Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin
Shawn J. Skerrett,1
H. Denny Liggitt,2
Adeline M. Hajjar,3
Robert K. Ernst,4
Samuel I. Miller,1,4 and
Christopher B. Wilson3,5
1Departments of Medicine, 2Comparative Medicine, 3Immunology, 4Microbiology, and 5Pediatrics, University of Washington School of Medicine, Seattle, Washington 98104
Submitted 4 February 2004
; accepted in final form 21 March 2004
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ABSTRACT
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To determine the role of respiratory epithelial cells in the inflammatory response to inhaled endotoxin, we selectively inhibited NF-
B activation in the respiratory epithelium using a mutant I
B-
construct that functioned as a dominant negative inhibitor of NF-
B translocation (dnI
B-
). We developed two lines of transgenic mice in which expression of dnI
B-
was targeted to the distal airway epithelium using the human surfactant apoprotein C promoter. Transgene expression was localized to the epithelium of the terminal bronchioles and alveoli. After inhalation of LPS, nuclear translocation of NF-
B was evident in bronchiolar epithelium of nontransgenic but not of transgenic mice. This defect was associated with impaired neutrophilic lung inflammation 4 h after LPS challenge and diminished levels of TNF-
, IL-1
, macrophage inflammatory protein-2, and KC in lung homogenates. Expression of TNF-
within bronchiolar epithelial cells and of VCAM-1 within peribronchiolar endothelial cells was reduced in transgenic animals. Thus targeted inhibition of NF-
B activation in distal airway epithelial cells impaired the inflammatory response to inhaled LPS. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.
lipopolysaccharide; cytokines; nuclear factor-
B; transgenic mice
THE INHALATION OF LPS RESULTS in acute, neutrophilic inflammation of the distal air spaces of the lungs. The molecular mechanisms underlying the inflammatory response to LPS involve the detection of LPS by pattern recognition receptors followed by the coordinated expression of cytokines, chemokines, and adhesion molecules that direct the emigration of neutrophils across the endothelial and epithelial barriers that separate the bloodstream from the pulmonary air spaces (35, 39). The recognition of LPS is modulated by soluble factors, such as LPS binding protein and surfactant proteins, that are present in airway lining fluid and influence the presentation of LPS to membrane-bound CD14 (35, 38). Binding of LPS to CD14 triggers intracellular signaling that is mediated by Toll-like receptor 4 (TLR4) in association with a secreted cofactor, MD-2 (1, 2). LPS-induced signal transduction leads to activation of the NF-
B family of transcription factors, which in turn directs the expression of proinflammatory cytokines, chemokines, and adhesion molecules (30, 57). Early response cytokines, such as TNF-
and IL-1
, can amplify this response by stimulating the NF-
B-dependent induction of proinflammatory mediators in cells that lack components of the CD14/TLR4/MD-2 receptor complex and thus cannot respond directly to LPS (30, 57). Local production of CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) triplet establishes the chemotactic gradient for the emigration of neutrophils from the bloodstream (57). The upregulation of leukocyte integrins and the increased expression of adhesive counterligands on the surfaces of endothelial, interstitial, and epithelial cells permits neutrophil extravasation along the chemotactic gradient (39).
Murine models of LPS-induced lung inflammation support this paradigm. Bronchoalveolar accumulation of neutrophils after inhalation of LPS is markedly blunted in mice with mutations of TLR4 (34). Intrapulmonary activation of NF-
B follows airway challenge with LPS (8, 22, 40, 49), and LPS-induced lung inflammation is impaired in mice lacking the RelA component of NF-
B (3). Pulmonary neutrophil recruitment in response to LPS is also blunted in mice lacking the type 1 TNF receptor (TNFR1) (52) and in animals depleted of the ELR+ CXC chemokines KC or macrophage inflammatory protein-2 (MIP-2) (20, 50). Similarly, depletion studies have documented the roles of
1- and
2-integrins and their ligands in the airway inflammatory response to LPS (4, 10, 18, 28, 31, 46).
Although the importance of individual molecules in the bronchoalveolar inflammatory response to LPS has been recognized, the cellular participants in this cascade are incompletely understood. Multiple cells may be involved in the initiation and regulation of air space inflammation, including alveolar macrophages, epithelial cells, fibroblasts, and endothelial cells (57). There is strong evidence supporting the role of alveolar macrophages in the inflammatory response to inhaled LPS. Alveolar macrophages are sensitive to the presence of LPS in vitro (36) and are activated in response to airway challenge with LPS in vivo (22). Furthermore, depletion of alveolar macrophages results in diminished NF-
B activation, cytokine responses, and neutrophilic inflammation after inhalation of LPS (7, 24).
The role of alveolar epithelial cells in the regulation of lung inflammation is less clear. In vitro, respiratory epithelial cells can secrete proinflammatory mediators and upregulate adhesion molecules in response to bacterial stimuli or endogenous factors such as IL-1
and TNF-
(15). In vivo studies have provided evidence of NF-
B activation and increased ICAM-1 expression by bronchiolar and alveolar epithelial cells after airway challenge with LPS (4, 5, 9, 22). Moreover, targeted expression of KC or constitutive activation of NF-
B in airway epithelial cells is sufficient to elicit pulmonary inflammation in the absence of exogenous stimuli (48, 59). Furthermore, Poynter and colleagues (44) recently reported that targeted expression of a dominant negative I
B-
in proximal airway epithelial cells under the control of the rat CC10 promoter impaired lung inflammation after intranasal administration of LPS.
The goal of the present study was to evaluate the contribution of the distal airway epithelium to the regulation of lung inflammation induced by aerosolized LPS. Our strategy was to selectively block NF-
B activation in terminal bronchiolar and alveolar epithelial cells while leaving the signaling responses of proximal airway epithelial cells and alveolar macrophages intact. To achieve this end, we constructed transgenic mice in which a mutant I
B-
resistant to proteasomal degradation, because of alanine substitutions at Ser32 and Ser36 that block inducible phosphorylation (16), was targeted to the distal airway epithelium under the control of the human surfactant apoprotein C promoter (25). The mutant I
B-
functions as a dominant negative inhibitor of NF-
B translocation (dnI
B-
) in cells in which the transgene is expressed. We found that local cytokine responses and neutrophil recruitment in response to LPS were significantly reduced in the transgenic animals. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.
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MATERIALS AND METHODS
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Adenoviral vector-mediated transduction of A549 cells with dnI
B-
.
