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Airway Epithelium Controls Lung Inflammation and Injury through the NF-B Pathway¹

Dong-sheng Cheng^*, Wei Han^*, Sabrina M. Chen^*, Taylor P. Sherrill^*, Melissa Chont, Gye-Young Park^¶, James R. Sheller^*, Vasiliy V. Polosukhin^*, John W. Christman^,¶, Fiona E. Yull^2, and Timothy S. Blackwell^2,3,*,,,

^* Division of Allergy, Pulmonary and Critical Care Medicine, Department of Medicine, Department of Cancer Biology, and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; and Department of Veterans Affairs and ^¶ Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois, Chicago, IL 60605

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-B pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-B activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-B activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-B in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-B pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.

	Introduction

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The NF-B pathway impacts a number of key biological processes, including innate immunity, through transcriptional regulation of target genes. Following cell stimulation, IBs are phosphorylated on serine residues in the amino terminus by IB kinase 2 (IKK2),⁴ targeting them for destruction by the ubiquitin/proteasome (26S) degradation pathway (1). IB degradation allows NF-B nuclear translocation and transcriptional activation of a variety of genes, including cytokines, chemokines, and adhesion molecules (2, 3). In the lungs, many noxious/inflammatory stimuli have been shown to activate NF-B, including bacterial products, ozone and silica, as well as systemic inflammatory insults such as sepsis, trauma, and hemorrhage.

Innate immunity is critical for host defense against bacterial pathogens, but dysregulated or exaggerated immune responses can result in tissue injury. In the lungs, this form of host-derived tissue injury characterizes the acute respiratory distress syndrome (ARDS). ARDS is a common cause of morbidity and mortality in critically ill patients, resulting from local or systemic infection, trauma, or other inflammatory/injurious stimuli (4, 5). The inflammatory phenotype underlying the pathogenesis of ARDS includes neutrophilic alveolitis and increased levels of a number of cytokines and chemokines in the airways (4, 6). Improved understanding of critical cell types and biological pathways that regulate innate immunity in the lungs could be useful in designing therapies to limit or prevent lung injury in patients at risk for ARDS.

Airway epithelium provides a physical border between host and environment that protects from injurious and infectious stimuli that gain access to the respiratory tract through inspiration or aspiration. Well-recognized functions of airway epithelium include mechanical clearance of offending agents and production of antimicrobial agents; however, critical functions for coordinating the innate immune response or development of lung injury have not been identified. Airway epithelial cells express a number of TLRs, and we have recently shown that local and systemic inflammation results in prominent activation of the NF-B pathway in these cells (7, 8, 9). Therefore, we hypothesized that epithelial cells in the lung are important for transducing NF-B-dependent inflammatory signals and that prolonged NF-B activation in airway epithelial cells leads to a dysfunctional (injurious) inflammatory response culminating in lung injury. To evaluate whether airway epithelial cells critically regulate lung inflammation and injury, we generated inducible transgenic mice that express an activator or dominant inhibitor of the NF-B pathway in CC10-expressing airway epithelial cells. We then determined the effects of cell type-specific NF-B activation or inhibition on parameters of lung inflammation and injury. Our data indicate that activation of NF-B in airway epithelial cells is sufficient for generating acute lung injury, and inhibition of NF-B activation in airway epithelium abrogates lung inflammation and injury induced by Gram-negative bacterial LPS. These findings suggest a paradigm in which airway epithelial cells control parenchymal lung inflammation and injury via production of NF-B-dependent mediators.

