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Am J Physiol Lung Cell Mol Physiol 291: L496-L501, 2006. First published May 19, 2006; doi:10.1152/ajplung.00353.2005
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De novo ICAM-1 synthesis in the mouse lung: model of assessment of protein expression in lungs

Stephen M. Vogel, Janie Orrington-Myers, Michael Broman, and Asrar B. Malik

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


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{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-{kappa}B activation by electrophoretic mobility shift assay. TNF-{alpha} caused a progressive increase in NF-{kappa}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-{alpha} failed to induce NF-{kappa}B activation and ICAM-1 expression in lungs of NADPH oxidase-deficient mice lacking p47phox. 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-{kappa}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-{kappa}B; fluorigenic probe for oxidant species; tumor necrosis factor-{alpha}; endothelial cells

RECRUITMENT OF POLYMORPHONUCLEAR leukocytes (PMNs) to the site of infection is a complex process initiated by the binding between adhesive molecules on the endothelial cell surface and their ligands on the surface of leukocytes (13, 26, 28). Intercellular adhesion molecule-1 (ICAM-1), an inducible endothelial counterreceptor for beta2-integrins (CD11/CD18), is crucial in mediating stable adhesion of PMNs to endothelial cells. Engagement of ICAM-1 by beta2-integrins enables PMNs to migrate across the endothelial barrier in the region of infection. Although ICAM-1 is constitutively expressed in endothelial cells, the regulation of its expression level by proinflammatory cytokines is a critical innate immune defense mechanism during infection, since increased ICAM-1 expression is essential for recruitment of PMNs. Tumor necrosis factor-{alpha} (TNF-{alpha}), a proinflammatory cytokine, promotes adhesion of PMNs to vascular endothelium primarily by inducing the expression of ICAM-1 (5). The transcription factor NF-{kappa}B is a key regulator of ICAM-1 gene expression after TNF-{alpha} challenge of endothelial cells (14). Recent studies have indicated that NF-{kappa}B activation is regulated by NADPH oxidase-dependent pathways (22, 23). NADPH oxidase is an enzyme assembled in endothelial cell membrane that produces superoxide anion (O2–) via the reaction 2 O2 + NADPH -> 2 O2– + NADP+ + H+ (15–17, 25). The fully functional NADPH oxidase enzyme is a protein complex composed of a membrane-bound flavocytochrome b558 and its cytosolic activation factors Rac2, p40phox, p47phox, and p67phox. The membrane-bound flavocytochrome b558 consists of two heme redox centers and the subcomponents p22phox and gp91phox. The recruitment of cytoplasmic RhoGTPase Rac2, and p40phox, p47phox, and p67phox, to the flavocytochrome b558 membrane complex is considered the pivotal event in the activation of NADPH oxidase and subsequent generation of O2– (15–17, 25).

Oxidant signaling via NADPH oxidase is thought to regulate TNF-{alpha}-induced NF-{kappa}B activation in endothelial cells (8). TNF-{alpha}-induced activation of NF-{kappa}B results in increased expression of ICAM-1 (6, 7, 16). Because most of the studies addressing this regulatory mechanism have been done in cultured endothelial cells, we developed an isolated mouse lung preparation to study the expression of ICAM-1 gene and protein and its functional consequences in the proper cellular context of the organ. We also used mice genetically deficient in p47phox to address the role of the NADPH oxidase complex in signaling of ICAM-1 expression. We demonstrate the value of assessing gene and protein expression in vascular endothelial cells of the isolated-perfused lung preparation. Our results show that the robust ICAM-1 expression observed in lung endothelia in situ is dependent on NADPH oxidase-derived oxidant signaling and NF-{kappa}B activation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Reagents. 32P was purchased from ICN Pharmaceuticals (Costa Mesa, CA). ICAM-1 polyclonal goat blocking antibody (Ab), goat anti-actin Ab, and goat anti-mouse horseradish peroxidase-linked IgG were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse TNF-{alpha} was purchased from Cedarlane Laboratories (Ontario, Canada). Mouse ICAM-1 DNA primers were obtained from Integrated DNA Technologies (Coralville, IA). RPMI 1640 medium was supplied by Sigma (St. Louis, MO).

