Estriol sensitizes rat Kupffer cells via gut-derived
endotoxin
Nobuyuki
Enomoto1,
Shunhei
Yamashina1,
Peter
Schemmer1,
Chantal A.
Rivera1,
Blair U.
Bradford1,
Ayako
Enomoto2,
David A.
Brenner2, and
Ronald G.
Thurman1
1 Laboratory of Hepatobiology
and Toxicology and Department of Pharmacology, and
2 Division of Digestive Diseases
and Nutrition and Department of Medicine, University of North
Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
 |
ABSTRACT |
The relationship
between gender and alcohol-induced liver disease is complex; however,
endotoxin is most likely involved. Recently, it was reported that
estriol activated Kupffer cells by upregulation of the endotoxin
receptor CD14. Therefore, the purpose of this work was to study how
estriol sensitizes Kupffer cells. Rats were given estriol (20 mg/kg
ip), and Kupffer cells were isolated 24 h later. After addition of
lipopolysaccharide (LPS), intracellular
Ca2+ concentration was measured
using a microspectrofluorometer with the fluorescent indicator fura 2, and tumor necrosis factor-
was measured by ELISA. CD14 was evaluated
by Western analysis. One-half of the rats given estriol
intraperitoneally 24 h before an injection of a sublethal dose of LPS
(5 mg/kg) died within 24 h, whereas none of the control rats died.
Mortality was prevented totally by sterilization of the gut with
antibiotics. A similar pattern was obtained with liver histology and
serum transaminases. Translocation of horseradish peroxidase was
increased about threefold in gut segments by treatment with estriol.
This increase was not altered by treatment with nonabsorbable
antibiotics. On the other hand, endotoxin levels were increased to
60-70 pg/ml in plasma of rats treated with estriol. As expected,
this increase was prevented (<20 pg/ml) by antibiotics. In isolated
Kupffer cells, LPS-induced increases in intracellular
Ca2+ concentration, tumor necrosis
factor-
production, and CD14 were increased, as previously reported.
All these phenomena were blocked by antibiotics. Therefore, it is
concluded that estriol treatment in vivo sensitizes Kupffer cells to
LPS via mechanisms dependent on increases in CD14. This is most likely
due to elevated portal blood endotoxin caused by increased gut permeability.
lipopolysaccharide; tumor necrosis factor-
; CD14; intracellular
calcium
 |
INTRODUCTION |
ENDOTOXIN [lipopolysaccharide (LPS)] is a
component of the outer wall of gram-negative bacteria that causes many
biological effects, including lethal shock and multiple organ failure.
Kupffer cells, resident macrophages in the liver, not only remove
gut-derived endotoxin but are also activated during the process to
produce chemical mediators [i.e., eicosanoids, interleukin
(IL)-1, IL-6, tumor necrosis factor-
(TNF-
), superoxide, and
nitric oxide]. Kupffer cells contain voltage-dependent
Ca2+ channels (13), and
intracellular Ca2+ is an important
second messenger in the production and release of chemical mediators
(7, 17). Indeed, Ca2+ channel
blockers increased graft survival after transplantation (24) and
reduced liver injury due to alcohol (15), most likely by preventing
activation of Kupffer cells.
Recently, sensitization of Kupffer cells to ethanol was shown to be
caused by upregulation of CD14, the receptor for LPS/LPS binding
protein (LBP), via mechanisms dependent on gut-derived endotoxin (9). Importantly, pathology is greater in female rats exposed
to ethanol via an enteral protocol than in male rats. Moreover, Kupffer
cells from estrogen-treated animals expressed more endotoxin receptor,
CD14, than controls (16). However, it is unclear how estrogen increases
CD14. One possibility is that it increases gut-derived endotoxin.
Alternatively, estrogen could have direct or indirect effects on
Kupffer cells. The purpose of this study, therefore, was to evaluate
these possibilities.
 |
MATERIALS AND METHODS |
Estrogen treatment in vivo.
