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*
Department of Pharmacology, Laboratory of Hepatobiology and Toxicology, University of North Carolina, Chapel Hill, NC 27599; and
Division of Molecular Medicine, North Shore University Hospital, Cornell University Medical College, Manhasset, NY 11030
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, TGF-
, and
TNF-
were increased significantly in wild-type mice fed ethanol but
not in the CD14 knockout. Thus, chronic ethanol feeding caused more
severe liver injury in wild-type than CD14 knockouts, supporting the
hypothesis that endotoxin acting via CD14 plays a major role in the
development of early alcohol-induced liver
injury. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs and was prevented in TNF
receptor-1 knockout mice (8, 9).
CD14, a GPI-anchored glycoprotein that is highly expressed on the
surface of human monocytes and most macrophages (10), has
been demonstrated to be part of a specific cellular LPS binding site
(11) and is a key mediator of septic shock induced by
endotoxin (10, 12, 13). Circulating LPS binds to
LPS-binding protein
(LBP)3 and activates
monocytes/macrophages via binding to the CD14 receptor (14, 15). Kupffer cells, the resident liver macrophages, are the
major population of the monocyte-macrophage lineage (16)
and contain CD14 receptors (4, 17). Recent studies show
that alcohol exposure increases the expression of CD14 in Kupffer cells
(18) and estrogen sensitizes Kupffer cells to LPS via
increases in CD14. The interaction of LPS with CD14 triggers a
signaling cascade and results in induction of cytokines (IL-6, IL-1,
and TNF-) that are known to participate in liver injury
(2). It has been shown previously that CD14-deficient mice
do not produce significant levels of these cytokines even when exposed
to high levels of LPS (12). To test the hypothesis that
CD14 is involved in early alcohol-induced liver injury, the response of
CD14-deficient mice to ethanol was compared with that wild-type mice by
evaluating parameters of hepatic injury using a murine enteral ethanol
feeding model (9). After 4 wk of continuous enteral
ethanol feeding, not only was steatosis observed, but inflammation and
necrosis also occurred in livers of wild-type mice. However, in mice
lacking CD14 receptors, hepatic pathology due to alcohol was largely
blocked. These studies show that CD14, presumably on Kupffer cells,
plays a critical role in early alcohol-induced liver injury and further
support a role for endotoxin in this disease. Preliminary accounts of
this study have been reported elsewhere (19).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female CD14-deficient mice generated as described elsewhere (12, 20) were backcrossed 10 times with BALB/c mice (The Jackson Laboratory, Bar Harbor, ME). Age- and sex-matched wild-type BALB/c mice, weighing 21–24 g, served as controls. Mice were screened for the presence of the CD14 receptor mRNA (+/+) in wild-type mice and the absence of the CD14 receptor (-/-) mRNA in knockout mice by RNase protection assay. All animals received humane care in compliance with institutional guidelines. Body weight was measured before surgery and at necropsy after 4 wk of continuous delivery of control or ethanol-containing diet.
Surgery
The principle surgical procedures were similar to methods previously described by Tsukamoto et al. (21) in rats, with modifications based on the size of mice (9). Briefly, mice were anesthetized by injection of pentobarbital sodium (50 mg/kg; Abbott Laboratories, North Chicago, IL), and laparotomy was performed under sterile surgical conditions. A PE90 polyethylene tube (Becton Dickinson, Sparks, MD) was placed in the squamous part of the stomach. The tube was anchored to the stomach wall with Dacron and fixed to the abdominal wall. It was then tunneled s.c. to the dorsal aspect of the neck followed by closing of the abdominal wall with 7-0 prolene sutures. The tube was then pulled through a 250P polysulfone attachment mouse button (Instech Laboratories, Plymouth Meeting, PA) and spring coil. The button was fixed under the skin with its metal spring coil outside of the body to protect the tube. The feeding tube was attached to an infusion pump by means of a swivel, allowing complete mobility of the mouse within a metabolic cage. Animals were allowed to recover for 1 wk with free access to chow diet and water before starting alcohol-containing or control high-fat liquid diets.