Experimental animals. Male CD1 mice (Jackson Laboratory, Bar Harbor, ME) weighing 30–35 g were used. Breeder stock for p47phox-deficient (p47phox–/–) mice was obtained from Dr. Steven Holland [Laboratory of Host Defense, National Institutes of Health (NIH); see Ref. 11]. Mice were housed in pathogen-free conditions with free access to food and water at the University of Illinois Biological Resources Laboratory. All studies were made using approved institutional protocols conforming to institutional and NIH guidelines.

Methods

Mouse lung isolation and lung perfusion. Mice were anesthetized inside a bell jar using 3% halothane in room air, which was supplied at a flow rate of 2 l/min. With the animal lying in a supine position, anesthesia was continued by means of a nose cone. The trachea was cannulated with a 19-gauge stainless steel tube for constant positive pressure ventilation (pressure-controlled ventilator; Kent Scientific, Litchfield, CT) with the anesthetic gas mixture. Heparin (50 units) was injected in the jugular vein for anticoagulation. The abdominal cavity was opened to expose the diaphragm, which was ventrally punctured and cut free from the rib cage. A thoracotomy was performed, and the two halves of the rib cage were retracted to expose the heart and lungs. To make the pulmonary artery accessible for cannulation, the heart was caudally retracted with a silk suture (5–0; Ethicon, Somerville, NJ) through the apical musculature of the right ventricle. An incision was made in the right ventricle near the base of the pulmonary artery for introducing an arterial cannula. The cannula (PE-60) was maneuvered in the pulmonary artery via the pulmonic valve and secured by means of a suture encompassing the pulmonary artery and the underlying aorta. An incision was then made in the left atrium to insert a venous catheter (PE-60, with flared end) that was secured to the atrial appendage with 5–0 silk. The lungs were ventilated (120/min with end-expiratory pressure set at 2.0 cmH2O) and perfused in situ (2 ml/min, 37°C) with the aid of a peristaltic pump (Gilson, Middleton, WI). Venous pressure was set to +1 cmH2O. The perfusing liquid used was bicarbonate-buffered RPMI 1640 medium supplemented with 3 g/100 ml (3%) BSA. For control of solution pH, the lungs were ventilated with a gas mixture containing 20% O2 and 5% CO2 (balance 75% N2). Lung preparations were perfused for 20 min (i.e., the equilibration period). TNF-{alpha} was infused at of rate of 0.2 ml/min through a side port in the arterial cannula to achieve the final perfusate concentration of 2,000 U/ml. Experimental preparations received TNF-{alpha} for periods of 0, 0.5, 1, 2, or 3.5 h. Control preparations were treated identically except that TNF-{alpha} was excluded from the infusate. Stability of perfusate pH was verified at the end of all experiments; acceptable lung preparations had pH values of 7.35–7.45 in the venous effluent.

Oxidant generation by lung microvasculature. To measure oxidant production in pulmonary vessels, we used a fluorigenic reagent with up to 1,000 times greater sensitivity than conventional methods (4, 29). OxyBurst Green (H2HFF-BSA) consists of BSA coupled to the reduced probe, dihydro-2',4,5,6,7,7'-hexafluorofluorescein (H2HFF), which is not fluorescent. Mouse lungs were set up as described above. After a 20-min equilibration perfusion, 5 ml of albumin-Krebs solution containing the albumin-H2HFF conjugate (200 µg/ml) were recirculated at a flow rate of 1 ml/min. Serial 50-µl samples of perfusate were collected from the venous cannula at intervals of 8 min for a 60-min period. A test substance, e.g., TNF-{alpha}, was added to the perfusate after three baseline samples had been drawn. Control preparations were treated identically except that TNF-{alpha} was omitted. All samples were transferred to tightly stoppered glass tubes under 100% nitrogen. The fluorescence intensity of the samples was measured immediately using a spectrofluorometer set to the excitation and emission maxima of the oxidized probe (488 and 530 nm, respectively).

