1 Laboratory of Hepatobiology and Toxicology, Department of Pharmacology and 3 Department of Pathology, University of North Carolina, Chapel Hill, North Carolina 27599-7365; and 2 Department of Medicine, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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The purpose of
this study was to determine whether early alcohol-induced liver injury
(ALI) in females is associated with changes in CD14 on Kupffer cells,
activation of hepatic nuclear factor (NF)-B, and expression of tumor
necrosis factor (TNF)-
mRNA. Male and female rats were given
high-fat control or ethanol-containing diets for 4 wk using the
intragastric enteral protocol. Physiological parameters were similar in
both genders. Ethanol was increased as tolerance developed with higher
blood levels than previously observed, resulting in a fourfold increase
in aspartate aminotransferase (males 389 ± 47 IU/l vs. females 727 ± 66 IU/l). Hepatic pathology developed more rapidly and was nearly
twofold greater and endotoxin levels were significantly higher in
females after ethanol. Also, expression of CD14 on Kupffer cells was
1.5-fold greater and binding of transcription factor NF-
B in hepatic
nuclear extracts and TNF-
mRNA expression were threefold greater in
females. These data are consistent with the hypothesis that elevated
endotoxin after ethanol triggers more activation of Kupffer cells via
enhanced CD14 expression in females. NF-
B is activated in this
process, leading to increases in TNF-
mRNA expression in the liver
and more severe liver injury in females. It is concluded that gender differences in ALI are dependent on endotoxin and a signaling cascade
leading to TNF-
.
Kupffer cells; endotoxin
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INTRODUCTION |
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CLINICALLY, WOMEN DEVELOP alcohol-induced liver injury more rapidly and to a greater extent than men while consuming fewer grams of alcohol (3, 28). Although many hypotheses have been presented to explain gender differences in alcohol-induced liver injury, including alterations in absorption, disposition, and metabolism (14, 30, 49), mechanisms have remained unclear because of the lack of a clinically relevant animal model.
The establishment of a continuous intragastric enteral feeding protocol in the rat by Tsukamoto and French (13, 41) represented a major development in alcohol research. With this model, not only is steatosis observed, which is characteristic of several animal models, but inflammation and necrosis also occur in ~4 wk, and fibrosis begins to develop within 12-16 wk in male rats. With this model, it has been reported that intestinal sterilization with antibiotics (2) or lactobacillus feeding (25) diminishes plasma endotoxin levels and liver injury. Furthermore, destruction of Kupffer cells with GdCl3 prevents early alcohol-induced liver injury in males (1). These results are consistent with the hypothesis that Kupffer cells activated by gut-derived endotoxin play an important role in early alcohol-induced liver injury in males.
Ethanol increases plasma endotoxin levels and expression of the
lipopolysaccharide receptor CD14 on Kupffer cells in male rats (15,
34). Moreover, Kupffer cells activated by endotoxin produce reactive
oxygen species. These oxidants could activate transcription factors
such as nuclear factor (NF)-B, which regulates production of
inflammatory cytokines and adhesion molecules, leading to liver injury
(15). Tumor necrosis factor (TNF)-
, which plays a pivotal role in
the inflammatory cytokine cascade, is involved in early
alcohol-induced liver injury. Indeed, anti-TNF-
antibody reduces
inflammation and necrosis in the Tsukamoto-French model (16).
Furthermore, early alcohol-induced liver injury is diminished in TNF
receptor-1 knockout mice given intragastric enteral ethanol (47).
Previous work from this laboratory has shown that endotoxin levels in
plasma, intercellular adhesion molecule-1 (ICAM-1) expression in
sinusoidal lining cells, the number of infiltrating neutrophils in the
liver, and liver injury are significantly greater in females than in
males (15). Accordingly, the purpose of this study was to determine
whether CD14 expression on Kupffer cells, activation of NF-B, and
TNF-
mRNA in the liver are responsible for greater pathology in the
female. In the present study, both genders received progressively
increasing doses of ethanol by carefully challenging them as tolerance
developed over a 4-wk period, since alcohol-induced liver injury is
dependent on the dose of ethanol (9). This approach resulted in ethanol
delivery in both genders that was ~50% greater than in previous
studies (15, 27, 39). Under these conditions, the extent of early
alcohol-induced liver injury was more severe in both genders, yet the
injury in females developed earlier and was more extensive than in males.
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MATERIALS AND METHODS |
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Animals and experimental protocol.