The adenoviral vector directing the expression of human dnI
B-
, in which Ser32 and Ser36 have been mutated to alanine and a triple influenza hemagglutinin (HA) tag added to its 5' end, and the adenoviral vector directing expression of
-galactosidase, have been described previously (32). A549 cells (American Type Culture Collection CCL-185) were grown in RPMI 1640 medium plus 10% fetal calf serum (GIBCO BRL, Grand Island, NY) in 24-well tissue culture trays and transduced with one or the other adenoviral vector. Forty-eight hours later, cells were exposed to recombinant TNF-
(200 U/ml throughout the period of culture), Pseudomonas aeruginosa (strain PAK, 107 CFU/ml for 1 h, after which cells were washed and refed with fresh medium containing gentamicin at 20 µg/ml to prevent bacterial overgrowth), or medium alone. After 6 or 24 h, supernatants were harvested and stored at 80°C. Levels of immunoreactive IL-8 were measured by ELISA using reagents from R&D Systems (Minneapolis, MN).
Generation and characterization of surfactant protein C dnI
B-
transgenic mice.
The cDNA encoding the HA-tagged human dnI
B-
was identical to that used to make the recombinant adenovirus noted above (32). Transgenic mice were generated by inserting this cDNA into a vector under the control of the human surfactant protein C (SP-C) promoter, originally provided by Dr. Jeffrey Whitsett (Cincinnati Children's Hospital), as previously described (25, 55). Five lines of SPCdnI
B-
mice were generated, and all founders and their offspring were healthy. Tissue expression of the transgene was analyzed by Northern blot, as previously described (55), using probes for both the coding region and the simian virus 40 (SV40) 3 untranslated region. Results with both probes were similar, and those obtained with the SV40 probe are shown in RESULTS. Expression of transgene-encoded protein was determined by Western blot. Briefly, lungs were homogenized on ice in PBS containing Pefabloc (Roche Diagnostics, Indianapolis, IN), 50 µg of lung protein were electrophoresed on reducing 10% SDS-polyacrylamide gels and transferred to nitrocellulose, and HA-tagged dnI
B-
was detected using a rabbit polyclonal antibody to HA (Santa Cruz Biotechnology, Santa Cruz, CA) and the Amersham ECL system (Amersham Biosciences, Piscataway, NJ). The same antibody was used for transgene detection by immunohistochemistry, using an alkaline phosphatase-conjugated secondary antibody and the ABC development system (Vector Laboratories, Burlingame, CA) (55). Two lines (5990 and 6006) that exhibited strong, lung-specific transgene expression were used for further studies. SPCdnI
B-
mice were backcrossed a minimum of five times to C57BL/6 mice before they were used in the LPS challenge experiments. Transgene-negative littermates were used as controls in all experiments; locally bred C57BL/6 mice were used as supplemental controls where specified. All mice were bred at the University of Washington Animal Facility. Mice were housed under specific pathogen-free conditions in filtered cages and were permitted unlimited access to sterile food and water. All animal experiments were approved by the Animal Care Committee of the University of Washington.
Endotoxins.
Escherichia coli 0111:B4 LPS was purchased from Sigma (St. Louis, MO), reconstituted to 5 mg/ml in 20 mM EDTA, clarified in an ultrasonic water bath (Cole-Parmer, Vernon Hills, IL), aliquoted, and stored at 80°C. For each experiment, an aliquot was thawed, sonically dispersed, and diluted in sterile, endotoxin-free PBS (Mediatech, Herndon, VA) to 100 µg/ml. LPS was prepared from P. aeruginosa strain PAK by magnesium-ethanol precipitation, as described (14, 19). For aerosolization, the lyophilized material was reconstituted in sterile water, clarified ultrasonically, and diluted to 100 µg/ml.
Exposure to aerosolized LPS.
Mice were exposed to aerosolized LPS in a whole animal exposure system, as described (52). The animals were placed in wire mesh cages within a 55-l Plexiglas cylinder connected via 10-cm ducting to a 16-l aerosol chamber. Airflow through the system was maintained at 20 l/min by negative pressure. Aerosols were generated from twin jet nebulizers (Salter Labs, Arvin, CA), each containing 8 ml of LPS suspension and driven by forced air at 1518 psi. After 30 min, the chamber was purged with ambient air, and the mice were returned to their microisolator cages. Each experiment paired 1014 transgenic mice with an equal number of nontransgenic littermates. Control animals for aerosol experiments were either exposed to aerosolized PBS or were unexposed, as stated.
Bronchoalveolar lavage.
At each time point, 4 and 24 h after aerosol exposure, four to six transgenic mice and four to six nontransgenic control mice were killed with an overdose of pentobarbital (10 mg ip) and then exsanguinated by cardiac puncture. The trachea was exposed and cannulated with a 20-g polyethylene catheter before the chest was opened. The left hilum was clamped, and then the right lung was lavaged with four separate 0.5-ml volumes of 0.85% saline containing 0.6 mM EDTA, each retrieved after a 30-s dwell time. The aliquots from individual mice were combined and centrifuged at 300 g, and the supernatants were stored at 80°C. The cell pellets were resuspended in RPMI containing 5% heat-inactivated fetal calf serum (Hyclone, Logan, UT), and the cells were counted in a hemacytometer. Differentials were performed on slides prepared in a Shandon Cytospin (ThermoShandon, Pittsburgh, PA) and stained with a modified Wright-Giemsa technique (Diff-Quik; Dade Behring, Dudingen, Switzerland).
Myeloperoxidase activity.
Myeloperoxidase (MPO) activity was measured in lung tissue after a method used in the laboratory of Theodore Standiford (Univ. of Michigan) (11). The entire left lung or individual lobes of the right lung were flash-frozen in liquid nitrogen and stored at 80°C. The thawed tissue was homogenized in 2x protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and then diluted 1:1 with 2x MPO homogenization buffer composed of 3.7 mM hexadecyltrimethylammonium bromide, 0.5 mM EDTA, 0.44 M monobasic potassium phosphate, and 0.06 M dibasic potassium phosphate (all Sigma). The mixture was sonically dispersed and then centrifuged at 12,000 g for 15 min at 4°C. Supernatants were transferred in duplicate 10-µl volumes to 96-well plates and then mixed with 140 µl of MPO assay buffer composed of 88 mM monobasic potassium phosphate, 1.24 mM dibasic potassium phosphate, 0.005% hydrogen peroxide, and 0.53 mM O-dianisidine hydrochloride. MPO activity was read as the rate of change in absorbance at 490 nm over 2 min and corrected to lung weight.
