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
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ABSTRACT |
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We investigated the requirement
for tumor necrosis factor- (TNF-
) 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-
B activation was similar among the groups after
exposure to aerosolized LPS. After intraperitoneal injection of LPS,
NF-
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-
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-
B activation in liver is mediated
through TNF-
- and IL-1 receptor-dependent pathways, but, in the
lung, LPS-induced NF-
B activation is largely independent of these receptors.
sepsis; macrophage; neutrophil; cytokines; chemokines
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INTRODUCTION |
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GRAM-NEGATIVE BACTERIAL
SEPSIS evokes production of the early-response cytokines tumor
necrosis factor- (TNF-
) and interleukin (IL)-1
(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-
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-
. Previous studies using mice deficient in TNFR1 have shown that TNF-
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-
B
(NF-
B) signal transduction pathway, which leads to transcriptional
upregulation of a variety of inflammatory genes (9, 15,
21). The NF-
B pathway appears to be central to innate
immunity and mediates cellular responses to TNF-
and IL-1
, as
well as LPS. IL-1
activates NF-
B through the IL-1 receptor
(IL-1R), and LPS activates NF-
B through binding to TLR4 and CD14
(12, 13, 26, 27).
Our previous studies showed that NF-B is activated in liver and
lung in response to treatment with LPS and that inhibition of NF-
B
activation results in suppression of chemokine production in the lungs
and downregulation of neutrophilic alveolitis (3, 4, 7).
Because production of TNF-
and IL-1
is rapidly induced by LPS, we
sought to determine whether these cytokines contribute to NF-
B
activation in the lungs and liver after LPS treatment. We hypothesized
that, in LPS-induced inflammation, TNF-
and IL-1
act to amplify
inflammatory signaling through binding to TNFR1 and IL-1R and
activation of NF-
B. Therefore, in these experiments, we sought to
determine the degree to which NF-
B activation in response to LPS is
dependent on TNF-
and IL-1
. 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-
B activation was then measured by electrophoretic
mobility shift assays (EMSA) in lung and liver tissues at the time of
maximal NF-
B activation. We found that LPS-induced NF-
B
activation in the lungs was unaffected in these knockout mice after
aerosolized LPS and that NF-
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-
B activation was diminished in TNFR1 KO and TNFR1/IL-1R KO mice compared with WT controls. These findings suggest that NF-
B-dependent inflammation in
the lungs does not require amplification of the initiating signal
through production of the proximal cytokines TNF-
and IL-1
.
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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). [-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-
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-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-B and [
-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 -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-
B oligonucleotide and nonspecific
competition using an excess of unlabeled oligonucleotide that did not
contain an NF-
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-
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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RESULTS |
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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-B activation
and a significant neutrophil influx into the lungs at 4 h
(24). NF-
B activation was induced in lung tissue in all
three groups of mice at 4 h after aerosolized LPS at both doses.
Minimal NF-
B activation was found in lung tissue from untreated WT,
TNFR1 KO, and TNFR1/IL-1R KO mice (not shown). Figure 1A shows NF-
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-
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-
B (not shown). Although some
biological variability was present in the intensity of NF-
B binding
within groups, no differences were detected in NF-
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-
B activation among these groups
of mice were identified at either dose of inhaled LPS.
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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-B in liver and lung tissue (7). Mice
were harvested 90 min after intraperitoneal LPS, at which time point
NF-
B activation in lungs and liver is near maximal (7).
NF-
B activation in the liver is shown in Fig.
4. There was a marked induction of
NF-
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-
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-
B activation in
TNFR1 KO mice compared with WT mice (P < 0.01; Fig.
4C). Densitometry revealed a greater reduction in NF-
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-
B contains a transactivation domain and is thought
to stimulate the transcriptional activity induced by NF-
B binding
(1).
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DISCUSSION |
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In these studies, TNFR1 KO and combined TNFR1/IL-1R KO mice were
used to examine the role of TNF- and IL-1
in liver and lung
NF-
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-
-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-
and IL-1
in the lungs. We found that
pulmonary NF-
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-
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-
B-dependent manner. Alternatively, it is possible that our
global measurements of NF-
B activation in lung tissue did not detect
differences in NF-
B activation among specific lung cell types that
contribute disproportionately to chemokine production. In addition,
EMSA measurement of NF-
B activation does not detect differences in transcriptional activity of NF-
B dimers, which could account for
differences in production of NF-
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- 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-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-
B activation in WT, TNFR1 KO, and TNFR1/IL-1R KO mice.
Together, these studies suggest that TNF-
- and IL-1
-independent
pathways mediate activation of the NF-
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-
B pathway in target lung cells by
binding to cell surface CD14 and toll-like receptors (12, 26,
27), resulting in I
B phosphorylation and nuclear translocation of NF-
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-
and IL-1
) that result in upregulation of NF-
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-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-
B in the liver, but there was substantial
activation of NF-
B in the livers of WT mice 6 h after bacterial
inoculation. We found that hepatic NF-
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-
B activation induced by systemic LPS. Furthermore, the impairment of hepatic NF-
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-
B
activation were identified between WT and TNFR1/IL-1R KO mice,
suggesting that these receptors are required for maximal, sustained
NF-
B activation in the liver. Initiation of NF-
B activation in
liver cells, in contrast, may be a direct effect of systemic LPS. The
attenuation of the NF-
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-
B activation in lung tissue. This suggests
that activation of NF-
B in liver, in distinct contrast to lung,
occurs as the result of a TNF-
- and IL-1
-dependent pathway
through TNFR1 and IL-1R signaling. Interestingly, these observed
differences in NF-
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-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-B in
lung tissue, but activation of NF-
B in the liver in response to LPS
is partially mediated by TNF-
and IL-1
. On the basis of these
data, it is unlikely that strategies that are limited to inhibiting the
actions of TNF-
and IL-1
will be sufficient to abrogate multiple
organ inflammation and injury in the setting of endotoxemia.
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ACKNOWLEDGEMENTS |
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The authors thank T. Lasakow for editorial assistance.
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FOOTNOTES |
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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.
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