Western blot analysis of ICAM-1 from mouse lung homogenates. Control or TNF-{alpha}-challenged mouse lung preparations (see Mouse lung isolation and lung perfusion) were quick-frozen with liquid nitrogen, and, with the aid of a tissue grinder, homogenized in PBS (pH 7.4) containing protease inhibitor cocktail (60 µl/10 ml PBS; Sigma). The homogenates were centrifuged for 10 min at 14,000 revolutions/min (rpm) and 4°C. Supernatants were collected, and protein concentration of each sample was measured with a bicinchoninic acid assay kit using BSA as standard (Pierce, Rockford, IL). An equal amount of protein from each sample (125 µg) was resolved in 10% Tris-glycine SDS polycrylalmine gel. Protein bands were blotted to nitrocellulose membranes (Millipore, Bedford, MA). After incubation for 1 h in blocking solution (5% dry milk in Tris-buffered saline with Tween 20) at room temperature, the membrane was incubated for 24 h with anti-ICAM-1 polyclonal Ab (1:2,000) at 4°C. The membrane was then incubated with secondary Ab (horseradish peroxidase-conjugated goat anti-mouse Ab at 1:1,000 dilution) for 1–2 h at room temperature. Peroxidase labeling was detected with the enhanced chemiluminescence Western blotting detection system (Amersham Biosciences, Piscataway, NJ).

Nuclear extract preparation and electrophoretic mobility shift assay. Control or TNF-{alpha}-challenged mouse lungs (see Mouse lung isolation and lung perfusion) were minced in 0.5 ml of ice-cold buffer A consisting of (in mM) 10 HEPES (pH 7.9), 1.5 KCl, 10 MgCl2, 0.5 dithiothreitol (DTT), and 0.5 phenylmethylsulfonyl fluoride (PMSF), plus 0.1 g/100 ml IGEPAL CA-630 (Sigma). The minced tissue was homogenized using a Dounce homogenizer. The crude nuclear pellet was incubated for 30 min on ice (28°C) and then centrifuged for 10 min at 14,000 rpm and 4°C. The resulting pellet was resuspended in 0.5 ml of buffer B containing (in mM) 20 HEPES (pH 7.9), 1.5 MgCl2, 420 NaCl, 0.5 DTT, 0.2 EDTA, 0.5 PMSF, and 4 leupeptin, plus 25 g/100 ml glycerol, and incubated at 28°C for 30 min. The suspension was centrifuged for 30 min at 14,000 rpm and 4°C. The supernatant (nuclear protein) was collected and stored at –80°C. Protein concentration was determined using a bicinchoninic acid assay kit with BSA as standard (Pierce). Electrophoretic mobility shift assays were performed as described (21).

Recovery of vascular endothelial cells from mouse lung for immunofluorescence studies. Tissue from control or TNF-{alpha}-challenged lungs was placed in a sterile petri dish and diced into small fragments (~1 mm diameter) using surgical scissors. The lung fragments were transferred using a serological pipette to a sterile 15-ml centrifuge tube. A 10-ml solution of sterile, filtered collagenase A (1.75%; Roche, Indianapolis, IN) was added to each tube, and tubes were placed on a rocking shaker inside of a 37° C incubator for 30 min. The fragments were centrifuged at 1,000 g for 2 min at 22°C. The collagenase supernatant was removed and replaced with 4 ml of modified endothelial growth medium-2 (Cambrex, Baltimore, MD) supplemented with penicillin/streptomycin. The fragments were gently triturated two times using a serological pipette. Larger fragments were allowed to settle for ~2 s, and the supernatant containing cell clusters was immediately applied to gelatinized 1.5-cm glass cover slips in a 12-well dish. The islets were allowed 30 min to attach to the cover slip, gently washed with PBS, and then fixed with 3.7% formaldehyde in PBS for 20 min. The islets were permeabilized with 0.4% Triton X-100 in PBS. Cells were then blocked using 3% BSA plus 2% gelatin in Hanks’ balanced salt solution for 20 min, followed by incubation for 1 h with primary antibodies against ICAM-1 (mouse, G-5) and VE-cadherin (goat, C-19; Santa Cruz), to identify endothelial cells, at a dilution of 1:100 in blocking buffer. The cover slips were washed three times in PBS, then blocked again for 20 min, followed by incubation with fluorescent secondary Abs (Donkey anti-mouse Alexa 594- and anti-goat Alexa 488-labeled Abs; Molecular Probes, Eugene, OR) at a dilution of 1:200 in blocking buffer. The cover slips were washed three times in PBS, mounted in ProLong Antifade Medium (Molecular Probes), and analyzed with a confocal microscope (Carl Zeiss LSM 510).