Histopathology.
Lungs were inflated in situ to
20 cm of pressure with 4% paraformaldehyde and then removed en bloc and stored at 4°C in the same fixative. The tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin. A veterinary pathologist reviewed two to four sections from individual mice in a manner blinded to genotype and time from LPS exposure (54). The intensity of peribronchial and perivascular inflammation was scored on a scale of 04, with a score of 0 representing absence of cells and a score of 4 representing maximal accumulation relative to other sections. The ratio of mononuclear and polymorphonuclear cells in these inflammatory foci was noted. The degree of parenchymal inflammation was also scored on an all-inclusive scale of 04, with a score of 0 representing normal tissue and a score of 4 representing widespread interstitial and/or alveolar infiltration. Lung tissue harvested from unexposed mice representing each genotype served as controls.
Immunohistochemistry.
Immunohistochemical procedures were performed on frozen sections or on treated paraffin sections. Before lung tissue was frozen, individual lobes were inflated under gentle pressure with a mixture of PBS and Optimum Cutting Temperature (OCT) compound (Miles, Elkhart, IN). Lobes were then embedded in OCT and flash-frozen in isopentane chilled in liquid nitrogen. Sections were cut at
5 µm and placed on charged slides. After brief fixation in cold acetone, sections were incubated with appropriate antibodies overnight at 4°C. Paraffin sections of tissue fixed in 4% paraformaldehyde were used for detection of RelA. Antigen retrieval was performed by boiling slides for 10 min in 10 mM citrate buffer (pH 6.0), after which sections were washed and incubated with antibody overnight at 4°C. Bound antibodies were detected after being washed with appropriate Vectastain ABC kits labeled with either alkaline phosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate) or peroxidase (diaminobenzidine substrate; Vector). Goat anti-mouse TNF-
and goat anti-mouse VACM-1 were purchased from R&D Systems. Rabbit anti-mouse RelA was purchased from Research Diagnostics (Flanders, NJ). Antisera to irrelevant antigens were used as controls. To heighten contrast, no counterstains were used.
Cytokine assays.
Immunoreactive TNF-
, IL-1
, MIP-2, and KC were measured in lung homogenates by sandwich ELISA, using antibody pairs and recombinant standards purchased from R&D Systems. Freshly isolated left lungs were homogenized in PBS and then diluted 1:1 in lysis buffer containing 2x protease inhibitor cocktail. After 30 min of incubation on ice, the mixture was centrifuged at 1,200 g, and supernatants were stored at 80°C until assayed.
Data analysis.
Data are expressed as means ± SE. Statistical comparisons among groups for continuous variables measured at multiple time points were made by one-way ANOVA with Tukey's post hoc test. Comparisons of ordinal lung inflammation scores were made by Mann-Whitney's U-test. A P value of <0.05 was considered significant.
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RESULTS
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DnI
B-
inhibits IL-8 production in respiratory epithelial cells.
The dnI
B-
construct used to generate SPCdnI
B-
transgenic mice inhibits NF-
B activation in vitro (16), and we have previously shown that a recombinant adenovirus encoding dnI
B-
inhibits NF-
B activation, cytokine production, and inflammation in the liver in vivo (32). To determine whether this dnI
B-
construct could also block proinflammatory cytokine production by respiratory epithelial cells, we transduced type II-like A549 cells with a recombinant adenovirus encoding dnI
B-
or with a control adenovirus encoding
-galactosidase and then stimulated these cells with TNF-
or live P. aeruginosa. IL-8 production in response to these stimuli was abolished in cells transduced with the dnI
B-
adenovirus but not in cells transduced with the control
-galactosidase adenovirus (Table 1).
Characterization of SPCdnI
B-
transgenic mice.
DnI
B-
expressed under the control of the human SP-C promoter (Fig. 1A) was used to generate five lines of SPCdnI
B-
transgenic mice, two of which (lines 5990 and 6005) were selected for further study after initial screening. Transgene-positive mice from these lines expressed the transgene in their lungs but not in other tissues, as shown by Northern blot (Fig. 1B). The protein encoded by the transgene was detected in lung homogenates by Western blot (Fig. 1C) and by immunohistochemical staining of lung sections using a rabbit polyclonal IgG antibody to the HA tag (Fig. 2). In line 6006, there was substantial cytoplasmic and some nuclear staining in the majority of cells in the terminal bronchioles (Fig. 2, A and B). Staining of alveolar epithelial cells was detected but was not as distinct as the staining of the terminal airway epithelial cells (not shown). Expression of the transgene was not detected in other lung cells. Transgene-negative littermate controls showed minimal background staining (Fig. 2C). Similar results were obtained in the 5990 line, although the expression in the terminal bronchioles was not as intense (not shown). Thus dnI
B-
was selectively expressed in the distal lung epithelium of SPCdnI
B-
transgenic mice.

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Fig. 2. Detection of the transgene-encoded product in lung tissue by immunohistochemistry. A and B show results from a transgene-positive mouse from line 6006 and from a transgene-negative littermate control (C). Tissue sections were exposed to an antibody to the HA tag and processed as described in MATERIALS AND METHODS. Minimal background staining was evident in transgene-negative littermate controls (C) and in sections incubated with an irrelevant primary antibody (not shown). Original magnification, A = x20; B and C = x40.
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LPS-induced bronchiolar NF-
B activation is impaired in SPCdnI
B-
mice.
Nuclear translocation of NF-
B was detected by immunohistochemistry using an antibody to RelA. In unchallenged mice, RelA was not found in the nuclei of airway epithelial cells but was distributed diffusely and faintly in the cytosol (Fig. 3A). After inhalation of LPS, nuclear localization of RelA was evident in bronchiolar epithelial cells of transgene-negative littermate control mice (Fig. 3B), but RelA translocation was not observed in the airway epithelium of SPCdnI
B-
mice (Fig. 3C). Nuclear RelA was detected following LPS administration in some alveolar macrophages in both transgene-negative littermate controls and in SPCdnI
B-
mice (Fig. 3, B and C). It was technically difficult to localize RelA in alveolar epithelial cells by this method, which precluded determination of whether or not NF-
B activation occurred in type I or type II cells following inhalation of LPS. However, aerosolized LPS did not induce the nuclear translocation of RelA in proximal conducting airway epithelial cells of either control or transgenic mice (not shown). Minimal background staining was evident in sections incubated with the isotype control antibody (not shown). These findings indicate that inhalation of LPS was followed by NF-
B activation in bronchiolar epithelial cells and that dnI
B-
inhibited LPS-induced NF-
B translocation in these cells.
