Sequential recruitment of neutrophils into lung and bronchoalveolar lavage fluid in LPS-induced acute lung injury

1 Robert M. Berne Cardiovascular Research Center, Departments of 2 Physiology and Biological Physics, and 3 Biomedical Engineering, University of Virginia, Charlottesville, Virginia; and 4 Department of Anesthesiology and Intensive Care Medicine, University of Tübingen, Tübingen, Germany Submitted 23 December 2004 ; accepted in final form 9 June 2005 Infiltration of activated neutrophils [polymorphonuclear leukocytes (PMN)] into the lung is an important component of the inflammatory response in acute lung injury. The signals required to direct PMN into the different compartments of the lung have not been fully elucidated. In a murine model of LPS-induced lung injury, we investigated the sequential recruitment of PMN into the pulmonary vasculature, lung interstitium, and alveolar space. Mice were exposed to aerosolized LPS and bronchoalveolar lavage fluid (BAL), and lungs were harvested at different time points. We developed a flow cytometry-based technique to assess in vivo trafficking of PMN in the intravascular and extravascular lung compartments. Aerosolized LPS induced consistent PMN migration into all lung compartments. We found that sequestration in the pulmonary vasculature occurred within the first hour. Transendothelial migration into the interstitial space started 1 h after LPS exposure and increased continuously until a plateau was reached between 12 and 24 h. Transepithelial migration into the alveolar air space was delayed, as the first PMN did not appear until 2 h after LPS, reaching a peak at 24 h. Transendothelial migration and transepithelial migration were inhibited by pertussis toxin, indicating involvement of G i-coupled receptors. These findings confirm LPS-induced migration of PMN into the lung. For the first time, distinct transmigration steps into the different lung compartments are characterized in vivo. polymorphonuclear leukocytes; pulmonary circulation; chemokines; lipopolysaccharide; flow cytometry; pertussis toxin Address for reprint requests and other correspondence: K. Ley, Univ. of Virginia Health System, Cardiovascular Research Center, PO Box 801394, Charlottesville, VA 22908-1394 (e-mail: klausley{at}virginia.edu )