Female Sprague-Dawley rats weighing 200-250 g were used for all
experiments. All animals were given humane care in compliance with
institutional guidelines. Rats were given estriol (20 mg/kg ip; Sigma
Chemical, St. Louis, MO) 24 h before experiments. All control rats
received an equivalent volume of saline vehicle. A sublethal dose of
LPS (5 mg/kg iv, Escherichia coli
0111:B4; Sigma Chemical) was injected via the tail vein, and survival
was assessed after 24 h. Some rats were treated for 4 days with
polymyxin B and neomycin to prevent growth of intestinal bacteria, the
primary source of endotoxin in the gastrointestinal tract (23). On the basis of results of preliminary experiments, 150 mg · kg
1 · day
1
of polymyxin B and 450 mg · kg
1 · day
1
of neomycin were given orally. Under these conditions, gut
sterilization was achieved (1).
Analytic methods.
Blood was collected from the portal vein in pyrogen-free heparinized
syringes and centrifuged, and plasma was stored at
20°C in
pyrogen-free glass test tubes until endotoxin was measured, as
described in detail elsewhere using the
Limulus amoebocyte lysate assay
(Whitaker Bioproducts, Walkerville, MD). Serum was stored at
20°C in microtubes, and aspartate transaminase (AST) and
alanine aminotransferase (ALT) were measured by standard enzymatic procedures (4). Livers were fixed in formalin, embedded in paraffin,
and stained with hematoxylin and eosin for assessment of inflammation
and necrosis.
Gut permeability.
Gut permeability was measured in isolated segments of ileum from
translocation of horseradish peroxidase, as described previously (5).
Briefly, 8-cm segments of ileum were everted, filled with 1 ml of Tris
buffer (125 mmol/l NaCl, 10 mmol/l fructose, 30 mmol/l Tris, pH 7.5),
and ligated at both ends. The filled gut segments were incubated in
Tris buffer containing 40 mg/100 ml of horseradish peroxidase (5).
After 45 min, gut sacs were removed and blotted lightly to eliminate
excess horseradish peroxidase, and the contents (~750 µl) of each
sac were collected carefully with a 1-ml syringe. Horseradish
peroxidase activity in the contents of each sac was determined
spectrophotometrically from the rate of oxidation of pyrogallol, as
described elsewhere (5).
Kupffer cell preparation and culture.
Kupffer cells were isolated by collagenase digestion and differential
centrifugation with use of Percoll (Pharmacia, Uppsala, Sweden), as
described elsewhere with slight modifications (22). Briefly, the liver
was perfused through the portal vein with
Ca2+- and
Mg2+-free Hanks' balanced salt
solution at 37°C for 5 min at a flow rate of 26 ml/min.
Subsequently, the liver was perfused with Hanks' balanced salt
solution containing 0.025% collagenase IV (Sigma Chemical) at 37°C
for 5 min. After the liver was digested, it was excised and cut into
small pieces in collagenase buffer. The suspension was filtered through
nylon gauze mesh, and the filtrate was centrifuged at 450 g for 10 min at 4°C. Cell pellets
were resuspended in buffer, parenchymal cells were removed by
centrifugation at 50 g for 3 min, and
the nonparenchymal cell fraction was washed twice with buffer. Cells
were centrifuged on a density cushion of Percoll at 1,000 g for 15 min, and the Kupffer cell
fraction was collected and washed with buffer again. Viability of cells determined by trypan blue exclusion was >90%. Cells were seeded onto
25-mm glass coverslips and cultured in DMEM (GIBCO Laboratories Life
Technologies, Grand Island, NY) supplemented with 10% fetal bovine
serum and antibiotics (100 U/ml of penicillin G and 100 µg/ml of
streptomycin sulfate) at 37°C with 5%
CO2. Nonadherent cells were
removed after 1 h by replacement of buffer, and cells were cultured for
24 h before experiments. Basically, Kupffer cells were incubated 24 h
after seeding. For intracellular
Ca2+ concentration
([Ca2+]i)
measurements, Kupffer cells were prepared after seeding for 24 h on
coverslips and loaded with fura 2 for 30 min. For TNF-
production,
Kupffer cells were prepared after seeding for 24 h on 24-well plates,
LPS containing medium was added, and samples were collected after 4 h
for measurement by ELISA. In the case of CD14, Kupffer cells were
prepared after seeding for 24 h on 6-cm culture dishes, and total
protein extracts were obtained as described above. These times are
optimal for each experiment. Time courses based on previous studies
showed that cell seeding for 24 h is also optimal.
