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B Activity Determine the Severity of Endotoxin-Induced Acute Lung Injury1
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,,
* Department of Cell and Developmental Biology,
Department of Medicine, Division of Allergy, Pulmonary, and Critical Care Medicine,
Department of Cancer Biology, Vanderbilt University School of Medicine, and
Department of Veterans Affairs, Nashville, TN 37232;
¶ Section of Pulmonary, Critical Care, and Sleep Medicine, University of Illinois, Chicago, IL 60612; and
|| Department of Immunology, Inflammation, and Pulmonary Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B pathway activation determine the outcome of acute lung inflammation using a novel line of transgenic reporter mice. Following a single i.p. injection of Escherichia coli LPS, transient NF-B activation was identified in a variety of lung cell types, and neutrophilic inflammation resolved without substantial tissue injury. However, administration of LPS over 24 h by osmotic pump (LPS pump) implanted into the peritoneum resulted in sustained, widespread NF-B activation and neutrophilic inflammation that culminated in lung injury at 48 h. To determine whether intervention in the NF-B pathway could prevent progression to lung injury in the LPS pump model, we administered a specific IB kinase inhibitor (BMS-345541) to down-regulate NF-B activation following the onset of inflammation. Treatment with BMS-345541 beginning at 20 h after osmotic pump implantation reduced lung NF-B activation, concentration of KC and MIP-2 in lung lavage, neutrophil influx, and lung edema measured at 48 h. Therefore, sustained NF-B activation correlates with severity of lung injury, and interdiction in the NF-B pathway is beneficial even after the onset of lung inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B pathway, which regulates transcription of a variety of proinflammatory mediators, is involved in generation of neutrophilic lung inflammation; however, it is unknown whether specific parameters of NF-B activation determine whether lung inflammation resolves or progresses to lung injury. Elucidating the relationships between NF-B activation, lung inflammation, and lung injury could provide important insights into the pathobiology of a variety of human lung diseases, including the acute respiratory distress syndrome (ARDS).4
In the lungs, many noxious/inflammatory stimuli have been shown to activate NF-B, implicating the NF-B pathway as a focal point for induction of lung inflammation. In vivo activators of NF-B in the lungs include intact bacteria, Gram-negative bacterial LPS, ozone, and silica delivered directly to the airways, as well as systemic inflammatory insults such as sepsis, hemorrhage, and direct liver injury (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). In rodent models of lung inflammation induced by LPS, pretreatment with relatively nonspecific inhibitors of NF-B activation has been found to diminish lung inflammation (11, 12, 13). Additionally, mice deficient in RelA (the transactivating subunit of NF-B) and TNFR type 1 have impaired neutrophil recruitment to the lungs in response to LPS compared with wild-type controls and TNFR1-deficient mice (14). Together, these studies indicate that NF-B plays an important role in initiation of inflammatory signaling in the lungs in response to LPS, a prototypical inflammatory stimulus. After establishment of an inflammatory response, however, it has been suggested that NF-B has a role in resolution of inflammation through antiapoptotic effects and expression of proteins that function to limit inflammation (15). The importance of NF-B in regulating ongoing lung inflammation or progression to lung injury is unknown.
We hypothesized that specific parameters of NF-B activation in the lungs, including cellular distribution, intensity, and/or duration of NF-B activity, determine whether lung inflammation is self-limited or progresses to injury. Further, we proposed that focused intervention to inhibit NF-B activity could limit lung injury and convert an injurious stimulus to a phenotype of transient inflammation that resolves without significant tissue injury. To investigate this hypothesis, we generated novel transgenic NF-B reporter mice to allow cell-specific detection of NF-B activity. We determined parameters of NF-B activation in a mouse model of transient lung inflammation following i.p. injection of Escherichia coli LPS and a model of lung inflammation and injury following prolonged delivery of LPS via an osmotic pump implanted into the peritoneum. We then intervened using a selective inhibitor of IB kinase (IKK) to down-regulate NF-B activation during a period in which we identified differential NF-B activation between the two models. Information from these studies provides evidence that inhibition of the NF-B pathway in vivo can ablate lung injury and identifies NF-B as a critical target for limiting ongoing, maladaptive inflammation in the lungs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Transgenic NF-B reporter mice were generated that contain four tandem copies of a 36-base enhancer from the 5' HIV-long terminal repeat (containing two NF-B binding sites, GGGACTTTCC) placed upstream of the HSV minimal thymidine kinase promoter. This enhancer-promoter construct was cloned into pEGFPluc (BD Clontech) for expression of an enhanced GFP-luciferase fusion protein. The 8xNF-B-GFP-luciferase construct was excised and purified using a GELase Agarose Gel-Digesting preparation kit following the manufacturer’s instruction (Epicentre Technologies). This construct was then injected at the Vanderbilt Transgenic/ES Cell Shared Resource to generate NGL (NF-B-GFP-luciferase construct) transgenic mouse lines on C57B6/DBA background. Founder animals were genotyped by Southern blot and then further generations were genotyped by PCR analysis for increased efficiency.
