Modulation of endotoxin-induced NF-kappa B activation in lung and liver through TNF type 1 and IL-1 receptors

M. Audrey Koay1, John W. Christman1,3, L. James Wudel2, Tara Allos2, Dong-Sheng Cheng1, William C. Chapman2, and Timothy S. Blackwell1,3

Departments of 1 Medicine and 2 Surgery, Vanderbilt University School of Medicine, Nashville 37232-2650; and 3 Department of Veterans Affairs Medical Center, Nashville, Tennessee 37203


    ABSTRACT
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ABSTRACT
INTRODUCTION
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We investigated the requirement for tumor necrosis factor-alpha (TNF-alpha ) and interleukin (IL)-1 receptors in the pathogenesis of the pulmonary and hepatic responses to Escherichia coli lipopolysaccharide (LPS) by studying wild-type mice and mice deficient in TNF type 1 receptor [TNFR1 knockout (KO)] or both TNF type 1 and IL-1 receptors (TNFR1/IL-1R KO). In lung tissue, NF-kappa B activation was similar among the groups after exposure to aerosolized LPS. After intraperitoneal injection of LPS, NF-kappa B activation in liver was attenuated in TNFR1 KO mice and further diminished in TNFR1/IL-1R KO mice; however, in lung tissue, no impairment in NF-kappa B activation was found in TNFR1 KO mice and only a modest decrease was found in TNFR1/IL-1R KO mice. Lung concentrations of KC and macrophage-inflammatory peptide 2 were lower in TNFR1 KO and TNFR1/IL-1R KO mice after aerosolized and intraperitoneal LPS. We conclude that LPS-induced NF-kappa B activation in liver is mediated through TNF-alpha - and IL-1 receptor-dependent pathways, but, in the lung, LPS-induced NF-kappa B activation is largely independent of these receptors.

sepsis; macrophage; neutrophil; cytokines; chemokines


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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GRAM-NEGATIVE BACTERIAL SEPSIS evokes production of the early-response cytokines tumor necrosis factor-alpha (TNF-alpha ) and interleukin (IL)-1beta (5). These proximal cytokines have a broad array of proinflammatory biological activities that are thought to contribute to the pathogenesis of severe sepsis (2, 11). Both cytokines are produced by mononuclear phagocytes in response to bacterial endotoxin [lipopolysaccharide (LPS)] (11, 14). TNF-alpha signals through type 1 (p55) and type 2 (p75) receptors. Type 1 receptors (TNFR1) are present on a wide variety of cell types and mediate most proinflammatory and cytotoxic effects of TNF-alpha . Previous studies using mice deficient in TNFR1 have shown that TNF-alpha signaling through this receptor plays an important role in bacterial host defense and survival after LPS injection in mice sensitized with D-galactosamine (21-23). Ligand binding to TNFR1, but not TNFR2, strongly activates the nuclear factor-kappa B (NF-kappa B) signal transduction pathway, which leads to transcriptional upregulation of a variety of inflammatory genes (9, 15, 21). The NF-kappa B pathway appears to be central to innate immunity and mediates cellular responses to TNF-alpha and IL-1beta , as well as LPS. IL-1beta activates NF-kappa B through the IL-1 receptor (IL-1R), and LPS activates NF-kappa B through binding to TLR4 and CD14 (12, 13, 26, 27).