Age-matched male and female Wistar rats were given high-fat control or
ethanol-containing diets for 4 wk using the intragastric enteral
protocol developed by Tsukamoto and French (13, 41). Daily caloric
intake reached 230 kcal · kg1 · day
1.
Ethanol initially was delivered at 10 g · kg
1 · day
1
(35% of total calories) and was increased 0.6 g · kg
1 · 2 days
1 until the end of the first week
and then 0.6 g · kg
1 · 4 days
1 until the end of the experiment
(final alcohol delivered = 17 g · kg
1 · day
1;
40% of total calories). All animals received humane care in compliance
with institutional guidelines, and alcohol intoxication was assessed
carefully to evaluate development of tolerance to ethanol using a
0-3 point scoring system (0, normal; 1, sluggish movement; 2, loss
of movement but still moving if stimulated; 3, loss of consciousness).
Ethanol was increased progressively to challenge animals based on this
assessment, allowing ~50% more ethanol to be delivered than in
previous studies by this and other laboratories (15, 27, 39).
Diets. A liquid diet described by Thompson and Reitz (36), supplemented with lipotropes as described by Morimoto et al. (24), was used. It contained corn oil as fat (37% of total calories), protein (23%), carbohydrate (5%), minerals and vitamins, plus ethanol (35-40% of total calories) or isocaloric dextrose (control diet) as described elsewhere (40).
Urine collection and ethanol assay.
Daily ethanol concentrations in urine, which are representative of
blood alcohol levels (4), were measured. Rats were housed in metabolic
cages that separated urine from feces, and urine was collected over 24 h in bottles containing mineral oil to prevent evaporation. Each day at
9 AM, urine collection bottles were changed and a 1-ml sample was
stored at 20°C in a microtube for later analysis. The
ethanol concentration was determined by measuring absorbance at 366 nm
resulting from the reduction of NAD+ to NADH by alcohol
dehydrogenase (7).
Clinical chemistry.
Blood was collected via the aorta at 4 wk and centrifuged. Serum was
stored at 20°C in a microtube until it was assayed. Aspartate aminotransferase (AST) was analyzed by standard enzymatic procedures (7). Estradiol was measured with no-extraction, solid-phase
125I-radioimmunoassay (Coat-A-Count Estradiol, Diagnostics
Products, Los Angeles, CA) based on antibody-coated tube technology
(21, 45). After incubation for 1 h at 37°C, separation of bound
from free estradiol was achieved by a decanting process. The remaining radioactivity was then counted in a gamma counter, with counts inversely related to the amount of estradiol present in the sample. The
quantity of estradiol was determined by using known standards and a
calibration curve.
Pathological evaluation. Rats underwent liver biopsy and autopsy at 2 and 4 wk of treatment with ethanol, respectively. Livers were formalin-fixed, 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 foci = 2+. Pathology was scored in a blinded manner by one of the authors and by an outside expert on rat liver pathology.
The number of neutrophils in liver sections was also determined after 4 wk by counting cells in three high-power fields (×400) per slide. Fat accumulation caused ballooning of hepatocytes and narrowing of the sinusoidal space. This could affect the number of hepatocytes and sinusoidal space in each field; therefore, the number of hepatocytes was also counted and the number of neutrophils was expressed per 100 hepatocytes. The mean value from three high-power fields was used for statistical analysis.Kupffer cell preparation and Western blotting for CD14.
Kupffer cells were isolated by collagenase digestion and differential
centrifugation using Percoll (Pharmacia, Uppsala, Sweden) as described
elsewhere with slight modifications (29). Cells were plated on plastic
culture dishes and cultured in RPMI 1640 media (GIBCO Life
Technologies, Grand Island, NY) supplemented with 25 mM HEPES, 10%
fetal bovine serum, and antibiotics (100 U/ml of penicillin G and 100 µg/ml of streptomycin sulfate). After 1 h of incubation, Kupffer
cells were scraped with a sterile cell scraper and pelleted by
centrifugation at 500 g for 7 min. Cell pellets were
resuspended in 250 µl of suspension buffer with Triton X-100,
agitated for 15 min at 4°C, and centrifuged at 12,000 g for
10 min at 4°C. The supernatant was removed, and the pellet was
resuspended in suspension buffer. Protein was stored at 20°C for subsequent Western blotting.