Reduced bronchoalveolar inflammatory response to aerosolized LPS in SPCdnI
B-
mice.
We used three complementary approaches to determine whether inhibition of NF-
B activation in the distal lung epithelium impaired pulmonary inflammation in response to inhaled LPS: enumeration of neutrophils in bronchoalveolar lavage (BAL) fluid, measurement of MPO activity in lung homogenates, and histopathological analysis of fixed lung tissue. As shown in Fig. 4, the inhalation of LPS resulted in prompt and sustained neutrophilic airway inflammation. However, the increase in the numbers of neutrophils and total cells in BAL samples harvested 4 h after inhalation of E. coli LPS was significantly blunted in SPCdnI
B-
mice from both the 5990 (Fig. 4A) and 6006 (Fig. 4B) transgenic lines compared with nontransgenic littermate controls. The increase in total cells after LPS exposure was attributable to the increase in neutrophils; there were no significant differences between transgenic and nontransgenic mice in the number of mononuclear cells in BAL specimens at either time point after inhalation of LPS.
We also measured the inflammatory responses to aerosolized P. aeruginosa LPS because P. aeruginosa is an important airway pathogen, particularly in the setting of cystic fibrosis, and because P. aeruginosa LPS has been shown to be less stimulatory than E. coli LPS in some settings (21). As with E. coli LPS, the accumulation of bronchoalveolar neutrophils in response to aerosolized P. aeruginosa LPS was significantly reduced in the SPCdnI
B-
mice (not shown). Thus the defective inflammatory response to inhaled endotoxin in SPCdnI
B-
mice was not limited to a particular type of LPS.
Lung MPO activity after inhalation of LPS is blunted in SPCdnI
B-
mice.
MPO activity, representing the total number of neutrophils present in lung tissue, was significantly diminished in SPCdnI
B-
mice compared with nontransgenic controls after inhalation of either E. coli LPS (Fig. 4C) or P. aeruginosa LPS (not shown). Thus the impaired accumulation of bronchoalveolar neutrophils in the SPCdnI
B-
mice reflects a reduction in the emigration of neutrophils from the blood, not merely defective migration across the epithelium.
Reduced lung tissue inflammation in response to LPS in SPCdnI
B-
mice.
No structural abnormalities or inflammation were evident in the lungs of unchallenged SPCdnI
B-
mice or transgene-negative controls as determined by histopathological examination of tissue sections (not shown). Inhalation of LPS induced robust neutrophilic inflammation in control animals, but this response was substantially reduced in the SPCdnI
B-
mice. Perivascular and peribronchial cuffs of leukocytes were less prominent and contained a lower proportion of neutrophils in SPCdnI
B-
mice (Fig. 5, B and D) than in the nontransgenic controls (Fig. 5, A and C). In more severe cases, the nontransgenic controls displayed peribronchiolar hemorrhage and, occasionally, small vessel thrombosis, features not observed in SPCdnI
B-
mice. Alveolar inflammation was mild in all animals but was less apparent in the transgenic mice. Engorgement of pulmonary lymphatics, a marker of edema formation, was more evident in sections from the nontransgenic mice than in sections from the SPCdnI
B-
mice.
The differences between SPCdnI
B-
and control mice in histological measures of inflammation are represented quantitatively in Fig. 6. The degree of perivascular and peribronchial cellular infiltration (Fig. 6A) and the ratios of neutrophils to mononuclear cells in these infiltrates (Fig. 6B) were significantly reduced both 4 and 24 h after inhalation of LPS in the SPCdnI
B-
mice. The alveolar inflammation scores were lower in the SPCdnI
B-
mice than in nontransgenic controls, but the trends did not reach statistical significance (Fig. 6C, P = 0.057 at 24 h).
Blunted intrapulmonary cytokine responses after inhalation of LPS in SPCdnI
B-
mice.
To explore mechanisms underlying the observed impairment in lung inflammation after inhalation of LPS in SPCdnI
B-
mice, we measured whole lung levels of early response cytokines known to be important in neutrophil recruitment, including the pluripotential mediators TNF-
and IL-1
and the ELR+ CXC chemokines MIP-2 and KC (Fig. 7). Low levels of these proteins were measured in lung tissue from unexposed animals. After exposure to aerosolized LPS, increased concentrations of TNF-
, IL-1
, MIP-2, and KC were detected in lung homogenates from both groups of mice 4 h later, but levels of all cytokines were significantly lower in lungs harvested from SPCdnI
B-
mice compared with nontransgenic animals. By 24 h after inhalation of LPS, pulmonary levels of these cytokines had declined to near the levels measured in unexposed mice. Thus the intrapulmonary cytokine response to LPS was significantly impaired in SPCdnI
B-
mice.
Impaired tissue expression of TNF-
and VCAM-1 after LPS in SPCdnI
B-
mice.
The defect in LPS-induced lung inflammation was paralleled by impaired induction of TNF-
in the bronchiolar epithelium and reduced adhesion molecule expression on lung vascular endothelium of SPCdnI
B-
mice. TNF-
was not detected by immunohistochemistry in the lungs of SPCdnI
B-
or littermate control mice before administration of LPS (not shown). However, after aerosol exposure to LPS, TNF-
was strongly expressed in bronchiolar epithelial cells of controls (Fig. 8A); this was not observed in SPCdnI
B-
mice (Fig. 8B). Similarly, lung vascular endothelial activation, as measured by the induction of VCAM-1, was clearly evident in the peribronchiolar vessels of controls following administration of LPS (Fig. 8C) but not in the peribronchiolar vasculature of SPCdnI
B-
mice (Fig. 8D). TNF-
expression by alveolar macrophages was evident in both transgenic and nontransgenic mice after LPS challenge (not shown).