Our previous studies showed that NF-kappa B is activated in liver and lung in response to treatment with LPS and that inhibition of NF-kappa B activation results in suppression of chemokine production in the lungs and downregulation of neutrophilic alveolitis (3, 4, 7). Because production of TNF-alpha and IL-1beta is rapidly induced by LPS, we sought to determine whether these cytokines contribute to NF-kappa B activation in the lungs and liver after LPS treatment. We hypothesized that, in LPS-induced inflammation, TNF-alpha and IL-1beta act to amplify inflammatory signaling through binding to TNFR1 and IL-1R and activation of NF-kappa B. Therefore, in these experiments, we sought to determine the degree to which NF-kappa B activation in response to LPS is dependent on TNF-alpha and IL-1beta . We studied mice deficient in TNFR1 [TNFR1 knockout (KO) mice] and mice deficient in both TNFR1 and IL-1R (TNFR1/IL-1R KO mice). These mice and wild-type (WT) controls were treated with LPS given by aerosol or as a single intraperitoneal injection. NF-kappa B activation was then measured by electrophoretic mobility shift assays (EMSA) in lung and liver tissues at the time of maximal NF-kappa B activation. We found that LPS-induced NF-kappa B activation in the lungs was unaffected in these knockout mice after aerosolized LPS and that NF-kappa B activation was only mildly inhibited in lungs of TNFR1/IL-1R KO mice after intraperitoneal LPS. Even though some decrease in lung chemokine concentrations was observed in knockout mice, no differences were found in lung neutrophil recruitment. After intraperitoneal LPS, however, liver NF-kappa B activation was diminished in TNFR1 KO and TNFR1/IL-1R KO mice compared with WT controls. These findings suggest that NF-kappa B-dependent inflammation in the lungs does not require amplification of the initiating signal through production of the proximal cytokines TNF-alpha and IL-1beta .


    METHODS
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INTRODUCTION
METHODS
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Materials. TNFR1 KO and TNFR1/IL-1R KO mice and appropriate controls (hybrid C57Bl/6 × 129Sv) were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained under specific pathogen-free conditions in a barrier facility. Escherichia coli LPS (serotype 055:B5) was obtained from Sigma (St. Louis, MO). [gamma -32P]ATP was obtained from NEN-DuPont (Boston, MA), and T4 kinase and T4 kinase buffer, used for oligonucleotide labeling, were purchased from New England Biolabs (Beverley, MA). For EMSA, the double-stranded consensus NF-kappa B motif 5'-GATCGAGGGGACTTTCCCTAAAAGC-3' was obtained from Stratagene (La Jolla, CA). Polyclonal antibodies to RelA (also called p65) and p50 used in performing EMSA supershifts were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Isoflurane (Fort Dodge Animal Health, Fort Dodge, IA) was used for animal anesthesia.

Animal model. Mice were maintained on a 12:12-h light-dark cycle, housed in filtered-air cages, fed standard chow pellet diet, and had free access to water. Adult mice weighing 25-30 g were used for all experiments. For aerosol exposure, lyophilized E. coli LPS was suspended in sterile saline (0.3 mg/ml or 1.0 mg/ml). LPS solution (7 ml) was delivered as a continuous aerosol with a driving flow rate of 8 l/min, which was generated by a small-volume nebulizer (Resigard II, Marquest Medical, Englewood, CO) over a standardized 30-min interval. Mice were placed in a sealed container for exposure to aerosolized LPS and were killed 4 h later. For intraperitoneal administration, lyophilized E. coli LPS was suspended in sterile saline, and LPS (5 µg/g) was administered by a single intraperitoneal injection. Mice were killed 45 or 90 min after intraperitoneal LPS injection for determination of liver and lung NF-kappa B activity and 4 h after intraperitoneal LPS for determination of cytokine levels. In our experiments, mice were euthanized by CO2 asphyxiation following the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

Bronchoalveolar lavage fluid and tissue harvesting. Bronchoalveolar lavage (BAL) fluid, serum, and tissue samples were collected after death. Mouse tracheas were cannulated with a 20-gauge blunt-tip needle attached to a 1-ml syringe, and the lungs were instilled with sterile pyrogen-free physiological saline until a total lavage volume of 3 ml was collected. Serum was obtained from the inferior vena cava. Lungs were harvested by resection, and tissues were immediately flash frozen in liquid nitrogen and stored at -70°C. Total and differential cell counts were measured in lung lavage fluid. After centrifugation at 500 g for 10 min, the cell pellet was resuspended in 1 ml of 1% BSA in sterile physiological saline. Total cell counts are determined using a grid hemocytometer. Differential cell counts were obtained by staining cytocentrifuge slides with a modified Wright's stain (Diff-Quik, Baxter) and counting 400 cells in a cross section.

