From the Laboratory of Hepatobiology and Toxicology, Curriculum of Toxicology, Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599
Received for publication, November 26, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Ethanol is known to cause both tolerance and
sensitization to endotoxin (lipopolysaccharide). It is also
known that ethanol modulates the expression and activity of several
intracellular signaling molecules and transcription factors in
monocytes and Kupffer cells, the resident hepatic macrophages.
Expression of CD14, the endotoxin receptor, is up-regulated following
chronic exposure to endotoxin and ethanol. Ethanol-induced oxidative
stress is important in the regulation of transcription factor
activation and cytokine production by Kupffer cells. Thus, it was
hypothesized that acute ethanol increases CD14 expression through a
mechanism dependent upon oxidant production. This hypothesis was tested by overexpression of superoxide dismutase via recombinant adenovirus. Mice were infected with adenovirus (3 × 109
plaque-forming units, intravenously) containing either Cu,Zn superoxide
dismutase (Ad.SOD1) or Ethanol is known to cause sensitization to endotoxin
(lipopolysaccharide (LPS)),1
yet the mechanisms responsible for this observation are not clearly understood. It is known that ethanol modulates both the expression and
activity of several intracellular signaling molecules and transcription
factors in monocytes and Kupffer cells, the resident hepatic
macrophages. For example, acute ethanol increases expression and
activity of the interleukin-1 receptor-associated kinase in mouse
Kupffer cells (IRAK) (1). Recently, the signaling pathways involved in
CD14 receptor activation have been described (2, 3). Because
CD14 is not a transmembrane-spanning receptor, it couples with the
toll-like receptor 4 (Tlr-4) and initiates intracellular signaling
cascades (2, 3). Specifically, the Tlr-4 protein activates the IRAK,
which in turn activates the NF It has been reported that ethanol also alters the subunit composition
of inflammatory transcription factor NF Enomoto et al. (11) have described the phenomena whereby
ethanol alters the response of Kupffer cells, the resident hepatic macrophages, to a subsequent insult by LPS in vivo. It was
also shown that pretreatment of animals with nonabsorbable antibiotics completely prevents tolerance and sensitization caused by ethanol, suggesting the mechanism involves gut-derived LPS and activation of
Kupffer cells (12). It was also reported that an increase in CD14
expression correlated with sensitization. Thus, it is hypothesized that
Kupffer cells are activated initially by LPS to produce oxidants via
NADPH oxidase, which activates redox-sensitive transcription factors
leading to an increase in cytokine production and CD14 expression. This
hypothesis was tested by overexpressing antioxidant Cu,Zn superoxide
dismutase (SOD) in liver using recombinant adenovirus. It is also
important to note that adenovirus transduces both hepatocytes and
Kupffer cells in vivo (8). Here, it is reported that ethanol
increases both NF Animals and Treatment--
Male C57Bl/6 mice (18-22 g, The
Jackson Laboratory) were used for these experiments except for studies
evaluating the role of NADPH oxidase and TNF Adenoviral Synthesis and Preparation--
Recombinant adenovirus
containing the transgene for either Cu,Zn-SOD (Ad.SOD1) or
Liver Cell Isolation--
Kupffer cells and hepatocytes were
isolated from naive Sprague-Dawley rats (250-300 g) or rats infected
with Ad.lacZ (1 × 109 pfu) 3 days earlier.
Briefly, livers were isolated following pentobarbital anesthesia (60 mg/kg, intraperitoneally) and perfused via the portal vein for
10 min with Krebs-Ringer-HEPES buffer containing 115 mM
NaCl, 5 mM KCl, 1 mM
KH2PO4, 25 mM HEPES, 1 mM CaCl2, and 0.016% collagenase (pH 7.4)
followed by 10 min of perfusion with calcium-free buffer containing 0.5 mM EGTA. Liver cells were dispersed by gentle shaking in
phosphate-buffered saline (pH 7.4, 4 °C), and the nonparenchymal
cell fraction was separated from parenchymal cells by centrifugation
through Percoll gradients based on a method developed by
Smedsrod and Pertoft (19).
Western Blot Analysis--
Either whole liver or isolated
hepatocytes and Kupffer cells were homogenized, and samples (30 µg)
were resolved by electrophoresis using 12% SDS-PAGE. Proteins were
blotted with either anti- Immunohistochemical Staining for
CD14--
Formalin-fixed, paraffin-embedded sections (6 µm) were
mounted on glass slides. Sections were deparaffinized, rehydrated, and
then stained with mouse anti-CD14 primary antibody (Santa Cruz
Biotechnology) for 30 min. The immunostaining was visualized using the
DAKO immunostaining kit. Slides were counterstained with hematoxylin.
Primary antibody dilutions were 1:500 in phosphate-buffered saline
containing 1% Tween 20.
Electomobility Shift Assay--
For studies in whole liver,
nuclear extracts were isolated as described by Dignam et al.
