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.
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