Extraction of nuclear proteins from tissue samples. Tissue nuclear proteins were extracted from whole lung by the method of Deryckere and Gannon (10). Tissue (50-100 mg) was mechanically homogenized in liquid nitrogen to which 4 ml of buffer A (150 mM NaCl, 1 M HEPES, 0.6% (vol/vol) NP-40, 0.2 M EDTA, and 0.1 M phenylmethylsulfonyl fluoride) were added. The homogenate was transferred to a 15-ml Falcon tube and centrifuged at 850 g for 30 s to remove cellular debris. The supernatant was then transferred to a 50-ml Falcon conical and incubated on ice for 5 min before centrifugation for 10 min at 3,500 g. The supernatant was collected as the cytoplasmic extract. The nuclear pellet was resuspended in 300 µl of buffer B (sterile water, glycerol, 1 M HEPES, 5 M NaCl, 1 M MgCl2, 0.2 M EDTA, 0.1 M phenylmethylsulfonyl fluoride, 1 M dithiothreitol, 10 mg/ml benzamidine, 1 mg/ml pepstatin, 1 mg/ml leupeptin, and 1 mg/ml aprotinin) and incubated on ice for 30 min. After 2 min of microcentrifugation at 14,000 rpm, the supernatant was collected as the nuclear extract and frozen at -70°C. Protein concentrations in nuclear and cytoplasmic extracts were determined using the Bradford assay (8).

Oligonucleotide labeling. Oligonucleotides were labeled using a double-stranded consensus sequence NF-kappa B and [gamma -32P]ATP. The reaction was catalyzed with T4 polynucleotide kinase and incubated in 10× kinase buffer at 37°C for 45 min. The reaction was ceased by heating at 65°C for 10 min. Labeled oligonucleotide was column purified on Sephadex G-25 columns (Amersham Pharmacia Biotech). The radioactivity of the labeled probe was assayed using a Beckman LS6500 scintillation counter and quantitated as counts per minute per microliter of probe.

EMSA. Nuclear protein (5 µg) was incubated with binding buffer on ice for 30 min (specific antibodies for p50 and RelA were added for supershift studies). Oligonucleotide labeled with gamma -32P (~100,000 cpm) was then added, and samples were incubated at room temperature for 1 h. Specificity of binding was ascertained using cold competition with an excess of unlabeled NF-kappa B oligonucleotide and nonspecific competition using an excess of unlabeled oligonucleotide that did not contain an NF-kappa B binding motif. Protein-DNA complexes were separated from the free DNA probe by electrophoresis through 6% polyacrylamide gels run at room temperature at 150 V for 3-4 h. Gels were dried under vacuum on Whatman filter paper in a Bio-Rad gel dryer and exposed to autoradiographic film. The intensity of NF-kappa B-to-DNA binding was quantified by densitometry of the RelA/p50 heterodimer band.

Cytokine ELISAs. Cytokine assays were performed on BAL or cytoplasmic extracts derived from whole lung homogenates. IL-6 and the neutrophil chemoattractants macrophage-inflammatory peptide 2 (MIP-2) and KC were assayed according to the manufacturer's instructions with commercially available ELISAs (R & D Systems, Minneapolis, MN). The minimum detectable concentrations of IL-6, MIP-2, and KC were <3.1, <1.5, and <2.0 pg/ml, respectively. Cytokine concentrations are reported as pg/ml of BAL fluid and serum and as pg/µg protein of lung tissue homogenates.

Myeloperoxidase assay. Total lung myeloperoxidase (MPO) assays were done as previously reported (6).

Statistical analysis. Statistical analyses were performed with InStat version 3.01 for Windows NT (GraphPad Software, San Diego, CA) using an unpaired ANOVA to identify differences among groups; P < 0.05 was considered significant.


