ARDS), pneumonia, endotoxin-induced lung injury, ventilator-induced lung injury and others. Even under control conditions in the absence of inflammation, neutrophils have a propensity to home to the lung through unknown mechanisms. Neutrophil recruitment is greatly enhanced by bacterial lipopolysaccharide (LPS). This project is designed to investigate the molecular mechanisms of neutrophil recruitment to the healthy and inflamed lung, using aerosolized, intratracheal and intraperitoneal administration of LPS to mice, which model inhalation injury, pneumonia and a systemic inflammatory response, respectively. Aerosolized LPS mimics exposure to airborne toxins, such as might be used in biological warfare or terrorist attacks, and minimizes the complexities of multi-organ inflammation introduced by the use of systemic LPS. We will study neutrophil homing and measure their accumulation in brochoalveolar lavage fluid (BAL), lung blood vessels, and in the interstitial spaces of the lung. We have developed methods to quantitatively determine the proportion of intravascular and interstitial leukocytes by flow cytometry. We will test whether and how selectins or Mac-1 are involved in neutrophil recruitment to the lung, and will assess the role of CD18 integrins on neutrophils and alveolar macrophages. To test whether endogenous chemokines are responsible for neutrophil recruitment, we will survey chemokine expression by superarray, multiplex RT-PCR and real time RT-PCR. To determine localization, we will use in situ hybridization and immunostaining. Flow cytometry will be complemented by MPO, whole mouse gamma imaging, light and electron microscopy. To test for chemotactic activity, we will neutralize the candidate chemokines KC and MIP-2 to gain mechanistic insights into the process. This work is complemented by using CXCR2-deflcient mice, which lack the receptor for KC and MIP-2. To test the role of adenosine A2A receptors in regulating chemokine expression and neutrophil recruitment in response to LPS, we will use A2A agonists, antagonists, A2A knockout mice, and chimeric mice generated by bone marrow transplantation, as well asconditional A2A knockout mice with selective A2A deficiency in myeloid cells (A2A[floxed]xlysM-cre), lymphocytes (A2A[floxed]xlck-cre) and endothelial cells (A2A[floxed]xtie2-cre). We will interact closely with other projects in the program to relate regulation of pulmonary chemokines and adhesion molecules to Neomycin and adenosine action (project by Linden)and chemokine and involvement in cardiopulmonary injury (project by French). Our studies are designed to identify the adhesion molecules and chemokines that regulate neutrophil recruitment to the lung at rest and during LPS-induced inhalation injury, pneumonia and widespread systemic inflammation.

中性粒细胞运输至正常和发炎的肺部。中性粒细胞向肺部募集至关重要 对于各种病理学的重要性，包括成人呼吸窘迫综合征（ARDS）、肺炎、内毒素引起的肺损伤、呼吸机引起的肺损伤等。即使在没有炎症的控制条件下，中性粒细胞也有通过未知机制回归肺部的倾向。细菌脂多糖 (LPS) 大大增强了中性粒细胞的募集。该项目旨在研究中性粒细胞募集到健康和发炎肺部的分子机制，通过对小鼠雾化、气管内和腹膜内施用LPS，分别模拟吸入性损伤、肺炎和全身炎症反应。雾化 LPS 模拟暴露于空气中 毒素，例如可能用于生物战或恐怖袭击的毒素，并最大限度地减少由于使用全身脂多糖引起的多器官炎症的复杂性。我们将研究中性粒细胞归巢并测量它们在支气管肺泡灌洗液 (BAL)、肺血管和肺间质中的积累。我们开发了通过流式细胞术定量测定血管内和间质白细胞比例的方法。我们将测试选择素或 Mac-1 是否以及如何参与中性粒细胞向肺部的募集，并将评估 CD18 整合素对中性粒细胞和肺泡巨噬细胞的作用。为了测试内源性趋化因子是否负责中性粒细胞的募集，我们将通过超阵列、多重 RT-PCR 和实时 RT-PCR 来调查趋化因子的表达。为了确定定位，我们将使用原位杂交和免疫染色。流式细胞术将得到 MPO、全鼠伽马成像、光学和电子显微镜的补充。为了测试趋化活性，我们将中和候选趋化因子 KC 和 MIP-2，以获得对该过程的机制了解。这项工作是通过使用来补充的 CXCR2 缺陷小鼠，缺乏 KC 和 MIP-2 受体。为了测试腺苷A2A受体在调节LPS趋化因子表达和中性粒细胞招募中的作用，我们将使用A2A激动剂、拮抗剂、A2A敲除小鼠和通过骨髓移植产生的嵌合小鼠，以及选择性A2A的条件A2A敲除小鼠骨髓细胞缺陷 (A2A[floxed]xlysM-cre)、淋巴细胞(A2A[floxed]xlck-cre) 和内皮细胞 (A2A[floxed]xtie2-cre)。我们将与其他项目密切互动 将肺趋化因子和粘附分子的调节与新霉素和腺苷作用（Linden 的项目）以及趋化因子和参与心肺损伤（French 的项目）联系起来的计划。我们的研究旨在确定在休息时和 LPS 诱导的吸入性损伤、肺炎和广泛的全身炎症期间调节中性粒细胞向肺部募集的粘附分子和趋化因子。