Male and female NGL transgenic mice weighing between 20 and 25 g were used for these studies. E. coli LPS (serotype O55:B5) was obtained from Sigma-Aldrich. LPS was delivered by a single i.p. injection of 3 µg/g body weight (1 µg/µl solution in sterile PBS). For prolonged delivery of LPS, an osmotic pump (2001D Alzet pump; ALZA) was filled with LPS solution (1 µg/µl in PBS) and surgically implanted in the peritoneal cavity using sterile technique. The pump delivered 8 µg LPS (8 µl) per hour for 24 h. A priming dose of 3 µg of LPS/g body weight was injected i.p. following pump implantation.
A selective inhibitor of IKK, BMS-345541, was obtained from Bristol-Myers Squibb. The compound was formulated as a 7.5 mg/ml solution in 3% Tween 80 and sterile water. Body weight (75 µg/g) of this solution or an equivalent volume of vehicle (without BMS-345541) was administered by peroral gavage after anesthesia with inhaled isoflurane.
Neutrophil depletion was performed as previously reported (16). Undiluted rabbit antineutrophil Abs (200 µl; Accurate Chemical and Scientific) or control rabbit IgG (1 µg/µl; Sigma-Aldrich) were administered by i.p. injection daily for 2 days. On days 3 and 4, 300 µl of a 1/15 dilution of Ab preparations (in 1x PBS) were injected i.p. On day 5, peripheral white blood cell counts and differentials were measured to verify neutrophil depletion.
The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Vanderbilt University (Nashville, TN).
Bioluminescence imaging and photon count quantification
Mice were anesthetized and shaved over the chest before imaging. Luciferin (1 mg/mouse in 100 µl isotonic saline) was administered by i.v. injection and mice were imaged with an intensified charge-coupled device (ICCD) camera (IVIS 200; Xenogen). For the duration of photon counting, mice were placed inside a light tight box that housed the camera. Light emission from the mouse was detected as photon counts by the ICCD camera and customized with image processing hardware and software (Living Image software; Xenogen). The imaging duration (30 s) was selected to avoid saturation of the camera during image acquisition. Quantitative analysis was performed by defining a standard area over the mid-lung zone and determining the total integrated photon intensity over the area of interest. For presentation, a digital false-color photon emission image was generated for each captured image.
Bone marrow-derived macrophage experiments
Bone marrow-derived macrophages were generated from NGL mice as previously described (17). Adenoviral vectors expressing a dominant inhibitor of NF-B (IBdn), which represents a S36-40A mutant of the avian IB-
that cannot be phosphorylated or degraded, and 
-galactosidase have been previously reported (10, 18). Cells were treated with adenoviral vectors (multiplicity of infection = 300) 48 h before LPS treatment. LPS was added to cultures (200 ng/ml) and cells were harvested 4 h later. In separate experiments, cells were treated with BMS-345541 (20 µM) 30 min before LPS.
Histology and immunohistochemistry
After euthanasia, lungs were inflated with 1 ml of 10% neutral buffered formalin. Lungs were then removed en bloc after tracheal ligation and preserved in 10% neutral buffered formalin for 24 h at 4°C, and subsequently embedded in paraffin. Lung tissue sections (5 µm) were prepared in the Mouse Pathology Core Facility (Vanderbilt University). H&E stains were performed using standard protocol. For GFP immunohistochemistry, 5-µm sections were cut and placed on charged slides. Following paraffin removal, sections were rehydrated and placed in heated Target Retrieval Solution (high pH; (DakoCytomation) for 20 min. Tissues were incubated with rabbit anti-GFP 1/200 (BD Clontech) for 30 min. Sections without primary Ab served as negative controls. The Vectastain ABC Elite System (Vector Laboratories) and diaminobenzidine (DAB+; DakoCytomation) were used to produce localized, visible staining. Slides then were lightly counterstained with Mayer’s hematoxylin, dehydrated, and coverslipped.
