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This Article Full Text Full Text (PDF) All Versions of this Article: 291/3/L496    most recent 00353.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Vogel, S. M. Articles by Malik, A. B. Search for Related Content PubMed PubMed Citation Articles by Vogel, S. M. Articles by Malik, A. B.

De novo ICAM-1 synthesis in the mouse lung: model of assessment of protein expression in lungs

Department of Pharmacology and Center for Lung and Vascular Biology, The University of Illinois College of Medicine, Chicago, Illinois

Submitted 13 August 2005 ; accepted in final form 27 February 2006

Because most studies addressing the regulatory mechanisms of intercellular adhesion molecule (ICAM)-1 expression have used cultured endothelial cells, we set out to develop an isolated mouse lung preparation to study gene and protein expression in its proper cellular context in the organ. Lungs from CD1 mice were isolated and perfused (2 ml/min, 37°C) with a recirculating volume of RPMI 1640 solution supplemented with 3 g/100 ml albumin. Lungs maintained their isogravimetric state for 4 h. Tumor necrosis factor (TNF-; 2,000 U/ml) was added to the perfusate for 0.5, 1, 2, or 3.5 h to induce ICAM-1 expression or lungs received no treatment (control). After quick-freezing the lungs using liquid nitrogen at different time points, the prepared tissue homogenates were analyzed for ICAM-1 protein expression by Western blotting and NF-B activation by electrophoretic mobility shift assay. TNF- caused a progressive increase in NF-B activity after 0.5 h and ICAM-1 protein expression two- to threefold of basal after 2 h. Untreated lungs expressed a low and constant level of ICAM-1 between 0 and 3.5 h. TNF- failed to induce NF-B activation and ICAM-1 expression in lungs of NADPH oxidase-deficient mice lacking p47^phox. We disaggregated mouse lungs using collagenase and stained the cells for ICAM-1 and VE-cadherin (used as an endothelial marker) to assess the in situ endothelial-specific expression of ICAM-1. We observed that TNF- challenge resulted in increased ICAM-1 expression in endothelial cells freshly isolated from lungs. These data show the role of NADPH oxidase-derived oxidant signaling in the mechanism of NF-B activation and ICAM-1 expression in mouse lung endothelial cells. Moreover, the general method presented herein has potential value in assessing mechanisms of gene and protein expression in the isolated-perfused mouse lung model.

ex vivo mouse lung preparation; nuclear factor-B; fluorigenic probe for oxidant species; tumor necrosis factor-; endothelial cells