Diets
A basic liquid diet was prepared according to Thompson and Reitz (22) as described previously and supplemented with lipotropes as described by Morimoto et al. (23). The control diet (1.3 kcal/ml) contained corn oil as fat (34% of total calories), protein (23%), carbohydrate (43%), plus minerals and vitamins. For the isocaloric ethanol diet, dextrin-maltose was replaced by ethanol (9). The liquid diet was fed continuously at the rate of 11 ml/day to achieve weight gain. Throughout the experimental period of liquid diet delivery, mice had free access to cellulose pellets as a source of fiber (Harlan Teklad, Madison, WI).
Experimental protocol
CD14-deficient and wild-type mice were randomly allocated into two experimental groups of five to seven mice each and were fed either ethanol-containing or an isocaloric high-fat control diet. Animals received diets by infusion through an intragastric cannula for up to 4 wk as described previously (9). Usually, the ethanol dose was 28–29 g/kg/day during the fourth week of feeding. CD14-deficient and wild-type mice were sacrificed after 4 wk and blood samples were collected via the inferior vena cava at necropsy. Serum was stored at -20°C until alanine aminotransferase (ALT) was analyzed by standard enzymatic procedures (24). Livers were removed and weighed and tissue samples were divided; some were fixed in Formalin, others were frozen in liquid nitrogen and stored at -80°C.
Urine collection and assay for ethanol
Concentrations of ethanol in urine are representative of blood alcohol levels (25). Mice were housed in metabolic cages that separated urine from feces and urine samples were collected over 24 h for each mouse. Ethanol levels in urine were determined daily by measuring absorbance at 366 nm resulting from the reduction of NAD+ by alcohol dehydrogenase (24).
Pathological evaluation
Formalin-fixed liver samples were embedded in paraffin and stained with hematoxylin and eosin to assess steatosis, inflammation, and necrosis. Liver pathology was scored as described by Nanji et al. (26) as follows: steatosis (the percentage of liver cells containing fat): <25% = 1+; <50% = 2+; <75% = 3+; >75% = 4+; inflammation and necrosis: 1 focus per low-power field = 1+; 2 or more = 2+. One point was given for each grade of severity of histological abnormality and a total score was calculated for each liver.
RT-PCR amplification of CD14 cDNA
Mouse Kupffer cell RNA was extracted by standard phenol:chloroform extraction in guanadinium isothiocyanate (RNA-STAT60; Tel-Test, Friendswood, TX) followed by ethanol precipitation. mRNA (5 µg) was added to reverse transcription buffer containing 0.5 U of RNase (Promega, Madison, WI), 5 mM DTT, 2 mM dNTPs (Life Technologies, Rockville, MD), 1 µM random hexamer primer (Boehringer Mannheim, Indianapolis, IN), and 1 U of avian myleoblastosis virus reverse transcriptase (Promega). The reaction was incubated at 42°C for 45 min and terminated at 95°C for 2 min. An aliquot (5 µl) of the RT reaction was added to PCR amplification buffer containing 2 mM dNTPs (Life Technologies), 0.4 µM forward and reverse primers, and 0.5 U of Taq DNA polymerase (Boehringer Mannheim). PCR was initiated by a 7-min incubation at 95°C, followed by 20 cycles of 1 min at 95°C, 1 min at 48°C, and 2 min at 72°C. The reaction was followed by a 10-min incubation at 72°C. The amplification product was resolved on 1.5% agarose gel. PCR primers for CD14 which generated a 180-bp fragment were as follows: forward, 5'-GCAACTTCTCAGATCCGAAGCC-3'; and reverse, 5'-CAACAGTAAGCCGCTTTAAGAC-3'.