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DISCUSSION
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The major finding of these studies is that targeted inhibition of NF-
B activation in distal airway epithelial cells leads to blunted local cytokine responses and impaired pulmonary inflammation after inhalation of LPS. To investigate the role of epithelial cells in the regulation of lung inflammation in vivo, we developed novel transgenic mice in which dnI
B-
was expressed under the control of the human SP-C promoter. Expression of the transgene in these mice was lung specific and localized to the epithelial cells lining the terminal bronchioles and the alveoli. SPCdnI
B-
mice exhibited impaired activation of NF-
B in distal airway epithelial cells after exposure to aerosolized LPS compared with nontransgenic littermates. This defect was accompanied by diminished LPS-induced neutrophilic inflammation of the lungs. The blunted inflammatory response in the transgenic mice was associated with lower whole lung levels of TNF-
, IL-1
, MIP-2, and KC as well as diminished epithelial cell production of TNF-
and reduced VCAM-1 expression by lung vascular endothelial cells. These data constitute conclusive evidence that distal airway epithelial cell activation contributes to the regulation of lung inflammation in vivo.
Our findings support earlier investigations that predicted a role for respiratory epithelial cells in regulating the pulmonary inflammatory response to LPS. First, respiratory epithelial cells can produce proinflammatory cytokines, release neutrophil chemotactic factors, and upregulate adhesion molecules upon stimulation in vitro. Several investigators have shown that A549 cells, a type II-like cell line derived from a human alveolar cell carcinoma (33), release CXC chemokines and neutrophil chemotactic activity upon stimulation with TNF-
or IL-1
in vitro (29, 53, 56). Tracheobronchial epithelial cells also can express IL-8 and other cytokines and release neutrophil chemotactic factors when stimulated with TNF-
and IL-1
(12, 13, 29, 37, 41). It is less clear that respiratory epithelial cells respond directly to LPS. Some investigators have reported that A549 cells (43, 45, 56) and tracheobronchial epithelial cells (17, 37, 41) do not respond to LPS in vitro, whereas others have described cytokine and neutrophil chemotaxin release by A549 cells and tracheobronchial epithelial cells stimulated with high concentrations of P. aeruginosa or E. coli LPS, particularly in the presence of serum (26, 27, 43, 51). Rat type II cells in primary culture have been shown to express monocyte chemotactic peptide-1 (42) and to upregulate ICAM-1 (4) on exposure to E. coli LPS, IL-1
, or TNF-
in vitro, although their responsiveness to LPS is limited to stimulation of the apical surface (47). One factor in these discrepant reports concerns the expression of LPS recognition molecules by the target cells that were studied. Some investigators have found that A549 cells and tracheobronchial epithelial cells express mRNA for TLR4 (6, 51), the signaling component of the LPS receptor complex (2). Other investigators have been unable to detect TLR4 expression by A549 cells (60). Nonetheless, these studies indicate that respiratory epithelial cells have the capacity to participate in the inflammatory response to LPS either by direct recognition of LPS or by amplifying signals initiated by other cells, such as alveolar macrophages, that are more sensitive to the presence of LPS. Our studies provide compelling evidence that distal airway epithelial cells contribute to LPS-induced lung inflammation but do not establish whether the epithelial cells respond directly to LPS or to secondary signals in vivo.
Additional evidence that respiratory epithelial cells play an active role in the pulmonary inflammatory response to LPS has come from in vivo observations that respiratory epithelial cells are stimulated after airway deposition of LPS and that artificial activation of NF-
B in the respiratory epithelium is sufficient to elicit neutrophilic pulmonary inflammation. Intratracheal instillation of LPS induces upregulation of ICAM-1 expression by alveolar and bronchiolar epithelium (4, 5, 9), suggesting proinflammatory activation of these cells. Airway challenge with LPS also induces I
B-
degradation and nuclear accumulation of NF-
B in lung tissue (8, 40, 49), and there is evidence for epithelial cell involvement in whole lung NF-
B activation. Using an NF-
B-luciferase reporter mouse, Hubbard and colleagues (22) found increased luciferase expression in alveolar macrophages and bronchiolar epithelium after intrapulmonary challenge with LPS. The important role of alveolar macrophages in the inflammatory response to LPS has been demonstrated: depletion of alveolar macrophages resulted in diminished whole lung NF-
B activation and neutrophilic inflammation after exposure of mice to aerosolized LPS (7, 24). However, a role for parenchymal cells in pulmonary NF-
B activation after inhalation of LPS was suggested by Alcamo et al. (3), using neonatal mice lacking both RelA and TNFR1. These animals were protected from the embryonic lethality of RelA deficiency but exhibited a severe defect in bronchoalveolar neutrophil accumulation after exposure to aerosolized LPS that was not evident in neonatal mice lacking only TNFR1. Interestingly, replacement of the alveolar macrophage population of lethally irradiated wild-type mice with hematopoietic progenitor cells harvested from RelA- or RelA/TNFR1-deficient mice yielded animals in which the inflammatory response to LPS was not significantly impaired. These studies suggested that pulmonary inflammation after inhalation of LPS is not dependent on RelA expression by alveolar macrophages but did not identify the lung parenchymal cells in which NF-
B activation was required. Finally, Sadikot et al. (48) reported that transduction of airway epithelial cells in vivo by intratracheal administration of recombinant adenoviral vectors encoding inhibitor of NF-
B kinase 1 (I
1) or I
2 resulted in constitutive expression of NF-
B-dependent luciferase activity, increased cytokine expression, and neutrophilic alveolar inflammation. Observations in this model system indicated that direct activation of NF-
B in airway epithelium can drive pulmonary inflammation. Our data show that this is physiologically relevant and that activation of NF-
B in distal airway epithelial cells is an important component of the coordinated inflammatory response to inhaled LPS.
Poynter and colleagues (44), using an approach similar to our own, recently reported that transgenic mice expressing a dominant negative I
B-
under control of the rat CC10 promoter exhibited impaired airway inflammation in association with reduced levels of MIP-2 and TNF-
in BAL fluid after nasal challenge with LPS. The rat CC10 promoter is expressed in transgenic mice in epithelial cells lining the trachea, bronchi, and larger bronchioles (58), whereas transgenes controlled by the human SP-C promoter are expressed in the epithelium of the terminal bronchioles and alveoli (62). Although overlap in the sites of transgene expression in these two studies cannot be excluded, our observations and those reported by Poynter et al. (44) suggest that both proximal and distal airway epithelial cells actively contribute to the inflammatory response to LPS.