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ABSTRACT
INTRODUCTION
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Role of TNF and IL-1 receptors in regulating the lung inflammatory response to aerosolized LPS. We initially assessed the response of WT, TNFR1 KO, and TNFR1/IL-1R KO mice to two concentrations of LPS delivered by aerosol (0.3 and 1 mg/ml solution), which we have previously shown elicits NF-kappa B activation and a significant neutrophil influx into the lungs at 4 h (24). NF-kappa B activation was induced in lung tissue in all three groups of mice at 4 h after aerosolized LPS at both doses. Minimal NF-kappa B activation was found in lung tissue from untreated WT, TNFR1 KO, and TNFR1/IL-1R KO mice (not shown). Figure 1A shows NF-kappa B activation in nuclear protein extracts from lung tissue in WT, TNFR1 KO, and TNFR1/IL-1R KO mice treated with 1 mg/ml aerosolized LPS. NF-kappa B binding was identified as RelA/p50 and p50 homodimer bands, which were identified by separate supershift studies using specific antibodies to the RelA and p50 components of NF-kappa B (not shown). Although some biological variability was present in the intensity of NF-kappa B binding within groups, no differences were detected in NF-kappa B binding activity among the three groups of mice (WT, TNFR1 KO, and TNFR1/IL-1R KO) as assessed by laser densitometry of the RelA-containing band (Fig. 1B). No differences in NF-kappa B activation among these groups of mice were identified at either dose of inhaled LPS.


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Fig. 1.   A: electromobility shift assay (EMSA) demonstrating lung nuclear factor-kappa B (NF-kappa B) activation 4 h after aerosolized lipopolysaccharide (LPS) treatment (1 mg/ml). Four wild-type (WT) controls, 5 mice deficient in tumor necrosis factor (TNF) type 1 receptor (TNFR1 KO mice), and 5 mice deficient in TNF type 1 receptor and interleukin (IL)-1 receptor (TNFR1/IL-1R KO mice) are shown. Identity of RelA/p50 heterodimer and p50 homodimer bands was determined by supershift analysis with specific antibodies (not shown). B: laser densitometry performed on RelA-containing band. Mean densitometric measurement of RelA-containing band in each experiment was arbitrarily defined as 100%. Relative values for RelA-containing band from TNFR1 KO and TNFR1/IL-1R KO mice are shown. Values are means ± SE (n = 8-10).

At 4 h after aerosolized LPS, BAL was done to quantify the neutrophilic influx. At both doses of LPS, the majority of BAL cells were neutrophils. After 1 mg/ml aerosolized LPS, the percentage of neutrophils in lung lavage was 92 ± 1.5% in WT, 84 ± 1.3% in TNFR1 KO, and 94 ± 1.6% in TNFR1/IL-1R KO mice. The difference in total cell counts in BAL was not statistically different between TNFR1 KO or TNFR1/IL-1R KO mice (31.6 ± 4.6 × 104 and 75.2 ± 16.1 × 104, respectively) and WT mice (44.5 ± 6.7 × 104; Fig. 2). Similarly, neutrophils in BAL were comparable in all three groups after 0.3 mg/ml aerosolized LPS (89 ± 2% in WT, 84 ± 3% in TNFR1 KO, and 86 ± 2% in TNFR1/IL-1R KO mice). Neutrophils comprised <2% of BAL cells in all groups in the absence of LPS treatment.


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Fig. 2.   Total cells and neutrophils in bronchoalveolar lavage (BAL) 4 h after aerosolized LPS treatment (1 mg/ml) in WT, TNFR1 KO, and TNFR1/IL-1R KO mice. No significant differences were found in total or percent neutrophils. Values are means ± SE (n = 5).