(20) with minor modifications (21). Binding conditions for NF RNase Protection Assay--
Total RNA was isolated from liver
tissue using RNA-STAT 60 (Tel-Test, Friendswood, TX). RNase protection
assays for cytokine expression were performed using the RiboQuant
multiprobe assay system (Pharmingen). Briefly, [32P] RNA
probes were transcribed with T7 polymerase using the multiprobe template set mCK-3 (Pharmingen). For CD14, a cDNA fragment
amplified by reverse transcriptase PCR from mouse macrophage cDNA
library was subcloned into pCR-Topo (Stratagene, Cedar Creek, TX) by
standard cloning procedures. Prior to translation of the RNA probe, the pCR plasmid containing the CD14 insert was linearized by
HindIII restriction digest. The RNA probe was generated with
T7 polymerase using the transcription assay described above. RNA (20 µg) was hybridized with 4 × 105 cpm of probe
overnight at 56 °C. Samples were then digested with RNase followed
by proteinase K treatment, phenol:chloroform extraction, and ethanol
precipitation. Samples were resolved on a 5% acrylamide-bisacrylamide (19:1) urea gel. After drying, the gel was visualized by autoradiography.
The Overexpression of SOD in Nonparenchymal and Parenchymal
Cells--
To evaluate gene transfer of SOD in these studies, whole
liver extracts were evaluated by Western blot using antibodies against the human isoform of Cu,Zn-SOD (Fig.
1A). In the livers of animals 3 days after Ad.lacZ infection, low levels of human
Cu,Zn-SOD were detected, which was most likely caused by
cross-reactivity with the mouse isoform. In Ad.SOD1-infected mice,
however, significant Cu,Zn-SOD expression was observed.
It was demonstrated that recombinant adenovirus could transduce both
hepatocytes and Kupffer cells in vivo (8). Thus,
experiments were performed to determine whether SOD was overexpressed
in each cell type following Ad.SOD1 infection in mice. Mice were
infected with either Ad.lacZ or Ad.SOD1 as described above,
and Kupffer cells and hepatocytes were isolated 3 days after infection
as described under "Materials and Methods." Lysates from each cell type were separated by SDS-PAGE and immunoblotted for either Cu,Zn-SOD or Ethanol-induced Sensitization to LPS in Mouse Liver Is Blunted by
Overexpression of SOD--
It is known that ethanol enhances the
response of mice to LPS administration. The hypothesis that acute
oxidative stress plays a role in the mechanisms of sensitization was
tested here. First, C57Bl/6 mice were given either saline or ethanol (5 g/kg, intragastrically) followed by LPS (2.5 mg/kg, intravenously)
24 h later. Serum alanine transaminase (ALT) levels were measured
at 0, 1, 2, 4, and 8 h after LPS administration (Fig.
2A). As expected, serum ALT
levels were elevated in saline-pretreated animals at 8 h after LPS
at 110 ± 12 units/ml. However, in ethanol-pretreated mice, the
increase in serum ALT levels 8 h after LPS was significantly
potentiated by nearly 80% (186 ± 12 units/ml, p < 0.05, Repeated measures ANOVA) mice. These data support the
hypothesis that ethanol induces sensitization to LPS, which is
consistent with numerous reports.
Next, animals were infected with recombinant adenovirus (1 × 109 pfu/animal, intravenously) containing either
Cu,Zn-superoxide dismutase (Ad.SOD1) or Overexpression of SOD Blunts Acute Ethanol-induced
Redox-sensitive Transcription Factor Activation--
To understand how
oxidants participate in the mechanisms of ethanol-induced sensitization
to LPS, the hypothesis that oxidants are involved in activation of the
redox-sensitive transcription factors NF
To test the hypothesis that overexpression of Cu,Zn-SOD would blunt
acute ethanol-induced NF Cytokine Production Caused by Acute Ethanol Exposure Is
Blunted by SOD--
NF
To verify these findings, serum cytokine levels were measured in
mice following ethanol exposure. In animals infected with Ad.lacZ, a peak increase in serum TNF Acute Ethanol Increases CD14 Expression--
Recently it was
demonstrated that chronic ethanol-fed animals exhibited high levels of
CD14 expression on Kupffer cells (12, 25). The hypothesis is that
ethanol exposure causes an up-regulation of CD14, which "primes"
Kupffer cells to LPS causing an elevated inflammatory response.
Moreover, promoter elements for the CD14 gene have been shown to
contain several AP-1 consensus binding sites (26, 27). Thus, the
hypothesis that acute ethanol increased the expression of CD14 in whole
liver was tested. Animals were given ethanol (5 g/kg, intragastrically)
and sacrificed after 0, 1, 3, 12, and 21 h. CD14 mRNA,
measured by RNase protection assay, was elevated significantly at
3 h after ethanol administration (Fig.
5A), supporting the hypothesis
that ethanol increases CD14 expression in vivo.
To test the hypothesis that oxidants were involved in
ethanol-induced up-regulation of CD14, mice were infected with
Ad.SOD1 (1 × 109 pfu/animal) or
Ad.lacZ 3 days prior to ethanol (5 g/kg, intragastrically) or saline treatment, and CD14 mRNA was measured 3 h later.