Western blotting for CD14. Extracted proteins (10 µg) from each group were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked by Tris-buffered saline-Tween 20 containing 5% skim milk, probed with mouse anti-rat ED9 monoclonal antibody (Serotec, Oxford, UK), followed by horseradish peroxidase-conjugated secondary antibody as appropriate. Membranes were incubated with a chemiluminescence substrate (ECL reagent; Amersham Life Science, Little Chalfont, UK) and exposed to X-OMAT films (Eastman Kodak, Rochester, NY).
Nuclear protein extraction and gel mobility shift assay.
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. There are
alternative approaches available, such as fluorescence in end-labeled
oligonucleotides, to avoid use of radioactivity and transient
transfection of cells with a NF-B-dependent luciferase reporter gene.
RNA isolation and RT-PCR amplification.
Liver tissues were flash-frozen in liquid nitrogen and stored at
80°C until analysis. Samples (~50 µg) of liver tissue
were collected, and total cellular RNA was extracted using the Qiagen Rneasy kit (Qiagen, Chatsworth, CA) according to the manufacturer's instructions. For the synthesis of complementary DNA (cDNA), 1.0 µg
of total RNA from each sample was resuspended in a 20-µl final volume
of reaction buffer, which contained 25 mM Tris · HCl,
pH 8.3, 37.5 mM KCl, 10 mM dithiothreitol, 1.5 mM MgCl2, 10 mM of each dNTP (Perkin Elmer Cetus, Norwalk, CT), and 0.5 mg
oligo(dT)12-18 primer (GIBCO BRL). After the reaction
mixture reached 42°C, 400 units of RT (GIBCO BRL) was added to each
tube and the sample was incubated for 45 min at 42°C. Reverse
transcription was stopped by denaturing the enzyme at 95°C. The
reaction mixture was diluted with distilled water to a final volume of
50 µl. Commercially available PCR primers for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from
Clontech Laboratories (Palo Alto, CA). Primers for TNF-
contained
the following sequences: TNF-
, sense
(5'-ATGAGCACAGAAAGCATGATG-3') and antisense
(5'-TACAGGCTTGTCACTCGAATT-3'); GAPDH, sense
(5'-TGAAGGTCGGAGTCAACGGATTTGGT-3') and antisense (5'-CATGTGGGCCATGAGGTCCACCAC-3'). The size of amplified PCR
products was 276 bp for TNF-
and 983 bp for GAPDH.
Statistics. ANOVA or Student's t-test was used for the determination of statistical significance as appropriate. For comparison of pathological scores, the Mann-Whitney rank sum test was used. A P value <0.05 was selected before the study as the level of significance.
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RESULTS |
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Body weight and ethanol concentrations in urine.
Liquid diets were initiated 1 wk after surgery to allow for complete
recovery (Fig. 1). All animals survived
throughout the enteral feeding period. Since animals were age matched,
males started the study heavier than females, as expected. Previous work has shown that age-matched or weight-matched animals respond similarly to ethanol (15). Weight gains during enteral feeding were
similar in males and females and were similar to rats fed chow in both
genders (data not shown). There were no differences in behavior scores
between males and females during alcohol exposure.
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Serum transaminase and estrogen levels.
At 3 wk of ethanol treatment, serum AST levels reached 149 ± 33 IU/l
in males and 224 ± 28 IU/l in females (data not shown). These values
were nearly identical to levels observed at 4 wk of ethanol treatment
with the standard enteral protocol (15). After 4 wk, however, values
reached 389 ± 47 IU/l in males and 727 ± 66 IU/l in females (Fig.
3). Thus transaminases were about twofold
greater in females than in males. Here, AST levels were nearly fourfold
higher than in a previous report from this laboratory in both genders
(15). Serum AST levels increased dramatically after 4 wk in this study
because rats received nearly 50% more ethanol.
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Pathological evaluation.
There were no pathological changes in male rats given the control diet
for 4 wk, whereas slight steatosis was observed in females (Fig.
4). Dietary ethanol dramatically increased
fatty infiltration after only 2 wk and caused mild inflammation and focal necrosis after 4 wk in ethanol-fed male rats. In contrast, the
micro- and macrovesicular pattern of steatosis was more severe and
panlobular in ethanol-fed female rats. The time course of changes in
hepatic pathology is summarized in Fig. 5.