Statistical analysis. Data are expressed as means ± SE. Comparisons between experimental groups were made by unpaired t-tests and ANOVA; post hoc comparisons among groups were made using the Neumann-Keuls test. The significance level was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We carried out experiments using intact mouse lungs to address the role of TNF-{alpha} activation of the transcription factor NF-{kappa}B in mediating the de novo synthesis of ICAM-1. Studies were made in the isolated-perfused lung model to assess ICAM-1 gene and protein expression in lung vascular endothelial cells in situ. Because this experimental strategy required stable lung preparations, we used an enriched perfusing solution (RPMI 1640) containing all 20 amino acids, which supported protein synthesis and extended the lifetime of lung preparations from ~1.5 h (9) to 4 h. The wet-to-dry ratio of six lung preparations perfused with RPMI medium for 4 h averaged 5.3 ± 0.3, a value comparable to the ratios determined in Krebs-perfused mouse lung preparations maintained for <1 h ex vivo (27).

The cytokine TNF-{alpha} activates NF-{kappa}B in endothelial cells in culture secondary to oxidant generation by NADPH oxidase (3, 24). We therefore evaluated the effects of TNF-{alpha} in perfused mouse lungs. We made serial measurements of oxidant release in the pulmonary venous effluent before (basal) and during a 30-min period in the absence (control group) or presence of TNF-{alpha} (2,000 U/ml) added to the perfusate. TNF-{alpha} produced an immediate rise in oxidized probe (see MATERIALS AND METHODS) recovered from perfusate, which continued to accumulate during the 30-min period (Fig. 1). The control (untreated) lungs showed only background fluorescence. To address the basis of oxidants elicited by TNF-{alpha} challenge, we studied lungs obtained from p47phox–/– mice, which lack the functional NADPH oxidase. TNF-{alpha} was ineffective in p47phox–/– mice in inducing oxidant production, indicating that NADPH oxidase was the exclusive source of the detected oxidants.


Figure 1
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  		Fig. 1. Tumor necrosis factor (TNF)-{alpha}-induced oxidant generation in wild-type (p47phox+/+) and p47phox-deficient (p47phox–/–) mice as measured by the fluorescence of albumin-conjugated dihydro-2',4,5,6,7,7'-hexafluorofluorescein (H2HFF) in perfused lungs. After lung isolation and equilibration for 20 min (see MATERIALS AND METHODS), preparations received the H2HFF-albumin conjugate (200 µg/ml) in a 5-ml volume of perfusate, which was recirculated at 1 ml/min and 37°C. Serial 50-µl samples of perfusate were taken from the venous cannula at intervals of 8 min for up to 1 h. The fluorescence intensity of the samples was measured using a spectrofluorometer set to excitation and emission maxima for oxidized probe (488 and 530 nm, respectively); reduced probe is nonfluorescent. TNF-{alpha} (2,000 U/ml, arrow) added to the perfusate produced an immediate increase in probe fluorescence because of generation of oxidants within lung vessels. This was not the case in lungs from p47phox–/– mice (triangles) lacking the ability to generate oxidants via NADPH oxidase. Time control experiments in normal mice (squares) showed that unstimulated preparations generate oxidants at a background level during the entire experimental period. *Significance with respect to corresponding time 0 value; error bars are SE (n = 3 preparations/group). **Statistical difference from the wild-type control (without drug) and p47phox–/– lungs (with TNF-{alpha}).

 
We next addressed the role of oxidant signaling in lungs in mediating the activation of NF-{kappa}B induced by TNF-{alpha}. Figure 2A shows the activation of NF-{kappa}B in the nuclear fraction of lung homogenates by gel-shift assay. TNF-{alpha} activated NF-{kappa}B within 15 min, and the activation was pronounced at 30 min (Fig. 2A, lane 2). A low basal level of activation was observed in control unchallenged lungs (Fig. 2A, lane 1). The effect of TNF-{alpha} was abrogated in lungs from p47phox–/– mice (Fig. 2A, lane 3); basal (unstimulated) NF-{kappa}B activation was lower in the p47phox–/– mouse lung than the control, implying that NADPH oxidase was constitutively active in the wild-type lung. We quantified NF-{kappa}B levels by densitometry (see Fig. 2A); results are presented in Fig. 2B. TNF-{alpha} significantly increased the NF-{kappa}B level in wild-type lungs, whereas this effect of TNF-{alpha} did not develop in lungs from p47phox–/– mice (Fig. 2B).