Our results and those of Poynter et al. (44) suggest that NF-
B activation in respiratory epithelial cells contributes to the lung inflammatory response to inhaled LPS through the induction of proinflammatory cytokines, which in turn act to upregulate the expression of adhesion molecules on the vascular endothelium. We found that intrapulmonary production of early response cytokines (TNF-
and IL-1
) and of ELR+ CXC chemokines (MIP-2 and KC) were significantly blunted in transgenic mice after inhalation of LPS. Furthermore, by immunohistochemical analysis, we detected TNF-
in the bronchiolar epithelial cells of control mice after LPS exposure but not in the epithelium of SPCdnI
B-
mice. This finding is consistent with reports that bronchial epithelial cells can secrete TNF-
in response to Haemophilus influenzae LPS in vitro (23) and that lung infection with H. influenzae induces the expression of TNF-
in airway epithelial cells in vivo in a TLR4-dependent manner (61). We have previously shown that pulmonary inflammation in response to inhaled LPS is partially dependent on TNFR1-mediated signaling (52, 55). TNF-
promotes inflammation, in part, by upregulating the expression of adhesion molecules, including ICAM-1 and VCAM-1, which are involved in leukocyte emigration from the vascular compartment into the air spaces of the lungs (4, 5, 9, 10, 18, 28, 31, 46). We observed that expression of VCAM-1 on the peribronchiolar vascular endothelium was markedly induced in response to LPS in control but not in SPCdnI
B-
mice. VCAM-1 is a vascular endothelial ligand for
1-integrins, which act in concert with
2-integrins and their ligands to mediate neutrophil migration into the air spaces in response to LPS (10, 18, 28, 31, 46). By contrast, only
1-integrins appear to be involved in neutrophil recruitment to the lungs in response to the CXC chemokine KC (46). Both KC and MIP-2 participate in pulmonary neutrophil recruitment in response to LPS (20, 50), and the low levels of these chemokines in SPCdnI
B-
mice after inhalation of LPS may have contributed to the blunted inflammatory response in these animals.
For several reasons, our findings are likely to underestimate the relative contribution of the respiratory epithelium in the pulmonary inflammatory response to inhaled LPS. First, NF-
B activation was impeded only in the bronchiolar and alveolar epithelial cells in which the transgene was expressed. The contribution of tracheobronchial epithelial cells to the inflammatory response to LPS would not have been detected by this approach. Second, the inhibition of NF-
B activation in the targeted epithelial cells is likely to have been incomplete. Finally, only events that are dependent on NF-
B activation would have been impaired. Despite these limitations, our studies provide strong support for the conclusion that the respiratory epithelium plays an active role in the pulmonary inflammation induced by inhalation of LPS.
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GRANTS
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This work was supported in part by National Institutes of Health Grants HL-65898, HL-69503, GM-37696, and HL-54972 and a grant from the Cystic Fibrosis Foundation.
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ACKNOWLEDGMENTS
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The authors thank Michele Timko, Catherine Brissette, Heidi Harowicz, Mac Durning, Heidi Jessup, and Brooke Fallen for technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. J. Skerrett, Div. of Pulmonary and Critical Care Medicine, Harborview Medical Center, Box 359640, 325 Ninth Ave., Seattle, WA 98104 (E-mail: shawn{at}u.washington.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.
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REFERENCES
|
---|
- Aderem A and Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 406: 782787, 2000.[CrossRef][ISI][Medline]
- Akira S. Mammalian Toll-like receptors. Curr Opin Immunol 15: 511, 2003.[CrossRef][ISI][Medline]
- Alcamo E, Mizgerd JP, Horwitz BH, Bronson R, Beg AA, Scott M, Doerschuk CM, Hynes RO, and Baltimore D. Targeted mutation of TNF receptor I rescues the RelA-deficient mouse and reveals a critical role for NF-
B in leukocyte recruitment. J Immunol 167: 15921600, 2001.[Abstract/Free Full Text]
- Beck-Schimmer B, Madjdpour C, Kneller S, Ziegler U, Pasch T, Wuthrich RP, Ward PA, and Schimmer RC. Role of alveolar epithelial ICAM-1 in lipopolysaccharide-induced lung inflammation. Eur Respir J 19: 11421150, 2002.[Abstract/Free Full Text]
- Beck-Schimmer B, Schimmer RC, Warner RL, Schmal H, Nordblom G, Flory CM, Lesch ME, Friedl HP, Schrier DJ, and Ward PA. Expression of lung vascular and airway ICAM-1 after exposure to bacterial lipopolysaccharide. Am J Respir Cell Mol Biol 17: 344352, 1997.[Abstract/Free Full Text]
- Becker MN, Diamond G, Verghese MW, and Randell SH. CD14-dependent lipopolysaccharide-induced
-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem 275: 2973129736, 2000.[Abstract/Free Full Text]
- Berg JT, Lee ST, Thepen T, Lee CY, and Tsan MF. Depletion of alveolar macrophages by liposome-encapsulated dichloromethylene diphosphonate. J Appl Physiol 74: 28122819, 1993.[Abstract]
- Blackwell TS, Lancaster LH, Blackwell TR, Venkatakrishnan A, and Christman JW. Differential NF-
B activation after intratracheal endotoxin. Am J Physiol Lung Cell Mol Physiol 277: L823L830, 1999.[Abstract/Free Full Text]
- Burns AR, Takei F, and Doerschuk CM. Quantitation of ICAM-1 expression in mouse lung during pneumonia. J Immunol 153: 31893198, 1994.[Abstract/Free Full Text]
- Burns JA, Issekutz TB, Yagita H, and Issekutz AC. The
4
1 [very late antigen (VLA)-4, CD49d/CD29] and
5