	Materials and Methods

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Transgenic mouse models

IKTA-transgenic mice. The pBSIIFlag-IKK2 plasmid containing FLAG-cIKK2, a constitutively active form of human IKK2 containing S177E and S181E mutations, was a gift from Dr. F. Mercurio (Signal Pharmaceutical, San Diego, CA). This plasmid was digested with BssHII to obtain a fragment containing the FLAG-IKK2. The ends of this fragment were filled in before ligation into the EcoRV site of a modified pBluescript II SK expression vector (pBSII KS/Asc). This vector contains a (tet-O)₇-CMV promoter that consists of seven copies of the tet operator DNA-binding sequence linked to a minimal CMV promoter together with bovine growth hormone polyadenylation sequences to ensure transcript termination. The final plasmid ((tet-O)₇-FLAG-cIKK2-BGH.poly(A)) was verified by sequencing. To prevent basal leakiness, we used a construct expressing a tetracycline-controlled transcriptional silencer (tTS) under control of the CC10 promoter (CC10-tTS-hGH-poly(A)) (10). The (tet-O)₇-FLAG-cIKK2 microinjection fragment was excised from the plasmid as a 3.3-kb XmnI-AscI fragment. We purified both CC10-tTS and (tet-O)₇-FLAG-cIKK2 constructs using a GELase Agarose Gel-Digesting preparation kit following the manufacturer’s instruction (Epicentre), and these constructs were coinjected at the Vanderbilt transgenic/ES cell shared resource to generate transgenic lines of mice (FVB background) that have cointegrated both the CC10-tTS and (tet-O)₇-FLAG-cIKK2 transgenes. Genotyping of founder animals was performed by Southern Blot and later stages of genotyping were performed by PCR analysis. Primers used for PCR of the (tet-O)₇-FLAG-cIKK2 transgene are as follows: 5' primer (located in the CMV minimal promoter) 5'-GAC GCC ATC CAC GCT GTT TTG-3'; and 3' primer (located in the constitutively active form of IB kinase 2 (cIKK2) coding region) 5'-CTT CTC ATG ATC TGG ATC TCC-3'. The product size is 452 bp. Primers used for identification of cc10-tTS transgene are as follows: upstream 5'-GAG TTG GCA GCA GTT TCT CC-3'; and downstream 5'-GAG CAC AGC CAC ATC TTC AA-3'. The product size is 472 bp. PCR protocols for both (tet-O)₇-FLAG-cIKK2 and CC10-tTS were as follows: 1 cycle 94°C for 2 min; 30 cycles at 94°C for 1 min, 56°C for 30 s, and 72°C for 1 min; and 1 cycle at 72°C for 10 min. Mice transgenic for CC10-tTS/(tet-O)₇-FLAG-cIKK2 were mated with cc10-rTTA homozygous mice (gift from Dr. J. A. Whitsett, University of Cincinnati, Cincinnati, OH) to obtain transgenic mice carrying all three transgenes, which were designated IKTA mice. IKTA mice from three separate founder lines were used for these studies.

IB-DN-transactivated (DNTA) transgenic mice. To tag the IB- dominant inhibitor (IB-DN) (8, 11, 12), a 1.35-kb BamHI/DraI fragment was excised from pCMX-pp40 and blunt-end ligated into BamHI/EcoRV-digested pEF4/Myc-HisA (Invitrogen Life Technologies). The IB-DN-Myc-His-tagged fragment was then excised by BamHI digest, fill-in of the overhanging ends and PmeI digestion. The resulting fragment was blunt-end ligated into the pBSII KS/Asc vector described above, which had been PstI digested and filled in. Plasmid constructs were verified by sequencing. A 2.1-kb AscI microinjection fragment was prepared and coinjected with the CC10-tTS microinjection fragment as described above at the Vanderbilt-transgenic/ES cell shared resource to generate transgenic lines of mice (FVB background) that have cointegrated both the CC10-tTS and (tet-O)₇-IB-DN-Myc-His transgenes. Genotyping of founder animals was performed by Southern blot analysis, and later stages of genotyping were performed by PCR analysis. Primers used for PCR of the (tet-O)₇-IB-DN-Myc-His transgene are as follows: sense primer, 5'-TGA GGA TGA GGA GAG CAG TGA ATC-3'; and antisense primer, 5'-CAC CCC CCA GAA TAG AAT GAC AC-3'. The product size is 422 bp. Primers used for identification of CC10-tTS transgene (as above). PCR protocols for (tet-O)₇-IB-DN-Myc-His was as follows: 1 cycle 95°C for 4 min; 30 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and 1 cycle at 72°C for 10 min. Mice transgenic for CC10-tTS/(tet-O)₇-IB-DN-Myc-His were mated with CC10-rTTA homozygous mice to obtain transgenic mice carrying all three transgenes, which were designated DNTA mice. DNTA mice from two separate founder lines were used for these studies.

Doxycycline (dox) treatment. All IKTA or DNTA mice (or appropriate controls) were maintained on normal water until transgene activation was desired. At that time, 2 g/L freshly prepared dox (Sigma-Aldrich) was added to the animals’ drinking water. Sucrose (2%) was also added to decrease the bitter taste of dox water. The bottle with dox and 2% sucrose water was wrapped with foil to prevent light-induced dox degradation, and dox water was replaced twice per week.