Figure 2
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  		Fig. 2. Suppression of TNF-{alpha}-induced NF-{kappa}B activation in p47phox–/– mouse lungs. A: lungs were isolated from p47phox–/– or wild-type mice and then challenged with or without TNF-{alpha} (2,000 U/ml) for 0.5 h. NF-{kappa}B binding activity was assayed by electrophoretic mobility shift assay (EMSA) as indicated (see MATERIALS AND METHODS). Results show the requirement for p47phox in mediating TNF-{alpha}-induced NF-{kappa}B activation in mouse lungs. Results are representative of at least 3 experiments/group. B: densitometric analysis of NF-{kappa}B activation. The effect of TNF-{alpha} was abrogated in p47phox–/– lungs. *Significance at P < 0.05 (n = 3–4 experiments/group). **Decrease of basal ("untreated") value compared with wild-type (WT).

 
We determined the time course of ICAM-1 expression after TNF-{alpha} challenge (0.5, 12, 2, and 3.5 h) in the mouse lungs in situ. Mean levels of ICAM-1 expression (based on densitometric analysis of Western blots) trended toward higher expression levels between 1 and 3.5 h, but the 3.5-h value was significantly higher than basal (Fig. 3A). Time-control experiments showed a low and constant expression level of ICAM-1 in untreated lungs from wild-type mice perfused for 3.5 h (Fig. 3B).


Figure 3
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  		Fig. 3. Western blot analysis of intercellular adhesion molecule (ICAM)-1 expression in mouse lungs. Lungs were perfused with RPMI 1640 solution for the indicated time period (between 0 and 3.5 h), with (experimental lungs) or without (control lungs) TNF-{alpha} (2,000 U/ml) added to the perfusate. Lungs were quick-frozen using liquid nitrogen, and homogenates were prepared for Western blotting. A: Western blots showing increased ICAM-1 expression after 3.5 h of TNF-{alpha} challenge in lungs obtained from wild-type but not p47phox–/– mice. A1–A2: quantification of Western blots by densitometry. Blot density (increase factor relative to time 0 value) was significantly elevated in wild-type lungs at 3.5 h (A1) but not in p47phox–/– lungs (A2). B: Western blots showing that control perfusion for up to 3.5 h did not alter basal ICAM-1 levels. B1: quantification of Western blots by densitometry. *Significance with respect to corresponding time 0 value; error bars are SE (n = 3 experiments/bar).

 
Because ICAM-1 expression observed in lungs in situ reflects expression in multiple cell types, we carried out immunofluorescent staining to identify whether endothelial cells from these lungs were responsive to TNF-{alpha} in terms of increased ICAM-1 expression. The lung preparations received TNF-{alpha} for 3 h, and a collagenase digest of the lung was immediately prepared (see METHODS). The obtained cells were plated and stained for VE-cadherin, an endothelial cell marker. VE-cadherin-positive cells were seen as uniform cell clusters (green fluorescence; Fig. 4). The cells were counterstained for ICAM-1 (red fluorescence; Fig. 4). TNF-{alpha} challenge of lungs caused a marked increase in ICAM-1 immunofluorescence only in the VE-cadherin-positive cells. ICAM-1 immunofluorescence was evident in the cell cytosol and perinuclear region in the majority of endothelial cells at 3 h after TNF-{alpha} challenge of lungs (Fig. 4), consistent with de novo synthesis of ICAM-1.