Compared with saline, ethanol caused a significant increase in CD14
mRNA in Ad.lacZ-infected animals. However, the increase
in CD14 induced by ethanol was dramatically blunted in animals infected
with Ad.SOD1 (Fig. 5B). These data strongly support the
hypothesis that oxidants are involved in acute ethanol-induced
increases in CD14 levels. The strong correlation between the inhibition
of redox-sensitive transcription factors and CD14 up-regulation
suggests that CD14 expression may be regulated by NF
Because it has been reported that CD14 can be expressed on hepatocytes
in addition to Kupffer cells, it was important to determine the
location of CD14 expression following acute ethanol exposure. To test
the hypothesis that ethanol caused an up-regulation of CD14 primarily
on Kupffer cells, liver sections from mice infected with either
Ad.lacZ or Ad.SOD1 and given either saline or ethanol were
stained immunohistochemically for CD14 (Fig.
6). Compared with results in
saline-treated mice, a significant increase in CD14 detection was
observed 24 h after ethanol administration. CD14 expression was
increased predominantly in sinusoidal cells (i.e. Kupffer
cells). Ethanol-induced CD14 expression was not significantly increased
over basal levels of expression in mice infected with Ad.SOD1. This is
an important finding confirming that an increase in CD14 mRNA
translates into increased protein expression and that ethanol-induced
expression is most likely limited to Kupffer cells within the
liver.
Inhibition of AP-1 Blunts Ethanol-induced Up-regulation of
CD14--
Because AP-1 regulatory sites are present in the promoter
region of CD14 (27), it was hypothesized that AP-1 regulates CD14 expression following acute ethanol administration. To test this hypothesis, animals were infected with an adenovirus (1 × 109 pfu/animal, intravenously) containing the transgene for
either NADPH Oxidase Is Necessary for Acute Ethanol-induced Increases in
CD14--
Because oxidants are involved in acute ethanol-induced CD14
expression in vivo, and CD14 is primarily
expressed on Kupffer cells, it is hypothesized that NADPH
oxidase, the primary source of oxidants in Kupffer cells, is important
in the mechanism. Thus, ethanol (5 g/kg, intragastrically) or saline
was given to either wild-type or NADPH oxidase-deficient mice
(p47phox TNF Role of CD14 in Ethanol-induced Liver Disease--
A recently
described polymorphism in the human CD14 promoter region, resulting in
higher levels of CD14, is associated with an increase in severity of
alcoholic liver disease (28). These human studies, along with several
animal studies, strongly support the involvement of CD14 in alcoholic
liver disease and suggest that CD14 is a risk factor for
ethanol-induced pathology (29). CD14, which is expressed largely on
Kupffer cells in the liver, is activated by circulating endotoxin that
enters the liver via the portal vein. CD14 is a membrane-associated
receptor but has no transmembrane-spanning region. Thus, it associates
with the toll-like receptor-4, which initiates intracellular signaling. Although clearly defined signaling pathways have not been identified, activation of CD14 results in the production of superoxide free radicals and toxic cytokines, including TNF Up-regulation of CD14 in Sensitization--
We and others have
shown that CD14 levels are elevated in liver following chronic exposure
to ethanol (1, 24, 25). This increase correlates with
ethanol-induced liver injury. Thus, steady increases in CD14 expression
because of ethanol exposure, resulting in increased cytokine
production, could exacerbate ethanol-induced injury. These
studies were done to understand the mechanisms involved in CD14
up-regulation due to ethanol. It is shown here that acute ethanol
administration leads to a rapid increase in CD14 expression in
vivo. These data are consistent with the observation that acute ethanol causes hypersensitivity to LPS, a mechanism known as
"priming" or "sensitization." It is hypothesized that ethanol
activates signaling mechanisms that result in the up-regulation of CD14 on Kupffer cells, which sensitizes animals to subsequent challenge with
LPS. Because it is known that ethanol activates redox-sensitive transcription factors NF Activation of AP-1 and Up-regulation of CD14--
To determine
whether AP-1 was directly responsible for the up-regulation of CD14,
studies were done to inhibit AP-1 using dominant-negative TAK
expression via adenovirus. Inhibition of AP-1, either indirectly by
overexpression of SOD or directly by dominant-negative TAK, blunted the
increase in CD14 expression due to acute ethanol in addition to
blunting AP-1 activation. TAK has been demonstrated to be an
"up-stream" kinase of AP-1, leading to its activation (18). These
data are consistent with the hypothesis that AP-1 is a critical
transcription factor involved in the up-regulation of CD14. This
finding is consistent with other reports demonstrating that the CD14
promoter contains several AP-1-responsive elements (26). Overexpression
of superoxide dismutase blunted ethanol-induced activation of NF
It is hypothesized that TNF
In conclusion, ethanol induces oxidant production through NADPH oxidase
in Kupffer cells leading to the activation of redox-sensitive transcription factors, which simultaneously up-regulates cytokine expression and CD14 expression. It has recently been shown that CD14 is
necessary for ethanol-induced liver injury in mice and that increased
levels of CD14 correlate with severity of alcoholic liver disease in
humans. Thus, it is very likely that acute doses of ethanol cause an
increase in CD14 and that progressive increases in CD14 underlie the
mechanisms of pathology. As ethanol exposure increases in CD14
expression, a hyper-responsiveness may lead to a significant and
sustained increase in oxidant and cytokine production, which disposes
the liver to greater injury. Although CD14 is required for
ethanol-induced liver injury, it is not clear whether basal levels of
CD14 are sufficient to mediate the pathological effects of ethanol and
endotoxin or whether the increase in CD14 expression is required. This
is an intriguing question because it represents a potential target for
therapy, to inhibit the up-regulation of CD14 in vivo.