RNase protection assay
Total RNA was isolated from hepatic tissue using RNA STAT60
(Tel-Test). Samples of total RNA (30 µg) were hybridized with
105 cpm of [-32P]UTP
(3000 Ci/mmol; Amersham, Arlington Heights, IL)-labeled probe (see
below) at 42°C overnight. RNase digestions were conducted with a
mixture of RNase A and RNase T1 (PharMingen, San Diego, CA)
at 37°C for 45 min, followed by proteinase K treatment for 15 min,
and phenol: chloroform extraction and ethanol precipitation. The
digested products were separated on a 6% acrylamide gel. The RNA probe
used to detect CD14-specific RNA was generated by in vitro
transcription of linearized pCR-rCD14 after cleavage with
HindIII. The pCR-rCD14 plasmid was generated by insertion of
the RT-PCR amplification fragment (180 bp) of mouse Kupffer cell cDNA
into the pCR-TOPO cloning plasmid (Invitrogen, San Diego, CA). The RNA
probes (PharMingen) used to detect GAPDH-specific RNA (as control), L32
as a ribosomal housekeeping gene, TNF-, lymphotoxin- (LT-),
macrophage migration inhibitory factor (MIF), and TGF- were
generated by in vitro transcription of mouse GAPDH template
(PharMingen). The protected RNA was visualized by autoradiography.
EMSA
A gel mobility shift assay was used in this study to assess the
amount of active protein involved in protein-DNA interactions. The
limitations of this method are the amount of nuclear protein needed for
assay as well as the number of cells isolated from the rat liver.
Binding conditions for NF-B were characterized and EMSAs were
performed as described in detail elsewhere (27).
Briefly, extracts (40 µg) from liver tissues were preincubated for 10
min on ice with 1 µg of poly(dI-dC) and 20 µg of BSA (both from
Pharmacia Biotech, Piscataway, NJ) in a buffer that contained 1 mM
HEPES (pH 7.6), 40 mM MgCl2, 0.1 M NaCl, 8%
glycerol, 0.1 mM DTT, 0.05 mM EDTA, and 2 µl of a
32P-labeled DNA probe (10,000 cpm/µl)
that contained 0.4 ng of double-stranded oligonucleotide. Mixtures were
incubated for 20 min on ice and resolved on 5% polyacrylamide (29:1
cross-linking) and 0.4x Tris-borate-EDTA gels. After electrophoresis,
gels were dried and exposed to Kodak film (Kodak, Rochester, NY). Data
were quantitated by scanning autoradiograms with GelScan XL
(Pharmacia).
Statistics
ANOVA was used for the determination of statistical significance as appropriate. For comparison of pathological scores, the Mann-Whitney U rank sum test was used. Data are presented as mean ± SEM. A p < 0.05 was selected before the study as the level of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Liver and body weight were determined to assess the general health
of the animals. All animals survived surgery and gained weight during 4
wk of continuous delivery of high-fat liquid diets with or without
ethanol. There were no significant differences in body weight among the
groups studied. At necropsy, liver to body weight ratios in wild-type
mice receiving ethanol were significantly higher than in wild-type mice
fed control diet (Fig. 1
). In contrast,
the ratio in CD14-deficient mice given ethanol was significantly lower
than in wild-type mice fed ethanol. Thus, ethanol caused significantly
greater enlargement of livers in wild-type than in CD14 knockout
mice.
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Urine ethanol concentrations were monitored to index the degree of intoxication. As reported previously in studies with rats (28) or C57BL mice (7, 9), urine ethanol levels also fluctuated in the CD14-deficient or wild-type BALB/c mice in a cyclic pattern from 18 to >500 mg/dl likely due to thyroid hormones and stress as was recently reported in rats (29). Average urine ethanol concentrations during 4 wk of ethanol exposure were 158 ± 25 mg/dl in wild-type mice and 162 ± 26 mg/dl in the knockouts, values which were not statistically different.
Serum transaminases
As a marker of liver injury, serum ALT was monitored throughout
the study. Before continuous administration of liquid diets, the serum
level of ALT in both wild-type and CD14 knockout mice was 44 ± 5
U/L. In wild-type mice, 4 wk of ethanol exposure significantly
increased the serum ALT 
3-fold (126 ± 27 U/L; Fig. 2); however, ethanol exposure did not
increase ALT in CD14-deficient mice.
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Fig. 3 shows representative
photomicrographs of livers from wild-type and CD14 knockout mice after
4 wk of exposure to control or ethanol diets. There were no
pathological changes seen in wild-type or CD14 knockout mice receiving
high-fat control diet (Fig. 3, A and B).