Figure 4
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  		Fig. 4. Increased expression of ICAM-1 in mouse lung endothelial cells after TNF-{alpha} challenge. Fluorescent images in A-C show a representative endothelial cell cluster from a control mouse lung digest (see MATERIALS AND METHODS). VE-cadherin staining decorates endothelial cell borders (A), and ICAM-1 staining (B) is low during the basal (unstimulated) period. Images in D-F depict a similar mouse endothelial cell cluster obtained after 3 h of TNF-{alpha} challenge of isolated perfused lung. These endothelial cells show marked increase in ICAM-1 protein expression (E). C and F give merged images of VE-cadherin (green), ICAM-1 (red) fluorescence, and nuclei (4',6-diamidine-2-phenylindole stain, blue). Data shown are representative of 3 similar runs in different lung preparations. Bars = 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The key observation made in the present study was the induction of de novo ICAM-1 protein expression by TNF-{alpha} in endothelial cells of the intact mouse lung preparation. We chose TNF-{alpha} for this study because it is a cytokine appearing in the circulation under conditions of acute inflammation and septic shock (1) and it is known to induce adhesiveness of the endothelium for leukocytes required for leukocyte extravasation by diapedesis (10, 22, 23). Our understanding in molecular terms of the role of TNF-{alpha} in inducing expression of adhesion proteins is based primarily on in vitro studies utilizing vascular endothelial cell monolayers that exhibit NF-{kappa}B-dependent regulation of ICAM-1 gene and protein expression accompanied by increased endothelial adhesivity and transendothelial migration of added PMNs (10, 22, 23). Thus we used the perfused lung preparation to address the physiological significance of these observations. The lung preparation was advantageous by allowing use of cell-free perfusing liquid at constant temperature and flow, and lung isolation allowed assessment of direct pulmonary vascular effects of TNF-{alpha} without the complication of secondary effects of TNF-{alpha} on nonpulmonary organs and tissues (including blood cells). Moreover, the isolated lung method lends itself to a variety of rigorous physiological assessments not possible in vivo (e.g., vascular permeability and indium-labeled PMN binding to the vessel wall) and biochemical assays. In the present study, we measured oxidant generation, NF-{kappa}B activity, and ICAM-1 protein expression under uniform experimental conditions, and, with the aid of suitable genetic mouse models, we established clearly the role of NADPH oxidase-derived oxidant generation in the regulation of ICAM-1 gene expression. Specifically, the blots in Fig. 2 demonstrate the important role of oxidants in regulating ICAM-1 gene expression at the level of the transcription factor NF-{kappa}B, which was not activated in response to TNF-{alpha} in lungs obtained from p47phox–/– mice. Furthermore, our results demonstrate the absence of ICAM-1 expression in NADPH oxidase null lungs. By contrast, oxidant generation in wild-type lungs was immediate and persistent over the study period in response to TNF-{alpha}, and NF-{kappa}B activation was observed 15–30 min later. These results suggest that TNF-{alpha} sequentially activates NADPH oxidase and NF-{kappa}B, thereby inducing de novo ICAM-1 expression in the lung vasculature. Oxidant signaling-mediated activation of NF-{kappa}B has been demonstrated in cultured endothelial cells (7, 23). TNF-{alpha} induced ICAM-1 expression in endothelial cell monolayers within 2–4 h (12, 22), which is in the range of ICAM-1 expression observed in the intact lung vascular bed in the present study.

A concern in the assessment of ICAM-1 expression in the intact lung is that the result may not be ascribed to the expression of protein in endothelial cells. Thus we isolated endothelial cells from lungs after TNF-{alpha} challenge (see METHODS). We used anti-VE-cadherin Ab and carried out immunostaining to identify the vascular endothelial cells. The isolated endothelial cells positive for VE-cadherin also showed robust expression of ICAM-1, indicating that endothelial cells were activated in situ after the TNF-{alpha} challenge.

The feasibility of the experiments depended on the development of the ex vivo mouse lung model, permitting us to extend its useful lifetime from ~1.5 to 4 h after isolation. This is an important consideration since significant de novo protein synthesis can occur in a time frame of >3 h, as seen with ICAM-1 protein expression. The key modification was use of an enriched perfusing liquid (RPMI 1640) containing albumin as osmotic balancer to replace albumin-Krebs solution. RPMI perfusion gave stable pulmonary arterial pressure without any sign of atelectasis or lung edema during the period of lung perfusion. The ex vivo mouse lung preparation has advantages for gene expression studies. First, the preparation can be used to assess the role of alterations in shear stress suggested to affect gene expression in cultured endothelial cells (2, 3). Second, the role of protein expression and certain aspects of acute lung injury response can be investigated (e.g., ischemia, increase in PMN adhesion to the vessel wall, and increase in lung vascular permeability) during an ex vivo experiment. However, the important caveat is the need for a stable lung preparation capable of maintaining fluid balance, since development of lung edema may trigger proteolysis (18–20), which may then injure the subendothelial matrix and endothelial cells, and thus interfere with protein expression.