We also evaluated the production of the NF-kappa B-dependent, neutrophil chemotactic chemokines MIP-2 and KC in BAL and lung tissue. Exposure of WT mice to LPS aerosol resulted in a dose-dependent increase in KC and MIP-2 production at 4 h (Fig. 3). Interestingly, at the lowest dose of LPS, no differences were found between groups in MIP-2 or KC levels in BAL (Table 1) or tissue homogenates (Fig. 3); however, at the higher LPS dose, MIP-2 and KC were present in the lungs in lower concentrations in TNFR1 KO and TNFR1/IL-1R KO mice than in WT controls (P < 0.05). No MIP-2 or KC was identified in lung homogenates or BAL from untreated mice.


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Fig. 3.   KC and macrophage-inflammatory peptide 2 (MIP-2) concentration in lung tissue homogenates from WT, TNFR1 KO, and TNFR1/IL-1R KO mice 4 h after exposure to 0.3 and 1 mg/ml aerosolized LPS. KC and MIP-2 concentrations were increased (P < 0.05) in samples from WT mice exposed to 1 mg/ml LPS compared with samples from WT mice exposed to 0.3 mg/ml LPS. After exposure to 1 mg/ml LPS, KC and MIP-2 concentrations were lower in TNFR1 KO and TNFR1/IL-1R KO than in WT mice. Values are means ± SE (n >=  5). *P < 0.01 and **P < 0.05 compared with WT mice treated with the same dose of aerosolized LPS.


                              
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Table 1.   Cytokine concentrations in BAL

We interpret these finding to indicate that direct lung exposure to LPS is sufficient to initiate the NF-kappa B activation pathway without involvement of TNFR1 or IL-1R. Congruent with the lack of effect on NF-kappa B activation, no changes in LPS-induced neutrophilic influx were observed. At the highest dose of LPS, some differences in chemokine production were identified, possibly occurring through a non-NF-kappa B-dependent mechanism.

Regulation of the systemic response to intraperitoneal LPS through TNFR1 and IL-1R. We reasoned that lung inflammation induced by intraperitoneal LPS likely results from direct effects of systemic LPS on the lungs and an indirect pathway involving production and release of cytokines from the liver and other organs. In these studies, mice were treated with a single intraperitoneal injection of 5.0 µg/g LPS. On the basis of previous work, this dose of LPS is beyond the threshold that results in activation of NF-kappa B in liver and lung tissue (7). Mice were harvested 90 min after intraperitoneal LPS, at which time point NF-kappa B activation in lungs and liver is near maximal (7). NF-kappa B activation in the liver is shown in Fig. 4. There was a marked induction of NF-kappa B binding activity in nuclear protein extracts of liver tissue from WT mice, but this was attenuated in liver extracts from TNFR1 KO mice (Fig. 4A). A further reduction in LPS-induced NF-kappa B activation was found in liver extracts from TNFR1/IL-1R KO mice (Fig. 4B). Densitometric evaluation of the RelA/p50 heterodimer band revealed a 41 ± 8% reduction in liver NF-kappa B activation in TNFR1 KO mice compared with WT mice (P < 0.01; Fig. 4C). Densitometry revealed a greater reduction in NF-kappa B activation of the RelA/p50 band on EMSA from TNFR1/IL-1R KO mice (82 ± 6% reduction, P < 0.001; Fig. 4C). We measured the RelA/p50 band, because the RelA component of NF-kappa B contains a transactivation domain and is thought to stimulate the transcriptional activity induced by NF-kappa B binding (1).


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Fig. 4.   NF-kappa B activation determined by EMSA from livers were obtained from mice 90 min after treatment with intraperitoneal LPS (5 µg/g). A: liver NF-kappa B activation in WT and TNFR1 KO mice. B: liver NF-kappa B activation in WT and TNFR1/IL-1R KO mice. Each lane represents a sample from a separate mouse. C: laser densitometry of RelA-containing band. Mean densitometric measurement of RelA-containing band in livers from WT mice in each experiment was arbitrarily defined as 100%. Relative values for RelA-containing band from TNFR1 KO and TNFR1/IL-1R KO mice are shown. Values are means ± SE (n = 13 for TNFR1 KO mice, 16 for TNFR1/IL-1R KO mice, and 23 for WT controls). *P < 0.01 and **P < 0.001 compared with WT mice.