Accordingly, the pathology score of livers from wild-type and knockout
mice fed control high-fat diet was minimal (Fig. 4). However, marked fatty accumulation
and mild to moderate inflammation and necrosis were observed in
wild-type mice given ethanol (Fig. 3C), with an average
pathology score of 3.8 ± 0.6 (Fig. 4D). This value was
significantly greater than values observed in wild-type mice given
control diet (Fig. 4D). Fatty accumulation in wild-type mice
receiving ethanol was panlobular, with massive large droplets of fat in
pericentral areas and midzonal regions near central veins. In livers
from CD14-deficient mice, however, only mild fatty accumulation was
detected (Fig. 3D), with mild inflammation but no necrosis.
As a result, the pathology scores of livers from CD14 knockout mice
receiving ethanol diet were only 1.7 ± 0.4 (Fig. 4D),
values which were significantly lower than ethanol-treated, wild-type
mice (p < 0.05).
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An RNase protection assay was used to determine the tissue levels
of message for TNF-, LT-, MIF, and TGF-. Ethanol caused about
a 2.5-fold increase in tissue levels of TNF- mRNA in livers from
wild-type mice (Fig. 5) as well as the
inflammatory cytokine LT-. This phenomenon was not observed in CD14
knockout mice. TGF- and LT- were doubled after ethanol in the
wild-type but not in CD14 knockout mice (Fig. 5). MIF was detected in
all groups. To assess the involvement of NF-B after alcohol
treatment in both strains of mice, activation was measured by the EMSA
in liver (Fig. 6). A doubling of NF-B
activation occurred after ethanol treatment in wild-type mice. In CD14
knockout mice, however, activation of NF-B did not occur after
ethanol treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of monocytes and macrophages by endotoxin (LPS) from Gram-negative bacteria has been extensively studied to define the mechanisms that underlie innate immune responses against bacterial pathogens (30). Numerous studies have revealed that LPS, instead of being directly toxic to cells or organs, exerts its biological effects indirectly through stimulation of host cells to produce endogenous mediators which elicit a response that can lead to severe tissue injury and/or death. The mechanism through which endotoxin stimulates cells is a multistep process that includes the initial binding of endotoxin to a serum protein, LBP (14). The binding of endotoxin to LBP forms monomers and allows LPS to efficiently bind to CD14 on the surface of monocytes/macrophages (11, 14). The binding of LPS to CD14 triggers the release of endogenous inflammatory mediators including cytokines and free radicals (15, 30, 31, 32, 33), leading to inflammation and requires additional molecules for signal transduction, including Toll-like receptors (34, 35). It was very recently reported that alcohol-induced liver injury, using the same model used in this study, is blunted in the Toll-like receptor 4 mutant C3H/HeJ mouse (36). Taken together, this study and the work presented here support the hypothesis that a signaling pathway comprised of CD14 and Toll-like receptor 4 is required for development of pathology due to ethanol.
Ethanol and endotoxin
Studies demonstrating the strong resistance of
CD14-deficient mice to endotoxin suggest that CD14 plays a predominant
role in endotoxin shock (12, 37). The development of the
CD14 knockout mice provides a powerful tool for studies of the role of
endotoxin in various diseases. It is well known that ethanol exposure
increases circulating endotoxin (4). However,
ethanol-induced hepatic injury was minimized significantly here in
CD14-deficient mice (Fig. 4
). This indicates that cells of the immune
system respond to LPS through a pathway involving the CD14 receptor
(13, 38) and supports the hypothesis that elevated
circulating endotoxin by excessive alcohol intake activates residential
hepatic macrophages (Kupffer cells) to release endogenous inflammatory
mediators via activation of NF-B (Figs. 6 and 7).
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B by enteral ethanol treatment in the rat
is known (39). It has been shown that NF-B activation
is not increased after 1 wk of ethanol treatment (40).
This suggests that longer exposure to ethanol is critical as was
observed here (Fig. 6). The increase in NF-B activation is
consistent with the hypothesis that endotoxin binds to CD14, activates
the Kupffer cells, and elicits the production of reactive oxygen
species which increases NF-B (Fig. 7).