Measurement of neutrophil influx
For tissue neutrophil quantification, H&E-stained lung tissue sections were used to count the number of neutrophils per high power field. For each slide, neutrophils were counted in a blinded fashion on 10 sequential, nonoverlapping high power fields (magnification, x400) of lung parenchyma beginning at the periphery of the section. Three separate H&E-stained sections were evaluated per mouse, and the mean number of neutrophils per high power field was reported.
Lung lavage neutrophil counts
For bronchoalveolar lavage (BAL) neutrophil quantification, BAL cells were collected after lavage with 3 aliquots of 1 ml of sterile PBS. BAL was combined and centrifuged at 400 x g for 10 min to separate cells from supernatant. Supernatant was saved separately and frozen at –70°C. The cell pellet was suspended in PBS with 1% BSA, and total cell counts were determined on a grid hemocytometer. Differential cell counts were determined by staining cytocentrifuge slides with a modified Wright stain (Diff-Quick; Baxter Scientific Products) and counting 200–300 cells in complete cross-section.
Tissue luciferase assay
Lungs were removed en bloc and homogenized in 1 ml of lysis buffer (Promega) using a dounce homogenizer. After pulse centrifugation, luciferase activity was measured in a Monolight 3010 Luminometer (Analytical Luminescence Laboratory) after adding 100 µl of freshly reconstituted luciferase assay buffer to 20 µl of lung tissue homogenate. Results were expressed as relative light units (RLU) normalized for protein content, which was measured by Bradford assay (Bio-Rad).
Western blots
One hundred micrograms of protein from lung tissue homogenates were separated on a 10% acrylamide gel, transblotted, and immunodetected using phosphospecific Abs to an epitope of IKK1 containing phosphorylated serine 176 and serine 180 (BioSource International). The blots were also probed with Abs to MAPK p44 and p42 (Cell Signaling Technology), then stripped and reprobed for total IKK1 (Santa Cruz Biotechnology).
Lung wet to dry ratio
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.
MIP-2 and KC quantification
MIP-2 and KC levels in BAL fluid were measured using a specific ELISA according to manufacturer’s instructions (R&D Systems).
Statistical analysis
To assess differences among groups, analyses were performed with GraphPad Instat software using an unpaired t test or one-way ANOVA. Results are presented as the mean ± SEM. Two-tailed values of p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B activation
After testing a variety of NF-B-driven promoter constructs, we determined that basal expression and inducibility characteristics were optimal with a construct that contained four tandem copies of a 36-base enhancer from the 5' HIV-long terminal repeat (containing two NF-B binding sites, GGGACTTTCC). This NF-B-dependent promoter was placed upstream of GFP-luciferase expression cassette, and the construct was tested in vitro using multiple cell lines (A549, RAW 267.4, NIH-3T3, HeLa) to ensure reliable expression in a wide range of cell types (data not shown). Subsequently, the NF-B reporter construct was excised, purified, and microinjected at the Vanderbilt Transgenic/ES Cell Shared Resource to produce transgenic mice (NGL mice).
To investigate regulation of the reporter expression in primary cells, we obtained bone marrow macrophages and treated them in vitro with E. coli LPS (200 ng/ml) for 4 h to stimulate NF-B activation. Adenoviral vectors expressing a transdominant inhibitor of the NF-B pathway (Ad-IBdn) (10, 18) were used to specifically block NF-B activation in these cells. Cells were infected with adenoviral vectors 48 h before experimentation. Expression of the GFP-luciferase reporter was determined by luciferase assays and fluorescence microscopy (Fig. 1, A and B). By luciferase assay, no difference in reporter activity was detected between untreated and Ad-IBdn-treated macrophages (1015.2 ± 67.2 vs 1257.3 ± 174.17 RLU, respectively). Compared with untreated and Ad-IBdn-treated macrophages, LPS stimulation resulted in a significant increase in luciferase activity (5450.0 ± 265.59 RLU, p < 0.05); however, infection of cells with Ad-IBdn before LPS blocked induction of luciferase expression (1841.3 ± 171.1 RLU). Infection of macrophages with control adenoviral vectors did not affect LPS-induced luciferase activity (data not shown). Using fluorescence microscopy to detect GFP expression before cell harvest, we corroborated the results obtained by luciferase assays (Fig. 1B). In addition to Ad-IBdn, we treated macrophages from NGL mice with a specific inhibitor of the NF-B pathway, BMS-345541 (19) (Fig. 1C). In these studies, cells were treated with BMS-345541 (20 µM) or vehicle 30 min before addition of LPS (200 ng/ml) and cells were harvested 4 h later. Similar to IBdn, BMS-345541 treatment blocked LPS-induced luciferase activity in macrophages. Together, these findings confirm that LPS-induced expression of GFP-luciferase reflects NF-B activation in NGL cells.