In contrast to the liver, LPS-induced lung NF-kappa B activation was mildly attenuated only in the TNFR1/IL-1R KO mice (Fig. 5). Lung NF-kappa B activation was identified in the lungs of WT and TNFR1 KO mice 90 min after intraperitoneal LPS (Fig. 5A) and in lungs of WT and TNFR1/IL-1R KO mice 90 min after intraperitoneal LPS (Fig. 5B). The density of the RelA-containing band on the mobility shift assays using protein extracts from lung of all three groups is shown in Fig. 5C. Lung NF-kappa B activity was 34 ± 11% less in TNFR1/IL-1R KO than in WT mice (P < 0.05).


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Fig. 5.   NF-kappa B activation determined by EMSA from lungs obtained from mice 90 min after treatment with intraperitoneal LPS (5 µg/g). A: lung NF-kappa B activation in WT and TNFR1 KO mice. B: lung NF-kappa B activation in WT and TNFR1/IL-1R KO mice. Each lane represents a sample from a separate mouse. C: laser densitometry of RelA-containing band. Mean densitometric measurement of RelA-containing band in lungs from WT mice in each experiment was arbitrarily defined as 100%. Relative values for RelA-containing band from TNFR1 KO and TNFR1/IL-1R KO mice are shown. Values are means ± SE (n = 13 for TNFR1 KO mice, 15 for TNFR1/IL-1R KO mice, and 20 for WT controls). *P < 0.05 compared with WT.

There was a decrease in MIP-2 and KC concentrations in lung tissue from the TNFR1 KO and TNFR1/IL-1R KO mice 4 h after intraperitoneal LPS (Fig. 6), but no differences were found for IL-6. Compared with WT mice, decreased MIP-2 concentration was also detected in BAL fluid from TNFR1/IL-1R KO mice that were treated with intraperitoneal LPS (Table 1); however, there were no other differences among groups in BAL concentrations of MIP-2, KC, or IL-6 after exposure to intraperitoneal (or aerosolized) LPS. Serum MIP-2, KC, and IL-6 levels were similar in each group (data not shown). Lung neutrophils after intraperitoneal LPS were assessed by measuring lung MPO activity, because in this model few neutrophils are present in BAL at this time (7). No differences were seen in lung MPO activity 4 h after intraperitoneal LPS among the three groups (not shown).


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Fig. 6.   Cytokine levels measured by ELISA in lung homogenates from mice 90 min after treatment with intraperitoneal LPS (5 µg/g). MIP-2, KC, and IL-6 concentrations are shown for WT, TNFR1 KO, and TNFR1/IL-1R KO mice. Values are means ± SE. *P < 0.01 and **P < 0.05 compared with WT controls.

Because deficiency of TNFR1 and IL-1R inhibits activation of NF-kappa B in the liver 90 min after intraperitoneal LPS, we performed additional experiments at an earlier time point (45 min) to determine whether these receptors are required for initial activation of NF-kappa B by systemic LPS. Figure 7 demonstrates significant NF-kappa B activation in the liver by 45 min after intraperitoneal LPS. Interestingly, substantial NF-kappa B activation occurs in WT and TNFR1/IL-1R KO at this time point. Densitometry of the RelA-containing band in livers from TNFR1/IL-1R KO mice showed a reduction of 16 ± 4% compared with WT mice (P < 0.05). In these experiments, lung NF-kappa B activation was reduced by 18 ± 12% (not shown). Although mild attenuation of liver NF-kappa B activation was found 45 min after intraperitoneal LPS, the degree of inhibition was much less than at 90 min (82% reduction). From these experiments, we conclude that TNFR1 and IL-1R are primarily required for sustained, maximal NF-kappa B activation in the liver, rather than initial activation of NF-kappa B after intraperitoneal LPS.


