De novo ICAM-1 synthesis in the mouse lung: model of assessment of protein expression in lungs.
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 degrees 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-alpha; 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-kappaB activation by electrophoretic mobility shift assay. TNF-alpha caused a progressive increase in NF-kappaB 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-alpha failed to induce NF-kappaB 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-alpha 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-kappaB 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.
Vogel
Stephen M
SM
Department of Pharmacology and Center for Lung and Vascular Biology, The University of Illinois College of Medicine, Chicago, IL 60612, USA. vogel@uic.edu
Orrington-Myers
Janie
J
Broman
Michael
M
Malik
Asrar B
AB
D000818:Animals; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis*; D008168:Lung / Q000378:metabolism*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D019255:NADPH Oxidases / Q000235:genetics* / Q000502:physiology; D016328:NF-kappa B / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1