-galactosidase (Ad.lacZ), which caused significant expression of Cu,Zn-SOD in hepatocytes and Kupffer
cells. Three days post-infection, mice were given saline or ethanol (5 g/kg, intragastrically). A significant increase in CD14 mRNA was
observed 3 h after ethanol, and this increase was almost
completely blocked in mice overexpressing Cu,Zn-SOD. Additionally,
overexpression of SOD also blunted ethanol-induced activation of
redox-sensitive transcription factors NF
B and AP-1 and production of
cytokines. However, only inhibition of AP-1 with dominant-negative TAK1
but not NF
B by dominant-negative I
B
significantly blunted
ethanol-induced increases in CD14, suggesting that AP-1 is important
for CD14 transcriptional regulation. It is also shown here that NADPH
oxidase is important in the increase in CD14 due to ethanol.
Moreover, these data suggest that acute ethanol causes sensitization to
endotoxin through mechanisms dependent upon oxidative stress.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B signaling cascade including NIK,
IKK, I
B, and NF
B (4-6). Moreover, activation of Kupffer cell by
LPS leads to an increase in oxidant (i.e. superoxide and
nitric oxide) through NADPH oxidase and nitric-oxide synthase,
respectively. Interestingly, oxidant production is required for full
activation of LPS-induced NF
B (7), suggesting that oxidants are
involved in the signaling mechanisms. It was recently demonstrated that
overexpression of antioxidant superoxide dismutase in Kupffer cells
blunts LPS-induced NF
B activation and TNF
production in Kupffer
cells in vitro (8).
B by inducing the formation
of p50 homodimerization of NF
B (9). Independent studies have shown
that both IRAK and transcription factors NF
B and AP-1 are
redox-sensitive (10). Thus, it is conceivable that oxidant production
affects key signaling molecules and transcription factors and that
these alterations are involved in sensitization to LPS caused by acute ethanol.
B and AP-1 activity as well as cytokine production,
all of which are blunted by SOD overexpression. Ethanol-induced
increases in CD14 mRNA are blunted significantly by SOD
overexpression, consistent with the hypothesis that ethanol-induced sensitization is mediated in part by an increase in CD14, which is
dependent upon oxidant production. Finally, it is demonstrated that
inhibition of AP-1 with dominant-negative TAK1, an up-stream kinase for
c-jun, blunts ethanol-induced up-regulation of CD14. These
data clearly support the hypothesis that ethanol exposure results in
the up-regulation of CD14 through oxidant-dependent activation of AP-1.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in CD14 mRNA
regulation. For those studies, mice deficient in
p47phox, a required regulatory subunit of NADPH
oxidase, and mice deficient in TNF
were used. The
p47phox knockout mice were originally obtained
from Dr. Steve Holland (13). The TNF
knockout mice were purchased as
breeding pairs (The Jackson Laboratory), and colonies have been
maintained within IACUC (Institutional Animal Care and Use
Committee)-approved facilities and guidelines. C57Bl/6 were infected
with adenovirus (1 × 109 plaque-forming units/animal,
intravenously) 3 days prior to receiving either saline or ethanol (5 g/kg, intragastrically).
-galactosidase (Ad.lacZ) was prepared as described
elsewhere (14, 15). Briefly, the plasmid shuttle vector
pAd5.CMV.lacZ was constructed by standard cloning protocols as described by Sambrook et al. (16). The adenoviral shuttle plasmid was transfected into the permissive HEK-293 host cell line to generate recombinant Ad.lacZ adenovirus. Recombinant
adenovirus containing the transgene for human Cu,Zn superoxide
dismutase (Ad.SOD1) was the kind gift of Dr. John Engelhardt
(University of Iowa). Preparation of adenovirus containing the
transgene for hemagglutinin-tagged I
B
super-repressor (Ad.I
B)
has been described previously (17). The I
B
super-repressor is a
dominant-negative protein that contains Ser32
Ala and
Ser36
Ala mutations, which inhibit phosphorylation and
prevent NF
B dissociation and translocation into the nucleus.