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Fig. 7.   NF-kappa B activation determined by EMSA from livers of WT and TNFR1/IL-1R KO mice. Mice were untreated (lanes 1-4) or harvested 45 min after treatment with intraperitoneal LPS (5 µg/g, lanes 5-15). Each lane (1-13) represents a sample from a separate mouse. Cold (C) and nonspecific (NS) competition studies are shown (lanes 14 and 15).


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In these studies, TNFR1 KO and combined TNFR1/IL-1R KO mice were used to examine the role of TNF-alpha and IL-1beta in liver and lung NF-kappa B activation, cytokine production, and neutrophil recruitment in response to aerosolized or intraperitoneal LPS. Although it has been shown convincingly that ligand binding to TNFR1 can induce production of proinflammatory cytokines and adhesion molecules and that TNFR1 mediates TNF-alpha -induced recruitment of inflammatory cells to the lungs and other organs (19), the role of this receptor (and IL-1R) is less well defined in LPS-induced inflammatory states. In our studies, aerosolized LPS was expected to directly stimulate lung tissue, and any difference in inflammatory response between the groups of mice could be attributed to local production, release, and receptor binding of TNF-alpha and IL-1beta in the lungs. We found that pulmonary NF-kappa B activation was not reduced in TNFR1 KO and TNFR1/IL-1R KO mice compared with WT controls after direct exposure to aerosolization of LPS. However, concentrations of MIP-2 and KC were reduced in lung homogenates from TNFR1 KO and TNFR1/IL-1R KO mice. These findings seem to indicate that signaling through TNFR1 and IL-1R is not necessary for activation of NF-kappa B in the lungs, but these receptors are involved in finely tuning the production of CXC chemokines in response to direct exposure to LPS in a non-NF-kappa B-dependent manner. Alternatively, it is possible that our global measurements of NF-kappa B activation in lung tissue did not detect differences in NF-kappa B activation among specific lung cell types that contribute disproportionately to chemokine production. In addition, EMSA measurement of NF-kappa B activation does not detect differences in transcriptional activity of NF-kappa B dimers, which could account for differences in production of NF-kappa B-dependent chemokines. Regardless of the impact of TNFR1 and IL-1R deficiency on chemokine production, the differences in CXC chemokine production in the knockout mice were not sufficient to influence the abundant numbers of neutrophils that were recruited in the lavagable air space after exposure to LPS aerosol.

Although a previous study found that TNFR1 KO mice had fewer neutrophils and lower MIP-2 concentrations in BAL than WT control mice after aerosolized LPS, this study used lower LPS concentrations than were employed in our studies (25). Our findings are consistent with those of Peschon et al. (21), who reported that mice deficient in TNFR1 or TNFR1 and TNFR2 had comparable neutrophil accumulation in the lungs after intranasal LPS administration. Other direct airway stimuli, including heat-inactivated actinomycete from Micropolyspora faeni, induce a much more exuberant inflammatory response in WT than in TNFR1 KO mice, indicating that some lung inflammatory stimuli are more dependent on TNF-alpha than on LPS (21).

Studies using airway delivery of intact bacteria have investigated the role TNFR1 and IL-1R in the innate immune response in the lungs. Neutrophil influx into the lungs after Pseudomonas aeruginosa or E. coli instillation into the airway was found not to be impaired in TNFR1 KO mice but was somewhat diminished in TNFR1/IL-1R KO mice after airway E. coli instillation (16, 17, 25). However, intratracheal instillation of E. coli resulted in prominent activation of NF-kappa B binding activity in lung of WT, TNFR1 KO, and TNFR1/IL-1R KO mice (16, 17). We have extended these data to show that aerosolized E. coli LPS, a noninfectious stimulus, also results in similar NF-kappa B activation in WT, TNFR1 KO, and TNFR1/IL-1R KO mice. Together, these studies suggest that TNF-alpha - and IL-1beta -independent pathways mediate activation of the NF-kappa B pathway in lung cells in response to live E. coli or noninfectious E. coli LPS in the lung. It is likely that LPS in the lungs acts primarily by direct activation of the NF-kappa B pathway in target lung cells by binding to cell surface CD14 and toll-like receptors (12, 26, 27), resulting in Ikappa B phosphorylation and nuclear translocation of NF-kappa B (18). Alternatively, it is possible that LPS directly affects only certain cell types, such as alveolar macrophages, and these cells produce mediators (other than TNF-alpha and IL-1beta ) that result in upregulation of NF-kappa B activation in other lung cell types.