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B transcriptional activity.
Cellular distribution, intensity, and duration of NF-B activation in LPS-induced lung inflammation and injury
To model transient neutrophilic lung inflammation, a single dose of E. coli LPS (3 µg/g) was administered to NGL mice by i.p. injection (IP LPS treatment). Because established models of LPS-induced lung inflammation in mice do not produce consistent lung injury (20), we developed a model of LPS delivery into the peritoneal cavity over 24 h that produces severe lung inflammation and injury (Fig. 2A). In this model, an osmotic pump delivering E. coli LPS at 8 µg/h for 24 h was surgically implanted into the peritoneal space followed by a priming dose of 3 µg/g LPS by direct i.p. injection (LPS pump treatment). Although single-dose i.p. LPS produced only mild interstitial thickening and cellular infiltrate at 48 h, LPS delivered by osmotic pump caused lung inflammation and injury (edema, interstitial thickening, hemorrhage, and inflammatory cell influx). Persistent lung inflammation in the LPS pump model was confirmed by counting neutrophils on H&E-stained lung tissue sections. Although single-dose IP LPS treatment resulted in a transient influx of neutrophils at 4 h, LPS pump treatment resulted in persistent neutrophil influx through 48 h (Fig. 2B). The LPS pump model also resulted in a significant increase in lung edema as assessed by the wet to dry ratio at 48 h (3.81 ± 0.03 in untreated mice vs 4.65 ± 0.04 in the LPS pump group, p < 0.05), whereas the lung wet to dry ratio in the IP LPS group was similar to baseline. Delivery of LPS over 24 h into the peritoneum was required to induce this phenotype as treatment with an equal LPS dose by single i.p. injection or implantation of pumps alone did not induce sustained lung inflammation and injury (data not shown).
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B was differentially activated in these models. Expression of the NF-B-driven reporter in NGL mice was identified by bioluminescence imaging (following i.v. injection of 1 mg of luciferin) to detect luciferase activity in the lungs of intact mice and by immunohistochemistry for detection of GFP in lung sections. Consistent with our previous reports (10, 21), single-dose IP LPS treatment resulted in a transient increase in NF-B-driven luciferase activity by 4 h. However, by 24 h, photon emission from the lungs returned near baseline, indicating transient NF-B activation (Fig. 3A). In contrast, NGL mice treated with LPS delivered by osmotic pump showed sustained lung luciferase activity at 4, 24, and 48 h. Peak photon emission from the lungs at 4 h did not differ between the two models.
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B activity by immunohistochemistry for GFP on lung sections (Fig. 3B). At baseline, minimal GFP staining was identified in the lungs. LPS treatment (either IP LPS or LPS pump) resulted in widespread GFP staining in multiple lung cell types at 4 h, including airway epithelium, endothelium, and leukocytes (macrophages and neutrophils). GFP staining was also noted in alveolar epithelium, particularly type II cells. At 24 h after IP LPS treatment, GFP immunoreactivity in the lungs was limited to a few, scattered positive cells, and by 48 h, GFP staining returned to baseline. In the LPS pump model, widespread GFP staining persisted in the lungs through 48 h, consistent with bioluminescence detection of NF-B driven transgene expression. Together, these studies indicate that endotoxemia induces NF-B activation in a variety of lung cells. Although the cell-specific pattern of NF-B activity and peak intensity of lung NF-B intensity did not differ depending on the method of LPS delivery, prolonged duration of high-level NF-B activation in the lungs correlated with LPS-induced lung injury.
Early NF-B inhibition limits neutrophilic lung inflammation whereas late NF-B inhibition prevents lung injury
To evaluate the role of NF-B in determining the course of lung inflammation and injury, we used BMS-345541, which is a selective inhibitor of IKK1 and IKK2 (19). In prior studies, BMS-345541 at doses as high as 100 µg/g daily for 6 wk in mice showed no toxicologic changes, either by gross or by histopathologic evaluation, of major organs including the liver (22).