LPS models

Male and female mice weighing between 20 and 25 grams were used for these studies. Escherichia coli LPS (serotype 055:B5) was obtained from Sigma-Aldrich. For studies involving aerosolized LPS, 8 ml of a 1 µg/µl LPS solution in PBS was delivered by ultrasonic nebulization in a closed chamber for 30 min using a previously published methodology (13). To deliver systemic LPS, an osmotic pump (2001D Alzet pump; ALZA) filled with LPS solution (1 µg/µl in PBS) was implanted surgically in the peritoneal cavity using sterile technique (9). The pump delivered 8 µg of LPS (8 µl)/h for 24 h. In some experiments, osmotic pumps (1003D) were used. These pumps were filled with LPS solution (8 µg/µl in PBS) to deliver 8 µg/h (1 µl/h) over 72 h. A priming dose of 3 µg of LPS/g body weight was injected i.p. following pump implantation.

Implantation of carotid artery catheter and blood gas analysis

The common carotid artery was separated from the vagus nerve and muscle, and then, two 6-0 silk threads were passed under the artery. The cephalic thread was tied to prevent bleeding, and then, the artery was clamped by small bulldog clamp. A small incision was made just below the ligature, and the catheter was inserted into the lumen. The clamp was taken off, and the catheter was pushed in 10 mm. The catheter was fixed with a second thread and the thread previously used to prevent bleeding. A blunt 16-gauge needle was carefully inserted through the incision and pushed s.c. until the end protruded through the incision in the neck. The incision in the skin was then sutured, and the catheter was connected to a stainless steel tube. The implanted catheter was flushed with saline containing 200 IU heparin/ml and 1 mg ampicillin/ml every day. For blood gas analysis, 100 µl of arterial blood was collected via the catheter and immediately placed in ice. Blood gas analysis was done using an ABL-5 blood gas machine (Radiometer America) at 37°C. Before each measurement, the blood gas machine was calibrated with a standard solution.

Lung histology and immunohistochemistry

Lungs were inflated and fixed with 1 ml of 10% formalin and then were removed en bloc after tracheal ligation. For immunohistochemical analysis, 5-µm paraffin sections were deparaffinized, dehydrated, washed with PBS, treated with 0.05% trypsin, and incubated with 1% BSA in PBS for 20 min before incubation with rabbit polyclonal anti-FLAG Ab (Rockland) or rabbit polyclonal anti-myc Ab (Santa Cruz Biotechnology). After incubation with primary Ab, a standard avidin-biotin complex protocol (Vectastain ABC kit; Vector Laboratories) using anti-rabbit secondary Abs was used. TUNEL assays were performed using a commercially available kit in accordance with the manufacturer’s directions (In Situ Cell Death Detection kit; Roche Molecular Biochemicals). Semiquantitative scoring of TUNEL-positive cells was performed on histological specimens by a pathologist blinded to the genotype and treatment group. Ten sequential, nonoverlapping tissue fields of lung parenchyma were evaluated under x400 magnification. Each tissue field was scored using a 0–4 point system (0, no positive cells; 1, 1% positive cells; 2, 1–5% positive cells; 3, 5–10% positive cells; and 4, 10–25% positive cells). A mean score for all fields was calculated for each animal.

EMSA

Tissue nuclear proteins were extracted from whole lung tissue by the method described previously (14). After preparation of nuclear protein extract, EMSA for NF-B-binding activity was performed using oligonucleotides containing a consensus NF-B-binding sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3').

Western blot analysis

Protein extracts from tissue homogenates (100 µg) were separated on a polyacrylamide gel and transblotted for detection of FLAG-cIKK2 or IB-DN-Myc-His. For FLAG-cIKK2, proteins were separated on a 10% acrylamide gel, and anti-FLAG Abs (anti-FLAG conjugated with HRP M₂ mAb; Sigma-Aldrich) were used. HRP was detected by chemoluminescence using Lumi-Light^PLUS Western blotting substrate (Roche Diagnostics). For IB-DN-Myc-His, proteins were separated on a 12% polyacrylamide gel, and monoclonal anti-myc Abs (Sigma-Aldrich) were used for immunodetection. For detection of RelA in lung tissue nuclear protein fractions, nuclear proteins were prepared as previously described (14), 20 µg of protein was separated on a 10% acrylamide gel, and RelA was immunodetected using rabbit polyclonal anti-RelA Abs (Santa Cruz Biotechnology). TATA-binding protein was detected as a loading control using specific Abs (Santa Cruz Biotechnology).

RNA isolation and RNase protection assay (RPA)

Lung tissue was homogenized in TRIzol reagent (Invitrogen Life Technologies), and RNA was isolated following the manufacturer’s instructions. RPA using chemokine template mCK-5 was done with the RiboQuant multiprobe RPA system (BD Pharmingen) according to the manufacturer’s direction.