Deletion of the TNFR1 and TNFR1/IL-1R resulted in 41 and 82% reduction, respectively, in liver NF-kappa B activation 90 min after a single intraperitoneal injection of LPS. A similar observation was made by Mizgerd et al. (17) in studies using an E. coli pneumonia model. In this study, TNFR1/IL-1R KO mice showed no activation of NF-kappa B in the liver, but there was substantial activation of NF-kappa B in the livers of WT mice 6 h after bacterial inoculation. We found that hepatic NF-kappa B activation was markedly decreased in TNFR1 KO and TNFR1/IL-1R KO mice compared with WT control mice at a time of maximal NF-kappa B activation induced by systemic LPS. Furthermore, the impairment of hepatic NF-kappa B activation was much more severe in TNFR1/IL-1R KO than in TNFR1 KO mice. At an earlier time point (45 min), however, only small differences in liver NF-kappa B activation were identified between WT and TNFR1/IL-1R KO mice, suggesting that these receptors are required for maximal, sustained NF-kappa B activation in the liver. Initiation of NF-kappa B activation in liver cells, in contrast, may be a direct effect of systemic LPS. The attenuation of the NF-kappa B activation pathway that was observed in the livers of the TNFR1/IL-1R KO mice was associated with only a small downstream decrease in NF-kappa B activation in lung tissue. This suggests that activation of NF-kappa B in liver, in distinct contrast to lung, occurs as the result of a TNF-alpha - and IL-1beta -dependent pathway through TNFR1 and IL-1R signaling. Interestingly, these observed differences in NF-kappa B activation in the liver did not influence serum concentrations of MIP-2, KC, and IL-6.

Although it has been reported that deletion of TNFR1 in mice improves survival in a lethal systemic inflammation model in which mice are sensitized with D-galactosamine followed by LPS (21-23), no such protection occurs in these mice when challenged with lethal doses of LPS alone (21, 23). D-Galactosamine sensitizes mice to the effects of LPS and, in combination with LPS, induces fulminant hepatic injury that is TNFR1 dependent (20). The survival benefit in TNFR1 KO and TNFR1/IL-1R KO mice treated with D-galactosamine plus LPS may be due to inhibition of LPS-induced NF-kappa B activation in the liver that is deleterious in combination with D-galactosamine. The lack of survival benefit in these mice challenged with LPS alone is consistent with our findings that serum cytokines and lung MPO activity are similar among the three groups of mice after LPS treatment.

In summary, these data indicate that LPS directly activates NF-kappa B in lung tissue, but activation of NF-kappa B in the liver in response to LPS is partially mediated by TNF-alpha and IL-1beta . On the basis of these data, it is unlikely that strategies that are limited to inhibiting the actions of TNF-alpha and IL-1beta will be sufficient to abrogate multiple organ inflammation and injury in the setting of endotoxemia.


    ACKNOWLEDGEMENTS

The authors thank T. Lasakow for editorial assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-61419, HL-68121, and HL-66196 and by the Department of Veterans Affairs.

Address for reprint requests and other correspondence: T. S. Blackwell, Div. of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, T-1217 MCN, Nashville, TN 37232-2650 (E-mail: timothy.blackwell{at}mcmail.vanderbilt.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.

August 9, 2002;10.1152/ajplung.00036.2002

Received 23 January 2002; accepted in final form 29 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 283(6):L1247-L1254