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: 7062084
• 负责人: BRIAN R DULING
• 金额: $ 17.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7062084
• 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.

中性粒细胞运输到正常和发炎的肺。肺中性粒细胞招募至关重要 对于包括成人呼吸窘迫综合征（ARDS），肺炎，内毒素诱导的肺损伤，呼吸机诱导的肺损伤等各种病理的重要性。即使在没有炎症的情况下，在控制条件下，中性粒细胞也通过未知机制倾向于肺部。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的肺的分子机制，使用雾化，气管内和腹膜内LPS对小鼠的施用，分别模拟了吸入损伤，肺炎和全身性炎症反应。气溶解的LP模仿空气传播 毒素，例如在生物战或恐怖袭击中可能使用，并最大程度地减少了使用全身性LP引入的多器官炎症的复杂性。我们将研究中性粒细胞的归巢，并测量其在brocho肺泡灌洗液（BAL），肺血管以及肺部间隙空间中的积累。我们开发了通过流式细胞仪定量确定血管内和间质性白细胞比例的方法。我们将测试Selectins或Mac-1如何参与肺中性粒细胞募集到肺部，并评估CD18整联蛋白在中性粒细胞和肺泡巨噬细胞中的作用。为了测试内源性趋化因子是否负责中性粒细胞募集，我们将通过SuperArray，Multiplex RT-PCR和实时RT-PCR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。流式细胞仪将通过MPO，全小鼠伽马成像，光和电子显微镜补充。为了测试趋化活性，我们将中和候选趋化因子KC和MIP-2，以获得对这一过程的机械见解。这项工作是通过使用的补充 CXCR2-脱光度小鼠，缺少KC和MIP-2的受体。为了测试腺苷A2A受体在调节响应LPS的趋化因子表达和中性粒细胞募集中的作用，我们将使用A2A激动剂，拮抗剂，A2A敲除小鼠以及由骨髓移植产生的嵌合小鼠，以及A2A敲除A2A A2A Deepiation A2A Deepiperiation bone骨髓移植小鼠。 （A2A [Floxed] Xlysm-Cre），淋巴细胞（A2A [Floxed] XLCK-CRE）和内皮细胞（A2A [Floxed] Xtie2-Cre）。我们将与其他项目紧密互动 将肺趋化因子和粘附分子与新霉素和腺苷作用（Linden的项目）和趋化因子和趋化因子的调节以及参与心肺损伤（法语项目）的调节计划。我们的研究旨在鉴定粘附分子和趋化因子和趋化因子，这些因子在休息时和LPS引起的吸入损伤，肺炎和广泛的全身炎症中调节中性粒细胞募集。