Measurement of
[Ca2+]i.
[Ca2+]i
was measured fluorometrically using the
Ca2+ indicator dye fura 2 and a
microspectrofluorometer (PTI, South Brunswick, NJ) interfaced with an
inverted microscope (Diaphot, Nikon, Japan). Kupffer cells were
incubated in modified Hanks' buffer (115 mmol/l NaCl, 5 mmol/l KCl,
0.3 mmol/l
Na2HPO4,
0.4 mmol/l
KH2PO4,
5.6 mmol/l glucose, 0.8 mmol/l
MgSO4, 1.26 mmol/l
CaCl2, 15 mmol/l HEPES, pH 7.4)
containing 5 µmol/l fura 2-AM (Molecular Probes, Eugene, OR) and
0.03% Pluronic F-127 (BASF Wyandotte, Wyandotte, MI) at room
temperature for 60 min. Coverslips plated with Kupffer cells were
rinsed and placed in chambers with buffer at room temperature. Changes
in fluorescence intensity of fura 2 at excitation wavelengths of 340 and 380 nm and emission of 510 nm were monitored in individual Kupffer
cells. Each value was corrected by subtracting the system dark noise
and autofluorescence, assessed by quenching fura 2 fluorescence with
Mn2+, as described previously
(13).
[Ca2+]i
was determined from the following equation:
[Ca2+]i = Kd[(R
Rmin)/(Rmax
R)]/(Fo/Fs),
where
Fo/Fs
is the ratio of fluorescent intensities evoked by 380-nm light from
fura 2 pentapotassium salt loaded in cells with use of a buffer
containing 3 mmol/l EGTA and 1 µmol/l ionomycin (minimum
[Ca2+]i)
or 10 mmol/l Ca2+ and 1 µmol/l
ionomycin (maximum
[Ca2+]i),
R is the ratio of fluorescent intensities at excitation wavelengths of
340 and 380 nm, and Rmax and
Rmin are values of R at maximum and minimum
[Ca2+]i,
respectively. The values of these constants were determined at the end
of each experiment, and a dissociation constant
(Kd) of 135 nmol/l was used (12).
TNF-
detection.
Kupffer cells were seeded onto 24-well plates and cultured in DMEM
supplemented with 10% fetal bovine serum and antibiotics at 37°C
in the presence of 5% CO2. Cells
were incubated with fresh media containing LPS (100 ng/ml supplemented
with 5% rat serum) for an additional 4 h. Samples of media were
collected and kept at
80°C until assay. TNF-
in the
culture media was measured using an ELISA kit (Genzyme, Cambridge, MA),
and data were corrected for dilution.
Western blotting for CD14.