In the IP LPS treatment model, BMS-345541 (75 µg/g) or vehicle was administered to NGL mice by peroral gavage 2 h before injection of LPS and again 4 h after LPS treatment. By bioluminescence imaging, photon emission from the lungs was reduced at 8 h after IP LPS in the group that received BMS-345541 compared with controls treated with vehicle, although no significant difference between the groups was identified at 4 h (Fig. 4, A and B). At the time of harvest (8 h), luciferase activity in lung homogenates was also significantly reduced in mice treated with BMS-345541 before LPS (1347 ± 51 RLU/µg protein in the BMS-345541 group vs 2089 ± 134 RLU/µg protein in the control vehicle-treated group, p < 0.05) (Fig. 4C). Neutrophil influx was quantified on lung sections as a measure of lung inflammation. As shown in Fig. 4D, a marked reduction in lung neutrophils was identified in lungs treated with BMS-345541 before LPS compared with controls treated with vehicle before LPS. These experiments indicate that treatment with BMS-345541 inhibits lung NF-B activation and neutrophilic lung inflammation in response to IP LPS treatment.
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B activation that we observed between the IP LPS and LPS pump treatment models, we investigated whether sustained lung NF-B activation in the LPS pump model mediates lung injury. Therefore, we targeted the late phase of NF-B activation by beginning BMS-345541 after the establishment of lung inflammation in NGL mice treated with LPS via osmotic pump. Mice were treated with BMS-345541 or vehicle by peroral gavage beginning at 20 h after osmotic pump implantation. BMS-345541 dosing was repeated every 4 h for a total of five doses. Multiple doses were used because of the relatively short half-life of the compound in vivo (19). Bioluminescent imaging was performed at baseline, 20, 24, and 48 h after initiation of LPS to evaluate NF-B-dependent reporter gene expression (Fig. 5, A and B). In both, vehicle or BMS-345541 treatment groups, similar induction of photon emission from the lungs was observed at 20 h, before intervention. By 24 h, there was a trend toward lower chest bioluminescence in the group treated with BMS-345541, and at 48 h, a marked reduction in photon emission from the lungs was observed in the group treated with BMS-345541 compared with vehicle-treated controls. Treatment with BMS-345541 resulted in 
50% reduction in chest bioluminescence compared with the measurement before treatment (20 h). These findings were confirmed by postmortem measurement of lung tissue luciferase activity. Luciferase activity in BMS-345541 treated mice was 1061 ± 233 RLU/µg protein compared with 2521 ± 462 RLU/µg protein in the vehicle-treated group (p < 0.05) (Fig. 5C). To show that treatment with BMS-345541 reduces IKK activation in the lungs, we performed Western blot analysis for IKK1 using Abs that specifically identify the phosphorylated (activated) kinase. Fig. 5D indicates that IKK1 phosphorylation was reduced in lungs treated with BMS-345541 at 48 h after LPS pump implantation. Therefore, oral delivery of the IKK inhibitor was sufficient to reduce IKK activity and thereby block NF-B activation in the lungs.
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B, we performed immunostaining for GFP on lung tissue sections from NGL mice (Fig. 6). Interestingly, we found that BMS-345541 treatment broadly diminished GFP staining compared with vehicle-treated controls. Less GFP staining was observed in epithelium, endothelium, and leukocytes at 48 h after LPS pump implantation in BMS-345541-treated mice compared with vehicle-treated mice. By evaluation of H&E-stained lung sections, we observed a dramatic reduction in lung inflammation and injury in mice treated with the IKK inhibitor (Fig. 6). Mice treated with BMS-345541 showed preserved alveolar architecture with minimal edema, septal thickening, and hemorrhage.
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B activation after establishment of lung inflammation reduces lung inflammation and prevents injury.