Recombinant adenoviruses containing dominant-negative TAK, an inhibitor
of LPS-induced AP-1 activation (18), was provided by Dr. Richard Rippe
(University of North Carolina). The virus isolates were plaque-purified
and propagated in HEK-293 cells, isolated, concentrated, and titered by
plaque assay. Purified recombinant adenovirus (1 × 109 plaque-forming units) was suspended in normal saline
and injected into animals via the tail vein.
-galactosidase antibody (Roche Molecular
Biochemicals) or anti- Cu,Zn-SOD antibody (Oxis, Portland, OR),
followed by horseradish peroxidase-conjugated secondary antibody. The
anti-SOD antibody is cross-reactive with both endogenous rat Cu,Zn-SOD
and the human recombinant SOD. Protein was visualized by radiography
using ECL Western detection reagent (Amersham Biosciences).
B and
AP-1 were characterized and EMSA was performed as described elsewhere
(22, 23). Briefly, nuclear extracts (20 µg) from liver tissue were
preincubated with 1 µg of poly(dI-dC), 20 µg bovine serum albumin
(Amersham Biosciences) and 2 µl of a 32P-labeled DNA
probe (10,000 cpm/µl) containing 1 ng of double-stranded oligonucleotide in a total volume of 20 µl. Mixtures were incubated FOR 20 min on ice and resolved on 5% polyacrylamide (29:1
cross-linking) and 0.4× TBE (Tris borate-EDTA) gels. After
electrophoresis, gels were dried and exposed to X-Omat LS Kodak film.
The intensity of NF
B and NF
B binding was quantitated by scanning
autoradiograms with GelScan XL (Amersham Biosciences) and is expressed
as arbitrary densitometric units.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression of SOD in hepatocytes and Kupffer
cells. Mice were infected with either Ad.lacZ or
Ad.SOD1 (1 × 109 pfu). Three days after the
injection, livers were harvested. A, whole liver extracts
were separated by 12% SDS-PAGE, and SOD expression was
determined by Western blot (WB) analysis using
anti-Cu,Zn-SOD antibody. Each lane represents extract from individual
mice. B, hepatocytes and Kupffer cells were isolated from
mice infected with either Ad.lacZ or Ad.SOD1 as described
under "Materials and Methods." Extracts were separated by 16%
SDS-PAGE followed by immunoblotting with a monoclonal antibody against
-galactosidase or Cu,Zn-SOD. Lysates from human embryonic kidney
(HEK-293) cells infected with Ad.lacZ and Ad-SOD1 were used
as positive controls.
-galactosidase (Fig. 1B).
-Galactosidase was
detected in hepatocytes from Ad.lacZ-infected animals but
not in hepatocytes from Ad.SOD1-infected mice. Similarly,
-galactosidase expression was observed in Kupffer cells from
Ad.lacZ-infected animals but not in those from
Ad.SOD1-infected mice. As expected, Cu,Zn-SOD expression in hepatocytes
was significantly increased in mice infected with Ad.SOD1 compared with
mice infected with Ad.lacZ. Kupffer cells from
Ad.lacZ-infected mice expressed very little Cu,Zn-SOD.
However, Cu,Zn-SOD expression was significantly increased in Kupffer
cells from mice infected with Ad.SOD1, confirming that Ad.SOD1
transduces both hepatocytes and Kupffer cell in vivo, resulting in overexpression of SOD in each cell type.
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Fig. 2.
Overexpression of SOD blunts acute
ethanol-induced sensitization to LPS. A, mice (22-25 g,
C57Bl/6) were given LPS (2.5 mg/kg) 21 h after receiving ethanol
(5 g/kg, intragastrically) or saline. Serum was collected at intervals
of up to 8 h after LPS, and serum transaminase levels were
measured as described under "Materials and Methods." B,
mice (22-25 g, C57Bl/6) were infected with recombinant adenovirus
(1 × 109 pfu/animal) containing either Cu,Zn-SOD or
-galactosidase. Three days after infection, mice were given ethanol
(5 g/kg, intragastrically) or saline followed by LPS (2.5 mg/kg) after
21 h. Serum was collected at intervals of up to 8 h after
LPS, and serum transaminase levels were measured as described under
"Materials and Methods."
-galactosidase
(Ad.lacZ). Three days after infection, when transgene
expression is optimal, animals were given either saline or ethanol (5 g/kg, intragastrically), followed 24 h later by LPS (2.5 g/kg,
intravenously) or vehicle alone. The increase in serum ALT levels
caused by LPS alone was slightly blunted in Ad.SOD1-infected mice but
was not significantly different after 8 h compared with levels in
Ad.lacZ-infected animals (Fig. 2B). As expected,
ethanol pretreatment of Ad.lacZ-infected animals given LPS
resulted in a significant increase in serum ALT release over
Ad.lacZ-infected animals pretreated with saline.
Importantly, under these conditions, ethanol alone in both
Ad.lacZ- and Ad.SOD1-infected animals did not cause a
significant increase in ALT release. However, in animals infected with
Ad.SOD1, ethanol pretreatment had no effect on LPS-induced increase in
serum ALT levels. These data strongly support the hypothesis that
oxidative stress participates in the mechanisms of sensitization to LPS
induced by ethanol in mouse liver.