Total and differential cell counts and protein measurement in lung lavage

Lung lavage was performed with 3 aliquots of 800 µl of sterile normal saline. Fluid was combined and centrifuged at 400 x g for 10 min to separate cells from supernatant. Supernatant was stored at –70°C for cytokine and chemokine measurements. The total and differential cell counts were done as described previously (12). Protein concentration was quantified using the Bradford assay (Bio-Rad).

Lung wet/dry ratio measurement

Lungs were removed and the wet weight recorded. Lungs were then placed in an incubator at 65°C for 48 h, and the dry weight was determined.

Cytokine and chemokine measurements

Measurement of cytokines and chemokines in lung lavage fluid and cell culture supernatant was done using the Bio-plex mouse cytokine 23-plex kit (Bio-Rad) following the manufacturer’s direction and using Luminex technology. MIP-2 and KC levels were measured using a specific ELISA according to the manufacturer’s instructions (R&D Systems).

Tracheal epithelial cell culture

Mouse tracheal epithelial cell (MTEC) culture was done by following the previously published protocol with minor modification (15). After removing muscle and vessels, tracheas were incubated in Ham’s F-12 pen-strep containing 1.5 mg/ml pronase (Roche Molecular Biochemicals) for 18 h at 4°C to dislodge the epithelial cells. Cells were treated with 0.5 mg/ml crude pancreatic DNase I (Sigma-Aldrich) on ice for 5 min. After incubation in tissue culture plates (BD Biosciences) for 3–4 h in 5% CO₂ at 37°C to adhere fibroblasts, nonadherent cells were collected by centrifugation. Supported polycarbonate and polyester porous (0.4 µM pores) membranes (Transwell; Corning-Costar) were coated with type I rat tail collagen (BD Biosciences) in 0.02 N acetic acid for 18 h at 25°C. Membranes were seeded with cells and incubated with DMEM-Ham’s F-12 medium containing 15 mM HEPES, 3.6 mM sodium bicarbonate, 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.25 µg/ml Fungizone, 10 µg/ml insulin, 5 µg/ml transferrin, 0.1 µg/ml cholera toxin, and 25 ng/ml epidermal growth factor (BD Biosciences) and 30 µg/ml bovine pituitary extract, 5% FBS, and freshly added 0.01 µM retinoic acid, filling upper and lower chambers in 5% CO₂ at 37°C. Media were changed every 2 days until the transmembrane resistance (R_t) was >1000 · cm², as measured by an epithelial Ohm-voltmeter (World Precision Instruments). Media were then removed from the upper chamber to establish an air-liquid interface, and lower chambers only were provided fresh DMEM-Ham’s F-12 medium supplemented with 2% NuSerum (BD Biosciences) and 0.01 µM retinoic acid.

Membrane cultures were prepared for scanning electron microscopy, as described previously (16). Briefly, samples were fixed with 2.5% glutaraldehyde, stained with 1.25% osmium tetroxide, critical point dried under liquid carbon dioxide, gold sputter coated, and visualized on a Hitachi S-3000N microscope (Hitachi).

For immunofluorescent detection of FLAG-cIKK2 expression, membranes were fixed with 4% paraformaldehyde (pH 7.4) for 10 min at 25°C and washed in PBS. A piece of membrane was cut and used for staining. Nonspecific Ab binding was blocked using 5% nonspecific serum and 3% BSA in PBS for 30 min at 25°C. Samples were incubated for 18 h at 4°C with anti-FLAG M2-FITC conjugate Ab (Sigma-Aldrich) in blocking solution. Membranes were mounted on slides with VectaShield (Vector Laboratories) containing 4',6-diamidino-2-phenylindole to stain intracellular DNA. The microscopic images were obtained by using a Zeiss LSM 510 confocal microscope (Zeiss).

Statistical analysis

To assess differences among groups, analyses were performed with GraphPad Instat (GraphPad) using an unpaired t test or one-way ANOVA. Mortality differences were evaluated using a Fisher’s exact test. Results are presented as mean ± SEM. Two-tailed p values <0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Construction of transgenic mice with inducible activation of NF-B in airway epithelium

To achieve inducible NF-B activation using the tet-on system, we placed a FLAG-tagged cIKK2 (1) under control of the (tet-O)₇-CMV promoter (Fig. 1a). To prevent basal leakiness of transgene expression, a construct expressing tetracycline-controlled tTS under the control of the Clara cell-specific CC10 promoter (obtained from Dr. J. Elias, Yale University (New Haven, CT), with permission of A. Farmer, BD Clontech) was coinjected with (tet-O)₇-FLAG-cIKK2 to generate double transgenic mice. Unbound tTS interacts with tet-O sites and functions as a transcriptional repressor; however, binding of dox to tTS results in dissociation from DNA, allowing rtTA binding and promoter activation (10, 17, 18). Double transgenic mice were bred with transgenic mice expressing rtTA under the control of the rat CC10 promoter (obtained from Dr. J. A. Whitsett) to generate triple transgenic mice, which were designated IKTA (for cIKK2 transactivated).