1 (VLA-5, CD49e/CD29) integrins mediate
2 (CD11/CD18) integrin-independent neutrophil recruitment to endotoxin-induced lung inflammation. J Immunol 166: 46444649, 2001.[Abstract/Free Full Text]
- Chen SC, Mehrad B, Deng JC, Vassileva G, Manfra DJ, Cook DN, Wiekowski MT, Zlotnik A, Standiford TJ, and Lira SA. Impaired pulmonary host defense in mice lacking expression of the CXC chemokine lungkine. J Immunol 166: 33623368, 2001.[Abstract/Free Full Text]
- Cooper P, Potter S, Mueck B, Yousefi S, and Jarai G. Identification of genes induced by inflammatory cytokines in airway epithelium. Am J Physiol Lung Cell Mol Physiol 280: L841L852, 2001.[Abstract/Free Full Text]
- Cromwell O, Hamid Q, Corrigan CJ, Barkans J, Meng Q, Collins PD, and Kay AB. Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1
and tumour necrosis factor-
. Immunol 77: 330337, 1992.[ISI][Medline]
- Darveau RP and Hancock RE. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol 155: 831838, 1983.[ISI][Medline]
- Diamond G, Legarda D, and Ryan LK. The innate immune response of the respiratory epithelium. Immunol Rev 173: 2738, 2000.[CrossRef][ISI][Medline]
- DiDonato J, Mercurio F, Rosette C, Wu-Li J, Suyang H, Ghosh S, and Karin M. Mapping of the inducible I
B phosphorylation sites that signal its ubiquitination and degradation. Mol Cell Biol 16: 12951304, 1996.[Abstract]
- DiMango E, Zar HJ, Bryan R, and Prince A. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest 96: 22042210, 1995.[ISI][Medline]
- Doerschuk CM, Winn RK, Coxson HO, and Harlan JM. CD18-dependent and -independent mechanisms of neutrophil emigration in the pulmonary and systemic microcirculation of rabbits. J Immunol 144: 23272333, 1990.[Abstract/Free Full Text]
- Ernst RK, Yi EC, Guo L, Lim KB, Burns JL, Hackett M, and Miller SI. Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa. Science 286: 15611565, 1999.[Abstract/Free Full Text]
- Frevert CW, Huang S, Danaee H, Paulauskis JD, and Kobzik L. Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation. J Immunol 154: 335344, 1995.[Abstract/Free Full Text]
- Hajjar AM, Ernst RK, Tsai JH, Wilson CB, and Miller SI. Human Toll-like receptor 4 recognizes host-specific LPS modifications. Nat Immun 3: 354359, 2002.[CrossRef][ISI]
- Hubbard AK, Timblin CR, Shukla A, Rincon M, and Mossman BT. Activation of NF-
B-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol 282: L968L975, 2002.[Abstract/Free Full Text]
- Khair OA, Devalia JL, Abdelaziz MM, Sapsford RJ, Tarraf H, and Davies RJ. Effect of Haemophilus influenzae endotoxin on the synthesis of IL-6, IL-8, TNF-
and expression of ICAM-1 in cultured human bronchial epithelial cells. Eur Respir J 7: 21092116, 1994.[Abstract/Free Full Text]
- Koay MA, Gao X, Washington MK, Parman KS, Sadikot RT, Blackwell TS, and Christman JW. Macrophages are necessary for maximal nuclear factor-
B activation in response to endotoxin. Am J Respir Cell Mol Biol 26: 572578, 2002.[Abstract/Free Full Text]
- Korfhagen TR, Glasser SW, Wert SE, Bruno MD, Daugherty CC, McNeish JD, Stock JL, Potter SS, and Whitsett JA. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice. Proc Natl Acad Sci USA 87: 61226126, 1990.[Abstract]
- Koyama S, Rennard SI, Leikauf GD, Shoji S, Von Essen S, Claassen L, and Robbins RA. Endotoxin stimulates bronchial epithelial cells to release chemotactic factors for neutrophils. A potential mechanism for neutrophil recruitment, cytotoxicity, and inhibition of proliferation in bronchial inflammation. J Immunol 147: 42934301, 1991.[Abstract/Free Full Text]
- Koyama S, Sato E, Nomura H, Kubo K, Miura M, Yamashita T, Nagai S, and Izumi T. The potential of various lipopolysaccharides to release IL-8 and G-CSF. Am J Physiol Lung Cell Mol Physiol 278: L658L666, 2000.[Abstract/Free Full Text]
- Kumasaka T, Quinlan WM, Doyle NA, Condon TP, Sligh J, Takei F, Beaudet AL, Bennett CF, and Doerschuk CM. Role of the intercellular adhesion molecule-1 (ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice. J Clin Invest 97: 23622369, 1996.[Abstract/Free Full Text]
- Kwon OJ, Au BT, Collins PD, Adcock IM, Mak JC, Robbins RR, Chung KF, and Barnes PJ. Tumor necrosis factor-induced interleukin-8 expression in cultured human airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 267: L398L405, 1994.[Abstract/Free Full Text]
- Li Q and Verma IM. NF-
B regulation in the immune system. Nat Rev Immunol 2: 725734, 2002.[CrossRef][ISI][Medline]
- Li XC, Miyasaka M, and Issekutz TB. Blood monocyte migration to acute lung inflammation involves both CD11/CD18 and very late activation antigen-4-dependent and independent pathways. J Immunol 161: 62586264, 1998.[Abstract/Free Full Text]
- Lieber A, He CY, Meuse L, Himeda C, Wilson C, and Kay MA. Inhibition of NF-
B activation in combination with bcl-2 expression allows for persistence of first-generation adenovirus vectors in the mouse liver. J Virol 72: 92679277, 1998.[Abstract/Free Full Text]
- Lieber M, Smith B, Szakal A, Nelson-Rees W, and Todaro G. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer 17: 6270, 1976.[ISI][Medline]
- Lorenz E, Jones M, Wohlford-Lenane C, Meyer N, Frees KL, Arbour NC, and Schwartz DA. Genes other than TLR4 are involved in the response to inhaled LPS. Am J Physiol Lung Cell Mol Physiol 281: L1106L1114, 2001.[Abstract/Free Full Text]
- Martin TR. Recognition of bacterial endotoxin in the lungs. Am J Respir Cell Mol Biol 23: 128132, 2000.[Free Full Text]
- Martin TR, Mathison JC, Tobias P, Leturcq DJ, Moriarty AM, Maunder RJ, and Ulevitch RJ. Lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. Implications for cytokine production in normal and injured lungs. J Clin Invest 90: 22092219, 1992.[ISI][Medline]