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To evaluate whether reduction of neutrophil recruitment to the lungs accounts for prevention of lung injury resulting from NF-B inhibition, we depleted neutrophils and determined the impact on LPS-induced lung inflammation and injury. Neutrophil depletion was achieved by repeated i.p. injection of antineutrophil Abs using a previously published protocol (16). After 4 daily injections of antineutrophil Abs, we detected an 80% reduction in peripheral neutrophil counts compared with mice treated with control IgG (Fig. 8A). After documenting PMN depletion, LPS pumps were implanted and bioluminescent detection of NF-B-dependent luciferase expression was performed at 24 and 48 h (Fig. 8B). As shown, reduction of circulating neutrophils did not affect luciferase expression in NGL mice, implying that neutrophils do not make major contributions to the total lung NF-B activation in this model. At 48 h, neutrophils were found to be reduced in BAL (Fig. 8C) and the lung wet-to-dry ratio was significantly decreased in neutrophil-depleted mice compared with IgG-treated controls (Fig. 8D). However, the wet-to-dry ratio in the lungs of neutrophil-depleted mice treated with LPS pump was significantly greater than the ratio in untreated controls, indicating that the achieved degree of neutrophil depletion was only partially effective in preventing lung edema formation. By histological examination of lung sections, mice treated with antineutrophil Abs were found to have a reduction in lung neutrophil influx with a mild diminution in edema and evidence of lung injury compared with IgG-treated controls (data not shown). Accounting for the reduction in neutrophils, immunohistochemistry for GFP did not show any differences in intensity or distribution of GFP staining in the lungs of mice treated with antineutrophil Abs or control IgG before LPS pumps.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B activation in vivo, we developed a reporter system that allows identification of NF-B-positive cells using GFP detection, as well as quantification of NF-B activity in cells and tissue using luciferase detection methodologies. The NGL reporter construct was validated as an indicator of NF-B activation in cell lines, primary cells from transgenic mice, and intact animals. Following a single i.p. injection of LPS, the time course for increased bioluminescence over the chest was similar to our previous report with NF-B reporter mice that express luciferase under the control of the proximal promoter for the 5' HIV-long terminal repeat (21).
LPS activates NF-B in cells directly by interaction with cell surface molecules CD14 and TLR4. Although initial studies identified TLR4 on leukocytes, more recent studies have identified TLR4 expression in other cell types, including endothelium and bronchial epithelial cells, indicating that LPS may directly stimulate inflammatory signaling in a variety of cell types (24, 25). In addition to direct cellular effects, inflammatory signaling by LPS is amplified by rapid production and secretion of "proximal" cytokines, including TNF- and IL-, by macrophages. Through specific receptors, these molecules have the ability to activate NF-B signaling and amplify the host inflammatory response. In NGL mice, we showed that NF-B was efficiently activated throughout the lungs in response to systemic LPS. Within 4 h, most cells in the lungs were GFP+; however, in the absence of continued activation, inflammation resolved without substantial tissue injury.
We evaluated NF-B activation in the lungs in a model of transient lung inflammation (single-dose IP LPS treatment) and a model of LPS-induced lung inflammation that progresses to lung injury (LPS pump treatment). One limitation of previously reported models of LPS-induced lung inflammation in mice is that they produce relatively minor lung injury, even at near lethal doses; therefore, we chose to deliver LPS over 24 h by osmotic pump in a model that resembles subacute LPS release from i.p. infection. Using these two models, we observed clear differences in NF-B activation. Like other parameters of the inflammatory response, NF-B activation was transient in the IP LPS model but persisted in the LPS pump model up to 24 h after LPS delivery ceased. In the LPS pump model, obvious lung injury was present by 48 h after implantation of the osmotic pump. In our studies, mice were harvested at 48 h in the LPS pump model because substantial mortality was observed when mice were followed beyond this time point. The cellular distribution of NF-B activation as measured by GFP+ cells, and peak intensity of NF-B activation as determined by bioluminescence imaging of luciferase activity did not differ between the IP LPS and LPS pump treatment models, indicating that these parameters are not the critical determinants of progression to lung injury. However, widespread sustained cellular activation of NF-B correlated with lung injury.