The absence of activation of NF-B in the CD14 knockout mouse after
enteral ethanol strengthens this hypothesis.
Recently, it was reported that chronic ethanol increases mRNA for
TGF- expression in rats 2 fold after 2 wk of enteral ethanol
(41). In these experiments, mRNA for LBP and CD14 were not
increased by chronic endotoxin but were increased after acute
endotoxin. The authors conclude that down-regulation of mRNA for CD14
and LBP may occur as an adaptation in chronic alcohol abuse. The
results in mice presented here using a chronic model of ethanol
delivery provide an alternative explanation. TGF- protein is doubled
after ethanol treatment (see Fig. 5 and Results) in
wild-type mice but is not elevated in the CD14 knockout mouse fed
control or ethanol-containing diet, suggesting that CD14 is necessary
for TGF- activation and not for down-regulation.
Cytokines
TNF- is a central proinflammatory cytokine
(42). TNF- acting through its receptor-1 pathway plays
a predominant role in LPS-induced inflammatory diseases
(43) and mediates the lethal effects of endotoxin
(44). TNF- is increased in alcoholics with hepatitis
and levels correlate with survival (35) and appears to be
the principle mediator of early alcohol-induced liver injury since
injury was blocked in mice lacking the TNF receptor-1 (9).
In the present study, increased levels of TNF- (Fig. 5) along with
the most severe hepatic injury among the groups studied were observed
in the livers from wild-type mice fed ethanol, whereas these effects
were blocked in CD14-deficient mice. Therefore, these results are
consistent with the hypothesis that excessive intake of alcohol
increases circulating endotoxin which activates Kupffer cells via the
CD14 receptor to release TNF-, leading to liver injury (Fig. 7).
In this study, macrophage MIF was detected in all treatment
groups studied (Fig. 5) and is commonly expressed in liver and other
tissues (45) as an inflammatory cytokine that can also
induce TNF- (46). Studies with anti-MIF Ab
demonstrate a protective effect in models of endotoxin-induced injury
(47). After 4 wk of dietary treatment with or without
ethanol, a mild inflammatory infiltration was observed in mice
receiving a high-fat control diet. This is different from the results
of a previous study using mice on a C57BL/6J background
(9), where no inflammation was detected in animals
receiving a high-fat control diet. This phenomenon is possibly due to
the fact that BALB/c mice are more susceptible to inflammation
(48) involving, in part, the inflammatory cytokine
MIF.
In summary, the results of the present study are consistent with the hypothesis that CD14 receptors play a major role in vivo in the pathogenesis of alcohol-induced liver injury; thus, drugs or genes that target CD14 signaling pathways may prove beneficial in treating alcoholic hepatitis.
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Footnotes |
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2 Address correspondence and reprint requests to Blair U. Bradford, Department of Pharmacology, Laboratory of Hepatobiology and Toxicology, CB 7365, Mary Ellen Jones Building, University of North Carolina, Chapel Hill, NC 27599-7365.
3 Abbreviations used in this paper: LBP, LPS-binding protein; ALT, alanine aminotransferase; LT-, lymphotoxin-; MIF, migration inhibitory factor.
Received for publication November 6, 2000. Accepted for publication January 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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attenuate hepatic necrosis and inflammation due to chronic exposure to ethanol in the rat. Hepatology 26:1530.[Medline]
in alcohol-induced liver injury. Gastroenterology 117:942.[Medline]
production. J. Immunol. 162:4801.B: affinity, specificity, Zn2+ dependence, and differential half-site recognition. J. Biol. Chem. 266:252., IL-6, and IL-8 release by human monocytes and alveolar macrophages. J. Immunol. 150:2885.[Abstract]
and IL-6 in alcoholic liver disease. E. Wisse, and D. L. Knook, and R. S. McCuskey, eds. In Cells of the Hepatic Sinusoid Vol. 3:472.-475. Kupffer Cell Foundation, Leiden, The Netherlands.
prevents lethal endotoxin sepsis in adult rhesus monkeys. J. Lab. Clin. Med. 120:574.[Medline]
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