In conclusion, we have shown that TNF-{alpha} induces ICAM-1 protein expression in endothelial cells of the intact mouse lung. ICAM-1 expression required NADPH oxidase-dependent oxidant signaling and NF-{kappa}B activation. These results also show the potential value of using this isogravimetric preparation for studies of gene and protein expression in lung endothelia.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. M. Vogel, Dept. of Pharmacology, Univ. of Illinois College of Medicine, 835 South Wolcott Ave. (M/C 868), Chicago, IL 60612 (e-mail: vogel{at}uic.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bhatia M and Moochhala S. Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome. J Pathol 202: 145–156, 2004.[CrossRef][ISI][Medline]
  2. Chappell DC, Varner SE, Nerem RM, Medford RM, and Alexander RW. Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res 82: 532–539, 1998.[Abstract/Free Full Text]
  3. Chen XL, Zhang Q, Zhao R, and Medford RM. Superoxide, H2O2, and iron are required for TNF-{alpha}-induced MCP-1 gene expression in endothelial cells: role of Rac1 and NADPH oxidase. Am J Physiol Heart Circ Physiol 286: H1001–H1007, 2004.[Abstract/Free Full Text]
  4. Cohen HJ and Chovaniec ME. Superoxide production by digitonin-stimulated guinea pig granulocytes. The effects of N-ethyl maleimide, divalent cations; and glycolytic and mitochondrial inhibitors on the activation of the superoxide generating system. J Clin Invest 61: 1088–1096, 1978.[ISI][Medline]
  5. Diamond MS, Staunton DE, Marlin SD, and Springer TA. Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. Cell 65: 961–971, 1991.[CrossRef][ISI][Medline]
  6. Fan J, Frey RS, Rahman A, and Malik AB. Role of neutrophil NADPH oxidase in the mechanism of tumor necrosis factor-alpha-induced NF-kappa B activation and intercellular adhesion molecule-1 expression in endothelial cells. J Biol Chem 277: 3404–3411, 2002.[Abstract/Free Full Text]
  7. Frey RS and Malik AB. Oxidant signaling in lung cells. Am J Physiol Lung Cell Mol Physiol 286: L1–L3, 2004.[Free Full Text]
  8. Frey RS, Rahman A, Kefer JC, Minshall RD, and Malik AB. PKCzeta regulates TNF-alpha-induced activation of NADPH oxidase in endothelial cells. Circ Res 90: 1012–1019, 2002.[Abstract/Free Full Text]
  9. Gao X, Xu N, Sekosan M, Mehta D, Ma SY, Rahman A, and Malik AB. Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice. J Immunol 167: 2895–2901, 2001.[Abstract/Free Full Text]
  10. Haraldsen G, Kvale D, Lien B, Farstad IN, and Brandtzaeg P. Cytokine-regulated expression of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in human microvascular endothelial cells. J Immunol 156: 2558–2565, 1996.[Abstract]
  11. Jackson SH, Gallin JI, and Holland SM. The p47phox mouse knock-out model of chronic granulomatous disease. J Exp Med 182: 751–758, 1995.[Abstract/Free Full Text]
  12. Kluger MS, Johnson DR, and Pober JS. Mechanism of sustained E-selectin expression in cultured human dermal microvascular endothelial cells. J Immunol 158: 887–896, 1997.[Abstract]
  13. Lawrence MB and Springer TA. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65: 859–873, 1991.[CrossRef][ISI][Medline]
  14. Ledebur HC and Parks TP. Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells. Essential roles of a variant NF-kappa B site and p65 homodimers. J Biol Chem 270: 933–943, 1995.[Abstract/Free Full Text]
  15. Leusen JH, Fluiter K, Hilarius PM, Roos D, Verhoeven AJ, and Bolscher BG. Interactions between the cytosolic components p47phox and p67phox of the human neutrophil NADPH oxidase that are not required for activation in the cell-free system. J Biol Chem 270: 11216–11221, 1995.[Abstract/Free Full Text]