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Dox-induced expression of FLAG-cIKK2 is localized to lung epithelial cells and sufficient to activate NF-B. a, Schematic for construction of IKTA transgenic mice. b, Western blot analysis for FLAG-cIKK2 expression in tissue homogenates obtained from untreated IKTA mice, triple transgenic IKTA mice treated with dox for 3 days, or double transgenic (tet-O)7-FLAG-cIKK2/CC10-tTS mice treated with dox. Transgene expression is detected only in the lungs of IKTA mice following dox treatment. c, Immunohistochemistry for FLAG in lung tissue from an untreated IKTA mouse (left panel) or an IKTA mouse treated with dox for 3 days (right panel). FLAG-cIKK2 staining (brown stain) is localized exclusively in airway epithelial cells in dox-treated IKTA mice. d, EMSA for NF-B binding using lung nuclear protein extracts from WT control mice treated with dox (WT + dox), untreated IKTA mice (IKTA), and IKTA mice treated with dox for 3 days (IKTA + dox). Increased intensity of both NF-B bands (RelA/p50 and p50/p50) is present in the IKTA + dox group.

Sustained NF-B activation in airway epithelium results in neutrophilic lung inflammation and severe lung injury

Although untreated IKTA mice exhibited normal lung histology, IKTA mice showed progressive lung inflammation and injury after 3 and 7 days of dox treatment (Fig. 2a). After 3 days of dox treatment, lungs from IKTA mice showed evidence of edema and a cellular infiltrate consisting of neutrophils and macrophages. By 7 days of dox treatment, however, a massive infiltration of inflammatory cells into the lung parenchyma was present, along with septal thickening, edema, and alveolar hemorrhage. Other organs, including liver, spleen, and kidney, showed no evidence of inflammation or architectural abnormalities (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 2. NF-B activation in airway epithelium results in progressive lung inflammation and injury. a, H&E-stained lung sections from untreated IKTA mice and IKTA mice (line 3) treated with dox for 3 or 7 days. Although untreated IKTA mice have normal lung histology, a progressive inflammatory cell infiltrate is observed at 3 and 7 days after dox treatment, along with hemorrhage and edema at 7 days. b, Total neutrophils, macrophages, and lymphocytes in lung lavage from IKTA mice treated with dox for 3 or 7 days compared with WT mice treated with dox for 7 days (n = 3-4/group, *, p < 0.05 compared with WT, **, p < 0.05 compared with WT and IKTA mice treated with dox for 3 days). c, Lung lavage protein concentration in untreated WT and IKTA mice (day 0) and in both groups after 3 or 7 days of dox treatment (n = 4/group, *, p < 0.01 compared with WT). d, Multiprobe RNase protection assays for chemokines from lungs of IKTA or WT mice treated with dox for 7 days and IKTA mice without the addition of dox to drinking water. Each lane represents mRNA from a separate mouse.

Activation of NF-B in airway epithelium resulted in production of a variety of inflammatory mediators. Table I shows the profile of cytokines and chemokines up-regulated in lung lavage from IKTA mice treated with dox for 7 days compared with dox-treated WT mice and IKTA mice without dox treatment. Significantly increased levels of IL-1, IL-1, IL-5, IL-6, IL-12, IL-17, RANTES, MIP-2, KC, MCP-1, and G-CSF were observed in dox-treated IKTA mice. No differences in mediator production were identified between WT mice and IKTA mice in the absence of dox treatment. We used multiprobe RNase protection assays to confirm that mRNA expression of selected chemokines was increased in the lungs of dox-treated IKTA mice (Fig. 2d). Protein and mRNA measurements of mediators correlated well with the exception of MCP-1, which was increased in lung lavage fluid by Luminex assay, but increased mRNA expression was not identified in the lungs of dox-treated IKTA mice at this time point. Taken together, these studies show that sustained activation of NF-B in IKTA mice (in the absence of a specific inflammatory stimulus) results in a pattern of progressive lung inflammation and injury associated with production of a number of NF-B-regulated cytokines and chemokines.