- Massion PP, Inoue H, Richman-Eisenstat J, Grunberger D, Jorens PG, Housset B, Pittet JF, Wiener-Kronish JP, and Nadel JA. Novel Pseudomonas product stimulates interleukin-8 production in airway epithelial cells in vitro. J Clin Invest 93: 2632, 1994.[ISI][Medline]
- McCormack FX and Whitsett JA. The pulmonary collectins, SP-A and SP-D, orchestrate innate immunity in the lung. J Clin Invest 109: 707712, 2002.[Free Full Text]
- Mizgerd JP. Molecular mechanisms of neutrophil recruitment elicited by bacteria in the lungs. Semin Immunol 14: 123132, 2002.[CrossRef][ISI][Medline]
- Mizgerd JP, Scott ML, Spieker MR, and Doerschuk CM. Functions of I
B proteins in inflammatory responses to Escherichia coli LPS in mouse lungs. Am J Respir Cell Mol Biol 27: 575582, 2002.[Abstract/Free Full Text]
- Nakamura H, Yoshimura K, Jaffe HA, and Crystal RG. Interleukin-8 gene expression in human bronchial epithelial cells. J Biol Chem 266: 1961119617, 1991.[Abstract/Free Full Text]
- Paine R III, Rolfe MW, Standiford TJ, Burdick MD, Rollins BJ, and Strieter RM. MCP-1 expression by rat type II alveolar epithelial cells in primary culture. J Immunol 150: 45614570, 1993.[Abstract/Free Full Text]
- Peralta FM and Casale TB. Orientation and presence of epithelium are key to endotoxin-induced neutrophil migration. Eur Respir J 11: 10531059, 1998.[Abstract/Free Full Text]
- Poynter ME, Irvin CG, and Janssen-Heininger YM. A prominent role for airway epithelial NF-
B activation in lipopolysaccharide-induced airway inflammation. J Immunol 170: 62576265, 2003.[Abstract/Free Full Text]
- Pugin J, Schurer-Maly CC, Leturcq D, Moriarty A, Ulevitch RJ, and Tobias PS. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci USA 90: 27442748, 1993.[Abstract]
- Ridger VC, Wagner BE, Wallace WA, and Hellewell PG. Differential effects of CD18, CD29, and CD49 integrin subunit inhibition on neutrophil migration in pulmonary inflammation. J Immunol 166: 34843490, 2001.[Abstract/Free Full Text]
- Rose F, Guthmann B, Tenenbaum T, Fink L, Ghofrani A, Weissmann N, Konig P, Ermert L, Dahlem G, Haenze J, Kummer W, Seeger W, and Grimminger F. Apical, but not basolateral, endotoxin preincubation protects alveolar epithelial cells against hydrogen peroxide-induced loss of barrier function: the role of nitric oxide synthesis. J Immunol 169: 14741481, 2002.[Abstract/Free Full Text]
- Sadikot RT, Han W, Everhart MB, Zoia O, Peebles RS, Jansen ED, Yull FE, Christman JW, and Blackwell TS. Selective I
B kinase expression in airway epithelium generates neutrophilic lung inflammation. J Immunol 170: 10911098, 2003.[Abstract/Free Full Text]
- Sadikot RT, Jansen ED, Blackwell TR, Zoia O, Yull F, Christman JW, and Blackwell TS. High-dose dexamethasone accentuates nuclear factor-
B activation in endotoxin-treated mice. Am J Respir Crit Care Med 164: 873878, 2001.[Abstract/Free Full Text]
- Schmal H, Shanley TP, Jones ML, Friedl HP, and Ward PA. Role for macrophage inflammatory protein-2 in lipopolysaccharide-induced lung injury in rats. J Immunol 156: 19631972, 1996.[Abstract]
- Schulz C, Farkas L, Wolf K, Kratzel K, Eissner G, and Pfeifer M. Differences in LPS-induced activation of bronchial epithelial cells (BEAS-2B) and type II-like pneumocytes (A-549). Scand J Immunol 56: 294302, 2002.[CrossRef][ISI][Medline]
- Skerrett SJ, Martin TR, Chi EY, Peschon JJ, Mohler KM, and Wilson CB. Role of the type 1 TNF receptor in lung inflammation after inhalation of endotoxin or Pseudomonas aeruginosa. Am J Physiol Lung Cell Mol Physiol 276: L715L727, 1999.[Abstract/Free Full Text]
- Smart SJ and Casale TB. Pulmonary epithelial cells facilitate TNF-
-induced neutrophil chemotaxis. A role for cytokine networking. J Immunol 152: 40874094, 1994.[Abstract/Free Full Text]
- Smith S, Liggitt D, Jeromsky E, Tan X, Skerrett SJ, and Wilson CB. Local role for tumor necrosis factor
in the pulmonary inflammatory response to Mycobacterium tuberculosis infection. Infect Immun 70: 20822089, 2002.[Abstract/Free Full Text]
- Smith S, Skerrett SJ, Chi EY, Jonas M, Mohler K, and Wilson CB. The locus of tumor necrosis factor-
action in lung inflammation. Am J Respir Cell Mol Biol 19: 881891, 1998.[Abstract/Free Full Text]
- Standiford TJ, Kunkel SL, Basha MA, Chensue SW, Lynch JP III, Toews GB, Westwick J, and Strieter RM. Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung. J Clin Invest 86: 19451953, 1990.[ISI][Medline]
- Strieter RM, Belperio JA, and Keane MP. Cytokines in innate host defense in the lung. J Clin Invest 109: 699705, 2002.[Free Full Text]
- Stripp BR, Sawaya PL, Luse DS, Wikenheiser KA, Wert SE, Huffman JA, Lattier DL, Singh G, Katyal SL, and Whitsett JA. Cis-acting elements that confer lung epithelial cell expression of the CC10 gene. J Biol Chem 267: 1470314712, 1992.[Abstract/Free Full Text]
- Tsai WC, Strieter RM, Wilkowski JM, Bucknell KA, Burdick MD, Lira SA, and Standiford TJ. Lung-specific transgenic expression of KC enhances resistance to Klebsiella pneumoniae in mice. J Immunol 161: 24352440, 1998.[Abstract/Free Full Text]
- Tsutsumi-Ishii Y and Nagaoka I. Modulation of human
-defensin-2 transcription in pulmonary epithelial cells by lipopolysaccharide-stimulated mononuclear phagocytes via proinflammatory cytokine production. J Immunol 170: 42264236, 2003.[Abstract/Free Full Text]
- Wang X, Moser C, Louboutin JP, Lysenko ES, Weiner DJ, Weiser JN, and Wilson JM. Toll-like receptor 4 mediates innate immune responses to Haemophilus influenzae infection in mouse lung. J Immunol 168: 810815, 2002.[Abstract/Free Full Text]
- Wert SE, Glasser SW, Korfhagen TR, and Whitsett JA. Transcriptional elements from the human SP-C gene direct expression in the primordial respiratory epithelium of transgenic mice. Dev Biol 156: 426443, 1993.[CrossRef][ISI][Medline]