  16. Li JM, Mullen AM, Yun S, Wientjes F, Brouns GY, Thrasher AJ, and Shah AM. Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-alpha. Circ Res 90: 143–150, 2002.[Abstract/Free Full Text]
  17. Murphy HS, Yu C, and Quddus J. Functional expression of NAD(P)H oxidase p47 in lung microvascular endothelial cells. Biochem Biophys Res Commun 278: 584–589, 2000.[CrossRef][ISI][Medline]
  18. Negrini D, Passi A, de Luca G, and Miserocchi G. Pulmonary interstitial pressure and proteoglycans during development of pulmonary edema. Am J Physiol Heart Circ Physiol 270: H2000–H2007, 1996.[Abstract/Free Full Text]
  19. Negrini D, Passi A, De Luca G, and Miserochi G. Proteoglycan involvement during development of lesional pulmonary edema. Am J Physiol Lung Cell Mol Physiol 274: L203–L211, 1998.[Abstract/Free Full Text]
  20. Passi A, Negrini D, Albertini R, De Luca G, and Miserocchi G. Involvement of lung interstitial proteoglycans in development of hydraulic- and elastase-induced edema. Am J Physiol Lung Cell Mol Physiol 275: L631–L635, 1998.[Abstract/Free Full Text]
  21. Rahman A, Anwar KN, Uddin S, Xu N, Ye RD, Platanias LC, and Malik AB. Protein kinase C-delta regulates thrombin-induced ICAM-1 gene expression in endothelial cells via activation of p38 mitogen-activated protein kinase. Mol Cell Biol 21: 5554–5565, 2001.[Abstract/Free Full Text]
  22. Rahman A, Bando M, Kefer J, Anwar KN, and Malik AB. Protein kinase C-activated oxidant generation in endothelial cells signals intercellular adhesion molecule-1 gene transcription. Mol Pharmacol 55: 575–583, 1999.[Abstract/Free Full Text]
  23. Rahman A, Kefer J, Bando M, Niles WD, and Malik AB. E-selectin expression in human endothelial cells by TNF-{alpha}-induced oxidant generation and NF-{kappa}B activation. Am J Physiol Lung Cell Mol Physiol 275: L533–L544, 1998.[Abstract/Free Full Text]
  24. Sadikot RT, Zeng H, Yull FE, Li B, Cheng DS, Kernodle DS, Jansen ED, Contag CH, Segal BH, Holland SM, Blackwell TS, and Christman JW. p47phox deficiency impairs NF-kappa B activation and host defense in Pseudomonas pneumonia. J Immunol 172: 1801–1808, 2004.[Abstract/Free Full Text]
  25. Souza HP, Souza LC, Anastacio VM, Pereira AC, Junqueira ML, Krieger JE, da Luz PL, Augusto O, and Laurindo FR. Vascular oxidant stress early after balloon injury: evidence for increased NAD(P)H oxidoreductase activity. Free Radic Biol Med 28: 1232–1242, 2000.[CrossRef][ISI][Medline]
  26. Springer TA. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301–314, 1994.[CrossRef][ISI][Medline]
  27. Vogel SM, Gao X, Mehta D, Ye RD, John TA, Andrade-Gordon P, Tiruppathi C, and Malik AB. Abrogation of thrombin-induced increase in pulmonary microvascular permeability in PAR-1 knockout mice. Physiol Genomics 4: 137–145, 2000.[Abstract/Free Full Text]
  28. von Andrian UH, Chambers JD, McEvoy LM, Bargatze RF, Arfors KE, and Butcher EC. Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo. Proc Natl Acad Sci USA 88: 7538–7542, 1991.[Abstract/Free Full Text]
  29. Whitin JC, Chapman CE, Simons ER, Chovaniec ME, and Cohen HJ. Correlation between membrane potential changes and superoxide production in human granulocytes stimulated by phorbol myristate acetate. Evidence for defective activation in chronic granulomatous disease. J Biol Chem 255: 1874–1878, 1980.[Free Full Text]




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