Neutrophil Trafficking to Normal and Inflamed Lung

中性粒细胞向正常肺和炎症肺转移。中性粒细胞重新聚集到肺中是至关重要的 对于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。即使在没有炎症的控制条件下，中性粒细胞也有通过未知机制回到肺内的倾向。细菌脂多糖(LPS)能极大地促进中性粒细胞募集。本项目旨在通过雾化吸入、气管内和腹膜内注射脂多糖，分别建立吸入性损伤、肺炎和全身炎症反应的模型，研究中性粒细胞在健康和炎症肺中募集的分子机制。雾化内毒素模拟暴露在空气中 毒素，如可能用于生物战或恐怖袭击，并最大限度地减少复杂性的多器官炎症引起的系统性脂多糖。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聚。我们发展了用流式细胞术定量测定血管内和间质中白细胞比例的方法。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的募集，并将评估CD18整合素在中性粒细胞和肺泡巨噬细胞上的作用。为了验证内源性趋化因子是否与中性粒细胞募集有关，我们将通过超微阵列、多重RT-PCR和实时定量RT-PCR检测趋化因子的表达。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，以获得对这一过程的机械性见解。这项工作通过使用 CXCR2缺陷小鼠，缺乏KC和MIP-2受体。为了测试腺苷A2A受体在调节趋化因子表达和中性粒细胞募集方面的作用，我们将使用A2A激动剂、拮抗剂、A2A基因敲除小鼠和骨髓移植产生的嵌合体小鼠，以及选择性A2A缺乏髓系细胞(A2AxlysM-cre)、淋巴细胞(A2Axlck-cre)和内皮细胞(A2Axtie2-cre)的条件A2A基因敲除小鼠。我们将与其他项目密切互动。 计划将肺趋化因子和黏附分子的调节与新霉素和腺苷作用(林登项目)和趋化因子与心肺损伤的参与(法国项目)联系起来。我们的研究旨在确定在静息和脂多糖诱导的吸入性损伤、肺炎和广泛的全身炎症过程中，调节中性粒细胞向肺内募集的黏附分子和趋化因子。

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.