B and AP-1 following acute
ethanol was tested. First, control mice were given ethanol (5 g/kg,
intragastrically), liver nuclear extracts were isolated 0, 1, 3, and
6 h later, and the DNA binding activity of both NF
B and AP-1
was evaluated by EMSA (Fig. 3). Peak
NF
B DNA binding activity was observed ~3 h after ethanol
exposure (Fig. 3A). Using nuclear extracts isolated 3 h
after ethanol exposure, competition and supershift assays were performed to validate the specificity of NF
B DNA binding. The addition of unlabeled probe completely blocked DNA binding in extracts
from ethanol-treated mice. Moreover, the addition of antibodies against
mouse p50 and p65 subunits of NF
B caused retardation in NF
B gel
shift mobility, confirming the formation of active NF
B induced by
ethanol exposure. Similar studies were performed to evaluate the
activation of AP-1 following acute ethanol. The DNA binding activity of
AP-1 also peaked at ~3 h after ethanol (Fig. 3B). The
specificity of AP-1 DNA binding was also determined by competition and
supershift assays using antibodies against both c-Fos and
c-Jun.
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Fig. 3.
Acute ethanol-induced redox-sensitive
transcription factor activation is blunted by SOD. A,
mice were given ethanol (5 g/kg, intragastrically) and sacrificed after
0, 1, 3, and 6 h. Nuclear extracts (20 µg) prepared from whole
liver were used to evaluate NF B DNA binding activity by EMSA as
described under "Materials and Methods." Using nuclear extract
isolated from mice 3 h after ethanol exposure, NF
B competition
assay using 100-fold excess unlabeled oligo (comp), and
supershift assays using 4 µl of p50 and p65 antibodies (Santa
Cruz Biotechnology) were performed. B, AP-1 DNA binding
activity was determined by EMSA using nuclear extract as described
above. Competition was performed using excess unlabeled AP-1 oligo, and
supershifts were done using 4 µl of antibody against either
c-fos or c-jun (Santa Cruz Biotechnology).
Arrows refer to specific DNA binding; ss,
supershifted DNA-binding complex. C, mice were infected with
recombinant adenovirus (1 × 109 pfu/animal)
containing either
-galactosidase (Ad.lacZ) or Cu,Zn-SOD
(Ad.SOD1). Three days after infection, mice received ethanol (5 g/kg,
intragastrically) or saline (Con) and sacrificed 3 h
later. NF
B was measured by EMSA as described under "Materials and
Methods." D, AP-1 activity was measured 3 h after
ethanol administration in mice infected with either
Ad.lacZ or Ad.SOD1. Gels shown are representative
of three individual experiments.
B and AP-1 activation, mice were infected
with recombinant Ad.lacZ (1 × 109
pfu/animal) or Ad.SOD1 as described above. Three days after infection, mice were given 5 g/kg ethanol, and nuclear extracts were isolated from
liver 3 h later. Ethanol caused a significant increase in NF
B activity in Ad.lacZ-infected mice. Importantly,
ethanol-induced NF
B activation was markedly blunted in mice infected
with Ad.SOD1 (Fig. 3C). In animals infected with
Ad.lacZ, ethanol induced a significant increase in AP-1
activity, which was not observed in animals infected with Ad.SOD1 (Fig.
3D). These data strongly support the hypothesis that oxidant
generation caused by exposure to acute ethanol is important for
activation of redox-sensitive transcription factor AP-1. Moreover,
these data suggest that activation of NF
B and AP-1 may be important
in the sensitization to LPS caused by acute ethanol exposure.
B and AP-1 are known transcription factors
that regulate the expression of several inflammatory cytokines. Thus,
mRNA levels of cytokines were measured in livers of
Ad.lacZ- and Ad.SOD1-infected mice following
administration of either saline or ethanol (Fig. 4). Consistent with transcription factor
activation, mRNA levels for TNF
were significantly elevated in
Ad.lacZ-infected animals following ethanol injection. In
Ad.SOD1-infected mice, the ethanol-induced increase in TNF
mRNA
was blunted by >70%. Similarly, ethanol caused an increase in
interleukin-6 mRNA levels, which was nearly completely blocked in
Ad.SOD1-infected mice. These changes in mRNA, quantified by
phosphorimaging, clearly demonstrate that oxidants are involved in the
acute ethanol-induced increase in cytokine production.
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Fig. 4.