To evaluate whether a causal relationship exists between sustained lung NF-B activation and lung injury, we used a specific inhibitor of the NF-B pathway. BMS-345541 is a selective inhibitor of the catalytic subunit of IKK2 (IC50 = 0.3 mM) and IKK1 (IC50 = 4 µM) through binding to an allosteric site (19). Peroral administration of BMS-345541 inhibits serum TNF- production following LPS injection in a dose-dependent manner (19). Additional studies support the use of BMS-345541 as a selective NF-B inhibitor and anti-inflammatory agent, including findings of reduced joint inflammation and destruction in a collagen-induced arthritis model, reduced severity of colitis in a dextran sulfate sodium-induced colitis model, and improved graft survival in a murine model of cardiac graft rejection (22, 26, 27). In our experiments, we found that when administered before LPS, BMS-345541 treatment decreased NF-B activation and neutrophilic lung inflammation measured at 8 h. Subsequently, we treated mice with BMS-345541 during the period when NF-B activity was differentially up-regulated in the LPS pump model to investigate whether attenuation of NF-B activation would convert this model of lung injury to one of transient inflammation. We initiated treatment with BMS-345541 at 20 h after LPS pump implantation. At the time of harvest, NF-B activation, CXC chemokine production, and neutrophilic inflammation were markedly reduced in mice by BMS-345541 treatment. In addition, the histological appearance of lungs from BMS-345541-treated mice was improved and lung edema formation, as measured by wet-to-dry ratio, was eliminated. It appears that part of the benefit of NF-B depletion results from reducing the neutrophil influx into the lungs consequent to LPS pump implantation. Activated neutrophils produce a variety of oxidants and proteases that have the potential to disrupt the alveolar-capillary barrier and contribute to lung injury (28). By treatment with antineutrophil Abs, we were able to reduce lung neutrophil influx (measured in BAL) to a similar degree as that achieved by treatment with the NF-B inhibitor; however, the NF-B inhibitor had a much more profound benefit in protecting from lung injury. These findings imply that NF-B inhibition reduces lung injury in this model by reduction of neutrophil recruitment to the lungs and through other mechanisms (currently not well defined) that prevent disruption of the alveolar capillary barrier.
In contrast with our findings, a study by Lawrence et al. (15) found that NF-B played opposing roles in the onset and resolution of inflammation in rat carrageenin pleurisy and mouse carrageenin air pouch models. Using nonspecific inhibitors, decreased NF-B activation after establishment of inflammation was associated with protracted inflammation and prevention of leukocyte clearance, suggesting NF-B may not be a suitable target for therapeutic intervention to limit and/or resolve established inflammation. Our findings, however, indicate that NF-B could be an important target for limiting injury in the setting of ongoing inflammation in the lung parenchyma. The differences in our findings and those previously reported may be related to differences in the models of inflammation and/or the specificity of the agents used to inhibit the NF-B pathway.
In humans, lung injury resulting from pneumonia, systemic infection, or trauma can cause ARDS (29, 30). The inflammatory phenotype associated with ARDS supports a role for NF-B in the pathogenesis of this syndrome, including increased concentrations of a variety of NF-B-linked cytokines and chemokines in BAL fluid from patients with ARDS (31, 32, 33). Increased concentration of the NF-B-dependent CXC chemokine IL-8 correlates with neutrophilia in BAL, and prolonged neutrophilic alveolitis is associated with increased mortality in patients with ARDS (34, 35, 36, 37). In addition, NF-B activation has been identified in alveolar macrophages from humans with ARDS (38). Together, these data from human studies implicate the NF-B pathway as a potentially important determinant of lung injury in humans with ARDS. Based on our findings in relevant rodent models of lung inflammation and injury, NF-B may prove to be a beneficial target for therapeutic intervention to treat ongoing lung inflammation and prevent or limit lung injury. Although NF-B inhibition has the potential to reduce lung injury, innate immune functions that depend on NF-B may be required for adequate lung host defense against infection (39); therefore, application of these findings to human disease should proceed with caution. Hopefully, a better understanding of the intricacies of proinflammatory pathways that contribute to lung injury and host defense will lead to targeted therapies that limit injury while preserving lung defenses.
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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1 This work is supported by Grants HL61419 and HL66196 from the National Institutes of Health, by the U.S. Department of Veterans Affairs, Vanderbilt Ingram Cancer Center, by Grant BCTR02-1728 from the Susan G. Komen Foundation, and by Grant WX1XWH-04-1-0456 from the U.S. Department of Defense Breast Cancer Program. 
2 M.B.E. and W.H. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Timothy S. Blackwell, Division of Allergy, Pulmonary, and Critical Care Medicine, T-1218 Medical Center North, Vanderbilt University Medical Center, Nashville, TN 37232-2650. E-mail address: timothy.blackwell{at}vanderbilt.edu
4 Abbreviations used in this paper: ARDS, acute respiratory distress syndrome; IKK, IB kinase; BAL, bronchoalveolar lavage; RLU, relative light unit.
Received for publication September 19, 2005. Accepted for publication February 2, 2006.
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References |
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