After only 2 wk of ethanol treatment, the score for steatosis was
1.7-fold higher in females than in males. After 4 wk, inflammation and necrosis were also 1.4-fold higher in females than in males. The number
of infiltrating neutrophils in the liver was minimal and similar
between males and females in the absence of ethanol; however, significant increases were produced by 4 wk of ethanol treatment in
both genders. Importantly, values were about twofold greater in females
than in males (Fig. 6).
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Plasma endotoxin levels.
Basal endotoxin levels were near levels of detection (<10 pg/ml) and
not different in both genders (data not shown). In controls, endotoxin
levels were minimal over the 4-wk time course of this study (Fig.
7). Values were increased significantly by
enteral ethanol in both genders after 4 wk. Moreover, after 4 wk of
enteral feeding with higher delivery of ethanol in the final 2-4
wk period, endotoxin levels were 2.5-fold higher in this study than in
a previous study with the standard protocol (15), but values in females
were significantly higher than in males.
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Effects of chronic enteral ethanol and gender on expression of CD14
on Kupffer cells.
It was reported previously that CD14 expression was increased in the
liver from rats fed a high-fat control diet without ethanol in the
enteral feeding model (34). In cells from rats fed high-fat control
diets in this study, CD14 expression on Kupffer cells was minimal and
not different between males and females. However, enteral ethanol
caused an ~1.5-fold increase in expression of CD14 in both genders
after 4 wk, and values in females were significantly greater than in
males (Fig. 8).
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Effects of chronic enteral ethanol and gender on
NF-B in the liver.
The active form of the pleiotropic transcription factor NF-
B was
minimal in livers from female and male rats fed a high-fat control diet
for 4 wk (Fig. 9A). However,
chronic enteral ethanol increased NF-
B activity significantly,
nearly fivefold over control values after 4 wk. Moreover, this activity
was nearly threefold greater in females than in males.
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Effects of chronic enteral ethanol and gender on
TNF- mRNA expression in the liver.
TNF-
mRNA expression in livers from rats fed a high-fat control diet
for 4 wk was minimal in both genders (Fig.
10). However, ethanol treatment increased
TNF-
mRNA expression significantly, nearly fivefold over control
values after 4 wk. Moreover, expression was nearly threefold greater in
females than in males.
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DISCUSSION |
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Ethanol is a key factor in hepatic pathology in both genders.
In a previous weight-matched study, liver injury assessed from
transaminase release and pathology was greater in females than in males
(15). However, inflammation and necrosis were mild, and fibrosis was
not detected because the experiment was short term. Since the severity
of clinical alcoholic hepatitis and cirrhosis is related to the amount
of alcohol consumed (32, 38), here the enteral feeding model was
modified to increase ethanol delivery. This goal was achieved by
increasing alcohol levels in the diet based on behavioral assessment of
development of tolerance to ethanol. Accordingly, animals given ethanol
were observed frequently for signs of severe alcohol intoxication,
i.e., sluggish movement, relaxation of legs, and loss of consciousness,
using the 0-3 point scoring system detailed in MATERIALS AND
METHODS. In this manner, alcohol delivery in Wistar rats in the
300- to 350-g weight range could be increased to 17-18
g · kg1 · day
1,
yielding average blood alcohol levels in the 225- to 250-mg/dl range.
Thus alcohol delivery and alcohol levels were increased ~50% over
previous studies from this and other laboratories with adult rats in
the same weight range (13, 15, 42). As a result, serum transaminases
were about fourfold higher (Fig. 3) and pathology scores were elevated
significantly in both genders (Fig. 5). Moreover, steatosis,
inflammation, and necrosis were also greater in females than in males
in this study.
Role of endotoxin, CD14, and NF-B in gender
differences in early alcohol-induced liver injury.
Previous work has shown that intestinal sterilization with antibiotics
(2) and suppression of endotoxin production with lactobacillus feeding
(25) minimizes liver injury in the Tsukamoto-French model. Furthermore,
destruction of Kupffer cells with GdCl3 prevents hepatic
pathology as well (1). In the present study, circulating endotoxin was
significantly higher in females than in males after 4 wk of enteral
ethanol (Fig. 7). Based on these data, it is concluded that Kupffer
cells activated by gut-derived endotoxin are involved in early
alcohol-induced liver injury (see Fig.
11) (37). Activated Kupffer cells produce
reactive oxygen species (19). These oxidants could activate NF-
B and
induce transcription of inflammatory cytokines, such as TNF-
, which
is involved in early alcohol-induced liver injury. Indeed, TNF-
antibody reduces liver injury in the Tsukamoto-French model (8, 16).