View larger version (11K): [in this window] [in a new window]   FIGURE 3. Treatment of IKTA mice with dox results in hypoxemia and increased mortality. a, Serial arterial blood gas measurements were obtained from indwelling carotid artery catheters. PO2 and PCO2 were assessed in IKTA mice at baseline and up to 7 days of dox treatment (n = 3–4/time point, *, p < 0.01 compared with baseline). b, Mortality rates in mice from IKTA lines 3 and 31 compared with WT controls through 14 days of dox treatment. Although all WT mice survived, 60% of mice in IKTA line 31 and 70% of mice in IKTA line 3 died between day 7 and 14 (n = 10 mice/group).

Construction and characterization of transgenic mice that express a dominant inhibitor of NF-B in airway epithelium

We placed a Myc-His tagged dominant inhibitor of the NF-B pathway (IB-DN) under control of the (tet-O)₇-CMV promoter (Fig. 4a). IB-DN is an avian IB- with adenine substitutions at serines 36 and 40 that inhibit phosphorylation and degradation of the protein, therefore blocking NF-B nuclear translocation (8, 11, 12). Double transgenic mice containing (tet-O)₇-IB-DN-Myc-His and CC-10-tTS constructs were produced and cross-mated with CC10-rtTA mice to create triple transgenic mice with inducible expression of IB-DN-Myc-His in airway epithelium, which were designated DNTA (for IB-DN transactivated). As with IKTA mice, dox treatment resulted in transgene expression exclusively in the lungs (Fig. 4b). No leakiness of transgene expression was identified in the absence of dox. Immunohistochemistry for the Myc tag on IB-DN localized transgene expression to the airway epithelium in dox-treated mice (data not shown). Lungs of dox-treated DNTA mice were histologically normal.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. DNTA transgenic mice express a dominant inhibitor of the NF-B pathway in airway epithelium. a, Schematic for construction of DNTA transgenic mice. b, Western blot analysis for Myc-His-tagged IB-DN expression in tissue homogenates obtained from WT and DNTA mice and DNTA mice treated with dox for 7 days. Transgene expression is detected only in the lungs of DNTA mice following dox treatment. c, DNTA mice were crossed with IKTA mice (line 26) to create IKDNTA mice that express both transgenes. Total cell counts in lung lavage are shown for IKTA mice (line 26), DNTA mice, and IKDNTA mice following 7 days of dox treatment and IKDNTA mice without dox treatment. The inflammatory cell influx was inhibited in dox-treated IKDNTA mice, indicating that expression of IB-DN blocks cIKK2-induced inflammation (n = 4–8/group, *, p < 0.05 compared with other groups).

Prevention of lung inflammation and injury by blocking NF-B activation in airway epithelium

After demonstrating that expression of IB-DN in epithelial cells blocks NF-B activation, we undertook studies to identify the effects of inhibiting epithelial NF-B following treatment with E. coli LPS. Initially, WT and DNTA mice were treated with dox for 1 wk to induce transgene expression and then were administered aerosolized E. coli LPS (8 ml of a 0.1 µg/ml solution) as reported previously (13). At 4 h after LPS treatment, lungs were lavaged and harvested for determination of neutrophilic alveolitis and NF-B activation by EMSA. As shown in Fig. 5a, dox treatment of DNTA mice reduced nuclear translocation of RelA/p50 heterodimers (the transactivating component of NF-B) in lung tissue following treatment with aerosolized LPS. Neutrophil influx into the airways was also diminished in DNTA mice (Fig. 5b). These findings show that expression of IB-DN blocks NF-B activation and neutrophil recruitment induced by aerosolized LPS.

View larger version (48K): [in this window] [in a new window]   FIGURE 5. DNTA mice have reduced NF-B activation and neutrophil influx into the airways after aerosolized LPS. WT or DNTA mice were given dox (2 g/L) in drinking water for 1 wk, treated with aerosolized LPS, and lungs were harvested 4 h later. a, EMSA for NF-B binding using lung nuclear protein extracts indicates that induction of the RelA/p50 band is reduced in DNTA mice compared with WT in LPS-treated mice. b, Lung lavage cell counts show that LPS-induced neutrophil recruitment is inhibited in DNTA mice (n = 3/group, *, p < 0.05 compared with other groups).