Increase in cytokine mRNAs after acute
ethanol administration. A, whole liver RNA was isolated
from animals infected with Ad.lacZ or Ad.SOD1 (1 × 109 pfu/animal) 3 h after ethanol (5 g/kg,
intragastrically) or saline administration. Cytokine expression
was measured by RNase protection assay as described under "Materials
and Methods." B, image analysis was performed on an RNase
protection assay from the experiments described above using an
ImageQuant PhosphorImager. Data are expressed as means ± S.E. and
are representative of three experiments in each group. (*,
p < 0.05, two-way ANOVA, Tukey's post hoc
analysis).
levels was observed
6 h after ethanol administration. Ethanol caused a significant
increase in TNF
from basal levels of 28.1 ± 4.3 pg/ml to
46.7 ± 7.4 pg/ml (p < 0.05, two-way ANOVA,
Tukey's post hoc analysis). This increase in serum TNF
was blunted to 29.4 ± 6.3 pg/ml in mice infected with Ad.SOD1.
These data correlate with the ethanol-induced changes in TNF
mRNA levels in mice infected with Ad.lacZ and Ad.SOD. Surprisingly, interleukin-1 or interleukin-6 was not detected in serum
following ethanol exposure in either Ad.lacZ- or
Ad.SOD1-infected mice. Perhaps this is related to changes in localized
expression of these cytokines, which may not alter serum levels.
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Fig. 5.
Ethanol-induced CD14 expression is blunted by
SOD. A, mice were given ethanol (5 g/kg,
intragastrically) and sacrificed at 0, 1, 3, 12, and 21 h after
ethanol exposure. CD14 mRNA was then measured by RNase protection
assay. B, mice, treated as described in the legend for Fig.
4, were sacrificed at 3 h after ethanol or saline exposure. CD14
mRNA levels were measured by RNase protection assay as described
under "Materials and Methods." These data are representative of
three individual experiments.
B and/or
AP-1.
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Fig. 6.
Localization of CD14 expression.
Mice (22-25 g, C57Bl/6) were infected with recombinant
adenovirus (1 × 109 pfu/animal) containing either
-galactosidase (A and C) or Cu,Zn-SOD
(B and D). Three days after infection, mice were
given saline (A and B) or ethanol (C
and D) (5 g/kg, intragastrically). 24 h later, mice
were sacrificed and livers were harvested. Liver sections were stained
immunohistochemically for CD14 using antibodies against mouse CD14
(Santa Cruz Biotechnology). Data are representative of four individual
experiments.
-galactosidase, dominant-negative I
B
, an inhibitor of
NF
B, or dominant-negative TAK1 (Ad.
TAK), an up-stream activation
kinase for AP-1 (18). Three days after infection, animals were given 5 g/kg ethanol as above and sacrificed 3 h later, and CD14 mRNA levels were measured by RNase protection assay (Fig.
7). As expected, in
Ad.lacZ-infected animals, ethanol caused a significant
increase in CD14 mRNA compared with animals given saline.
Overexpression of dominant-negative I
B
, a specific inhibitor of
NF
B, had no effect on ethanol-induced up-regulation of CD14, despite
a clear inhibition of NF
B activation in vivo (data not
shown). However, the increase in CD14 mRNA levels following ethanol
was dramatically blunted in animals infected with Ad.
TAK, strongly
supporting the hypothesis that AP-1 activation is involved in the
up-regulation of CD14 under these conditions. Also, overexpression of
dominant-negative TAK significantly blunted ethanol-induced activation
of AP-1 DNA binding compared with controls (data not shown).
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Fig. 7.
Inhibition of AP-1 blunts ethanol-induced
CD14 up-regulation. A, mice were infected with
recombinant adenovirus (1 × 109 pfu/animal)
containing -galactosidase (Ad.lacZ),
dominant-negative I
B
super-repressor (Ad.I
B
), or
dominant-negative TAK (Ad.
TAK). Three days after
infection, mice received ethanol (5 g/kg, intragastrically) or saline
and were sacrificed 3 h later. CD14 mRNA was evaluated by
RNase protection assay as described under "Materials and Methods."
B, image analysis was performed using an ImageQuant
PhosphorImager. Data are expressed as means ± S.E. and are
representative of three experiments in each group. (*,
p < 0.05, two-way ANOVA, Tukey's post hoc
analysis).
/
), and CD14 expression was measured
3 h later. As expected, in wild-type mice, administration of
ethanol, as compared with saline, caused a significant increase in CD14
mRNA (Fig. 8A). However,
ethanol-induced CD14 expression was blunted by >75% in NADPH
oxidase-deficient mice. These data strongly suggest that NADPH oxidase
is central to oxidant production following acute ethanol and that it is
involved in the up-regulation of CD14, most likely through NF
B and
AP-1 transcription factors.
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Fig. 8.
NADPH oxidase, but not
TNF , is required for ethanol-induced increase
in CD14. Mice deficient in p47phox, a
critical regulatory subunit of NADPH oxidase
(p47phox
/
), TNF
(TNF
/
), or
wild-type control mice were given ethanol (5 g/kg, intragastrically) or
saline, and CD14 mRNA was evaluated 3 h later by RNase
protection assay as described under "Materials and Methods." Data
are expressed as means ± S.E. and are representative of three
experiments in each group. (*, p < 0.05, two-way
ANOVA, Tukey's post hoc analysis).