Furthermore, alcohol-induced liver injury is prevented in TNF
receptor-1 knockout mice given enteral ethanol (20). Here, chronic
enteral ethanol increased NF-
B activity much more in female than in
male liver (Fig. 10). ICAM-1 expression on sinusoidal lining cells and
the number of neutrophils in the liver were also significantly greater
in females than in males (15) (Fig. 6). Furthermore, TNF-
mRNA
expression in the liver was greater in females than males in this study
(Fig. 10). NF-
B activation could be a result of higher endotoxin
levels, leading to increased CD14 expression on Kupffer cells (Fig. 8). Increased inflammatory cytokine and adhesion molecule synthesis by
enhanced activation of NF-
B could be one explanation for the greater
liver injury observed in females (see Fig. 11). Alternatively, enhanced
NF-
B activation in female rats given ethanol may reflect an
antiapoptotic defensive response, since Rel A is known to be intimately
involved in this process (33).
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Possible role of estrogen in gender differences in early alcohol-induced liver injury. It is currently not clear why females have higher endotoxin levels in the blood. It is possible that gut permeability to endotoxin or endotoxin production by gut microflora is greater in females than in males, or that endotoxin clearance in females is compromised. Alternatively, estrogen might influence the gut microflora in females differently than in males, since some bacterial strains that have aryl steroid sulfatase activity for estrogen sulfates exist in rat and human intestine (43). Functional estrogen receptors exist in intestinal epithelial cells, suggesting that estrogen could affect the gut (35). Indeed, estrogen replacement increases gut permeability and endotoxin levels significantly in ovariectomized rats in the Tsukamoto-French model (46). During enteral feeding with or without ethanol, serum estrogen levels were higher in females than in males in this study, as expected (males 15.3 ± 5.0 pg/ml; females 50.0 ± 12.5 pg/ml). Thus these data are consistent with the hypothesis that the effect of estrogen on the gut is involved in the mechanism of gender differences in early alcohol-induced liver injury. On the other hand, estradiol treatment increases the number of hepatic low-density lipoprotein receptors, leading to increased lipoprotein clearance and decreased serum lipoprotein levels (44). Since lipoprotein binds endotoxin, this could contribute to the higher endotoxin levels observed in females.
The endotoxin receptor CD14, which is on the surface of the plasma membrane of Kupffer cells, plays an important role in activation of Kupffer cells (10). Pharmacological doses of estrogen treatment in vivo increase CD14 expression on Kupffer cells, and this increase was blunted by intestinal sterilization with antibiotics (11, 17). Importantly, CD14 expression on Kupffer cells was significantly greater in females than in males after ethanol treatment in this study (Fig. 8). Moreover, estrogen increased CD14 mRNA expression in livers from ovariectomized rats given enteral ethanol (46). Together, these results are consistent with the hypothesis that estrogen is involved in upregulation of CD14 expression on Kupffer cells during ethanol exposure (see Fig. 11). Importantly, a previous study from this laboratory has demonstrated that estrogen treatment in vivo increases intracellular calcium and cytokine production due to lipopolysaccharide in isolated Kupffer cells, suggesting that estrogen also increases sensitivity of Kupffer cells to endotoxin (17). Alternatively, it was reported that chronic endotoxin administration did not increase hepatic CD14 mRNA expression in rats (18). Furthermore, endotoxin can activate NF-Clinical implications. In addition to amount of alcohol consumed, risk factors for alcoholic hepatitis and cirrhosis include gender and being overweight (28). The Tsukamoto-French model has long been shown to have a strong nutritional component, with injury dependent on unsaturated fat in the diet (26). Moreover, it is now clear that the gender differences seen in the clinic are reflected in this model (Fig. 4) (15). Although the advantages and disadvantages of animal models have been discussed (23), this model exhibits three important characteristics similar to those observed clinically. Namely, injury in the Tsukamoto-French model is dependent on fat, gender, and alcohol. Thus it represents an ideal animal model to study mechanisms of alcohol-induced liver injury.
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ACKNOWLEDGEMENTS |
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Preliminary accounts of this work have appeared elsewhere (15).
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. U. Bradford, Laboratory of Hepatobiology and Toxicology, Dept. of Pharmacology, CB#7365, Mary Ellen Jones Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365.
Received 18 May 1999; accepted in final form 1 December 1999.
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