View larger version (40K): [in this window] [in a new window]   FIGURE 6. DNTA mice are protected from E. coli LPS-induced lung inflammation and injury. WT or DNTA mice were treated with dox (2 g/L) in drinking water for 1 wk, followed by i.p. implantation of osmotic pumps delivering LPS. a, Western blot analysis from lung nuclear protein extracts showing impaired nuclear translocation of RelA in DNTA mice at 4 h after LPS pump implantation (DNTA + LPS) compared with WT mice (WT + LPS). Samples from DNTA mice and WT mice without LPS pumps are shown as controls. TATA binding protein (TBP) was identified as a loading control. b, H&E-stained lung sections from WT and DNTA mice harvested 48 h after i.p. placement of pumps that deliver LPS continuously for 24 h. Lung inflammation and edema were markedly reduced in dox-treated DNTA mice. c, Lung lavage neutrophils obtained at 48 h after LPS pump placement (n = 10/group; *, p < 0.05). d, Lung wet/dry ratios for WT and DNTA mice treated with LPS pumps presented as increase above untreated controls (n = 10/group, *, p < 0.01). e, Lung lavage protein concentration in untreated WT mice and WT and DNTA mice at 48 h after treatment with LPS pump (n = 6/group, *, p < 0.05 compared with untreated mice and LPS pump-treated DNTA mice). f and g, Representative photomicrographs of TUNEL staining (brown nuclear stain) and scoring of TUNEL+ cells in lung parenchyma from WT and DNTA mice at 48 h after treatment with LPS pumps (n = 8/group, *, p < 0.05). h, Mortality rates in dox-treated WT and DNTA mice following i.p. implantation of osmotic pumps that deliver LPS at 8 µg/h over 72 h (n = 10/group, *, p < 0.05).

To determine whether reduced lung injury in DNTA mice could lead to improved survival, we performed peritoneal implantation of osmotic pumps that deliver LPS (8 µg/h) for 72 h into dox-treated WT and DNTA mice. As shown in Fig. 6h, delivery of LPS over 72 h resulted in 50% mortality in WT mice at day 7; however, all dox-treated DNTA mice survived.

	Discussion

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These studies describe the generation, characterization, and use of a novel modular transgenic system that enables regulation of NF-B activity in specific cell populations. By using this approach to specifically target the NF-B pathway in airway epithelium, we have defined a pivotal role for epithelial cells in controlling lung inflammation and injury. In DNTA mice, expression of a dominant NF-B inhibitor in CC10 expressing cells reduces neutrophilic lung inflammation resulting from airway or systemic delivery of LPS and diminishes lung injury and mortality following endotoxemia. In complementary studies using IKTA-transgenic mice, we show that IKK2 expression causes persistent activation of NF-B in airway epithelial cells, resulting in cytokine production, inflammatory cell recruitment, lung injury, hypoxemia, and high mortality. In IKTA mice, dox treatment induces progressive lung injury with inflammation and edema by 3 days, followed by impaired gas exchange and death after as few as 7 days. Since the onset of detectable transgene expression occurs by 24–48 h after adding dox to drinking water (data not shown), induction of lung injury in dox-treated IKTA mice occurs in a time frame similar to that observed in the systemic LPS model. Taken together, our studies suggest a paradigm in which inflammatory signaling in airway epithelium plays a critical role in orchestrating the lung’s response to LPS (and possibly other injurious stimuli) delivered either locally (via the airways) or systemically (via the bloodstream). In this model, the NF-B pathway in airway epithelium is a focal point for control of lung injury through regulated production of mediators that participate in recruitment/activation of inflammatory cells, induction of alveolar cell death, and disruption of the alveolar capillary barrier.

Although a large number of studies have investigated regulation of inflammatory responses to environmental stress through the NF-B pathway, the majority of these studies have focused on NF-B signaling in immune cells. In the lungs, NF-B is activated in macrophages early after LPS treatment (22), and macrophages are required for maximal activation of NF-B in the whole lung and the resulting neutrophil influx (23, 24). This information suggests that alveolar macrophages are required for initiation of LPS-induced inflammatory responses in the lungs, likely through activation of NF-B signaling. However, our data and another recent study (25) indicate that NF-B signaling in nonimmune cells is critical for determining the lung’s response to injurious stimuli. Kisseleva et al. (25) expressed a dominant inhibitor of the NF-B pathway in endothelial cells using the Tie2 promoter and found that NF-B blockade resulted in increased vascular permeability in the lungs, increased endothelial apoptosis, and increased mortality in response to systemic LPS. Based on these findings, LPS-induced NF-B activation in endothelium appears to be primarily protective through maintenance of vascular integrity. In contrast, NF-B signaling in airway epithelial cells leads to increased vascular permeability in the lungs, and inhibiting LPS-induced NF-B activation in these cells reduces lung inflammation, edema, and alveolar cell death. Therefore, targeting of NF-B pathway in specific cell types or compartments (like the airways) may be necessary to effectively reduce lung inflammation and injury.