Signaling Is Not Required for Ethanol-induced Increases in
CD14--
Because acute ethanol causes TNF
production and
also oxidative stress, we hypothesized that TNF
mediates AP-1
activation and up-regulation of CD14. To test this hypothesis,
mice deficient in TNF
(TNF
/
) and wild-type control mice were
given either saline or ethanol (5 g/kg, intragastrically). Three hours
later, the mice were sacrificed, and the level of CD14 mRNA was
measured by RNase protection assay (Fig. 8B). In the
wild-type controls, ethanol induced a significant increase in CD14
expression compared with wild-type mice receiving only saline.
Similarly, ethanol caused a significant increase in CD14 mRNA in
TNF
/
mice. Importantly, the increase in CD14 mRNA due to
ethanol was not different between wild-type and TNF
/
mice,
suggesting that TNF
signaling is not required for the increase in
CD14 expression under these conditions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, as well as a number of
other inflammatory responses. Mice deficient in CD14 are almost completely resistant to LPS-induced liver injury at physiological levels (i.e. less than 10-50 pg/ml). Moreover,
CD14-deficient mice do not exhibit any pathological changes due to
chronic ethanol exposure compared with wild-type mice. Together, these
findings demonstrate the importance of CD14 and endotoxin in
ethanol-induced liver injury. Activation of CD14 on Kupffer cells leads
to the production of superoxide and TNF
, which are involved in the
pathogenesis of alcoholic liver disease.
B and AP-1 and induces a rapid increase in
CD14 expression, it is hypothesized that oxidants activate either
NF
B or AP-1 and increase the synthesis of CD14. Moreover, it was
hypothesized that overexpression of SOD would blunt the increase in
CD14 due to acute ethanol. In these experiments it was clearly
demonstrated that SOD blunted the priming effect of ethanol in
vivo (Fig. 2B). However, it is difficult to understand whether SOD directly blunts the effects of ethanol on priming or
whether SOD blunts LPS signaling itself. Therefore, studies were done
to evaluate the role of oxidants in the priming effect of ethanol
alone. Consistent with the hypothesis that ethanol primes the liver,
acute ethanol activates NF
B and AP-1 (Fig. 3). Acute ethanol also
causes a transient increase in cytokine production similar to the
increase in CD14 expression (Figs. 4 and 5). Thus, it was also
hypothesized that activation of transcription factors NF
B and AP-1
played a role in the up-regulation of CD14.
B
and AP-1 and up-regulation of CD14, suggesting that these
redox-sensitive transcription factors are involved in the regulation of
CD14. However, it is likely that AP-1 and not NF
B is actually
involved because NF
B regulatory elements within the CD14 promoter
have not been identified. Moreover, inhibition of NF
B using the
dominant-negative I
B
super-repressor, although blocking
ethanol-induced NF
B activation, had no effect on the up-regulation
of CD14 following acute ethanol (Fig. 7). On the other hand, inhibition
of AP-1 activation using dominant-negative TAK completely and
significantly blunted the increase in CD14 expression (Fig. 7).
release by Kupffer cells in
response to acute ethanol activates the AP-1 pathway, leading to an
increase in CD14 expression. However, we demonstrate here that the
up-regulation of CD14 was not affected in mice deficient in TNF
(Fig. 8). These data suggest that TNF
signaling is not necessary for
ethanol to up-regulate CD14. This is an important point because it was
shown that TNF
is required for long-term ethanol induced liver
injury. Interestingly, deletion of NADPH oxidase in
p47phox mutant mice did inhibit CD14
up-regulation due to ethanol, supporting the hypothesis that NAPDH
oxidase is an early source of oxidants due to ethanol. This experiment
was performed to identify the potential source of oxidant production
that contributed to AP-1 activation and CD14 expression. The
p47phox subunit is most likely a critical
regulatory factor of NADPH oxidase, based on a number studies.
Therefore, these data suggest that NADPH oxidase is important for
oxidant production following acute ethanol. However, with the expanding
information about NADPH oxidase genes, it is becoming clear that
p47phox expression is not limited to
macrophages, unlike the catalytic subunit
gp91phox of NADPH oxidase, which is
predominantly expressed on macrophages. The role of
gp91phox in ethanol-induced oxidative stress in
liver is an important unresolved question.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institute on Alcohol Abuse and Alcoholism.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.
Published in memory of R. G. Thurman (1941-2001).
To whom correspondence should be addressed: CB 7365, Mary Ellen
Jones Bldg., University of North Carolina, Chapel Hill, NC 27599. Tel.:
919-966-1154; Fax: 919-966-1893; E-mail: wheelmi@med.unc.edu.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M212076200
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ABBREVIATIONS |
---|
The abbreviations used are:
LPS, lipopolysaccharide;
IRAK, interleukin-1 receptor-associated kinase;
TNF, tumor necrosis factor
;
SOD, superoxide dismutase;
TAK, TGF-
receptor-associated kinase;
pfu, plaque-forming
units;
EMSA, electrophoretic mobility shift assay;
ALT, alanine
transaminase;
ANOVA, analysis of variance.
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