Previous studies have suggested that lung epithelial cells impact neutrophil recruitment through NF-B pathway signaling. We found that intratracheal administration of adenoviral vectors expressing NF-B-activating transgenes in mice results in neutrophilic inflammation (11). Mice deficient in RelA, the primary transactivating component of NF-B, and TNFR1 exhibit a marked reduction of neutrophilic inflammation in response to airway delivery of LPS (26). In contrast, bone marrow chimeras in which the RelA/TNFR1 deficiency is limited to immune cells (including lung macrophages) have normal LPS-induced neutrophil recruitment, implicating non-bone marrow-derived cells in generation of neutrophilic inflammation in this model. In addition, transgenic mice constitutively expressing a NF-B inhibitor in lung epithelial cells have reduced neutrophil influx into the airways in response to intranasal instillation of E. coli LPS (27, 28). In the gastrointestinal tract, selective deletion of IKK2 in intestinal epithelial cells impairs NF-B activation and results in decreased lung and systemic inflammation in a gut ischemia-reperfusion model through reduction of TNF- expression (29). However, local tissue injury in the intestinal mucosa is exacerbated in this model related to increased apoptosis. In combination with the present study, these findings point to the NF-B pathway in epithelial cells as an important target for therapies designed to modulate inflammation-induced tissue injury.

In humans, evidence supports the role of NF-B-dependent mediators in inducing lung injury, although the cellular source of these mediators is not well defined. A variety of NF-B linked cytokines and chemokines has been reported to be increased in lung lavage fluid obtained from patients with ARDS, including TNF-, IL-1, IL-6, and IL-8 (6, 30, 31). Increased concentrations of IL-8, a NF-B-regulated CXC chemokine in humans, are found in lungs of at-risk patients who progress to ARDS, and high levels of IL-8 and neutrophils in lung lavage have been correlated with increased mortality in ARDS patients (32, 33, 34). Our mouse model indicates that prolonged activation of NF-B in epithelial cells is sufficient to produce an inflammatory profile similar to human ARDS, as well as the pathophysiological and histological abnormalities. These findings solidify the NF-B pathway as an important therapeutic target for interventions targeted to limit lung injury in ARDS. It is intriguing to note that the relatively small percentage of lung cells that constitute the airway epithelium appear to have the potential to play a powerful protective role against lung injury. Airway epithelial cells are relatively accessible to aerosolized agents, and specific inhibition of NF-B activity by this route could leave intact host defense mechanisms mediated by inflammatory cells in the lung.

In summary, our findings support three major conclusions. First, we have generated a modular transgenic system that can be used to efficiently modulate NF-B activity in specific cell types. These mice have the potential to be used in a broad range of future research endeavors. Second, our findings implicate the NF-B pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli. Indeed, persistent NF-B activation in epithelium may provide a common pathway for driving the dysregulated inflammatory response that culminates in ARDS. Third, while interventions that reduce inflammation by blocking NF-B activation in epithelium must be rigorously examined to define their effects on host defense, the airway epithelium may prove to be an important and feasible target for reducing or preventing lung injury in patients at risk for ARDS.

	Acknowledgments

 
We thank Dr. Jeffrey A. Whitsett of the University of Cincinnati College of Medicine for the donation of CC-10 rtTA-expressing transgenic mice used in these studies. We also thank the Vanderbilt University Mouse Metabolic Phenotyping Core, the Vanderbilt Transgenic/ES Shared Resource, and the Mouse Pathology Core for their valuable assistance.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by National Institutes of Health Grants HL61419, HL66196, and HL07123; the U.S. Department of Veterans Affairs; Vanderbilt Ingram Cancer Center; Susan G. Komen Foundation Grant BCTR02-1728; and Department of Defense Breast Cancer Program Grant WX1XWH-04-1-0456.

² F.E.Y. and T.S.B. contributed equally to this manuscript.

³ Address correspondence and reprint requests to Dr. Timothy S. Blackwell, Vanderbilt University School of Medicine, T-1218 MCN, Nashville, TN 37232. E-mail address: timothy.blackwell{at}vanderbilt.edu

⁴ Abbreviations used in this paper: IKK2, IB kinase 2; ARDS, acute respiratory distress syndrome; cIKK2, constitutively active human IKK2; DNTA, IB-DN-transactivated mice, transgenic mice expressing IB-DN under control of the CC10 promoter; dox, doxycycline; IB-DN, IB- dominant negative; IKTA, cIKK2-transactivated mice, transgenic mice expressing cIKK2 under control of the CC10 promoter; MTEC, mouse tracheal epithelial cell; RPA, RNase protection assay; rtTA, reverse tetracycline transactivator; tTS, tetracycline-controlled transcriptional silencer; WT, wild type.

Received for publication November 9, 2006. Accepted for publication March 1, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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