Total protein extracts of cultured Kupffer cells were obtained by
homogenizing samples in a buffer containing 10 mmol/l HEPES, pH 7.6, 25% glycerol, 420 mmol/l NaCl, 1.5 mmol/l
MgCl2, 0.2 mmol/l EDTA, 0.5 mmol/l
dithiothreitol, 40 mg/ml bestatin, 20 mmol/l
-glycerophosphate, 10 mmol/l 4-nitrophenylphosphate, 0.5 mmol/l Pefabloc, 0.7 mg/ml pepstatin
A, 2 mg/ml aprotinin, 50 mmol/l Na3VO4,
and 0.5 mg/ml leupeptin. Protein concentration was determined using the
Bradford assay kit (Bio-Rad Laboratories, Hercules, CA). Extracted
protein was separated by 10% SDS-PAGE and transferred to
polyvinylidene difluoride membranes. Membranes were blocked by
Tris-buffered saline-Tween 20 containing 5% skim milk and probed first
with mouse anti-rat ED9 monoclonal antibody (Serotec, Oxford, UK), then
with horseradish peroxidase-conjugated secondary antibody as
appropriate. Membranes were incubated with a chemiluminescence substrate (enhanced chemiluminescence reagent, Amersham Life Science, Buckinghamshire, UK) and exposed to X-OMAT films (Eastman Kodak, Rochester, NY).
Statistical analysis.
Values are means ± SE. Statistical differences between means were
determined using ANOVA or ANOVA on ranks as appropriate. P < 0.05 was selected before the
study to reflect significance.
 |
RESULTS |
Effect of estriol on mortality due to endotoxin.
To assess the effect of estriol on endotoxin shock, rats were given an
intraperitoneal injection of estriol 24 h before an intravenous
injection of a sublethal dose of endotoxin (LPS) via the tail vein.
Table 1 depicts mortality 24 h after LPS.
Obviously, all control rats survived for 24 h after a sublethal
injection of LPS (5 mg/kg); however, 50% mortality was observed in
rats given estriol 24 h earlier (20 mg/kg), confirming early work. Interestingly, mortality due to LPS in estriol-treated rats was prevented totally by gut sterilization with antibiotics, indicating that gut-derived endotoxin is involved in this phenomenon.
Effect of estriol and LPS on liver histology and serum
transaminases.
Liver specimens were collected for histology 24 h after administration
of LPS (5 mg/kg). Histology was normal in control rats (Fig.
1A),
whereas LPS caused focal necrosis and neutrophil infiltration in the
liver, as expected (Fig. 1B).
Twenty-four hours after estriol treatment, necrosis and neutrophil
infiltration due to LPS were increased dramatically (Fig.
1C). These histological changes due to estriol were blunted by treatment with antibiotics (Fig.
1D).

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Fig. 1.
Photomicrographs (hematoxylin and eosin) of liver tissue.
A: no treatment.
B: 24 h after lipopolysaccharide (LPS,
5 mg/kg iv, Escherichia coli serotype
0111:B4, Sigma Chemical). C: 24 h of
estriol exposure and 24 h of LPS. D:
antibiotics for 4 days (150 mg · kg 1 · day 1
of polymyxin B and 450 mg · kg 1 · day 1
of neomycin), estriol for 24 h, and LPS for 24 h. Original
magnification, ×100. Typical photomicrographs are shown.
|
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Twenty-four hours after estriol treatment, LPS (5 mg/kg) was injected
via the tail vein, and blood samples were collected 24 h later for
serum AST and ALT measurements (Fig. 2).
Mean values for AST and ALT in the control group were low, whereas
values were increased slightly to 71 ± 3 and 64 ± 7 IU/l,
respectively, with LPS treatment (5 mg/kg). In contrast, LPS increased
transaminases dramatically to >700 IU/l in estriol-treated rats. This
increase was also blunted significantly by antibiotics.

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Fig. 2.
Effect of estriol treatment on LPS-induced increases in serum aspartate
transaminase (AST) and alanine aminotransferase (ALT). Rats were
treated with estriol, and blood samples were collected 24 h after LPS
(5 mg/kg). Some rats were given antibiotics for 4 days before
experiments. Values are means ± SE for 4 rats/group.
* P < 0.05 vs. 5 mg/kg LPS.
# P < 0.05 vs. 20 mg/kg estriol + 5 mg/kg LPS by ANOVA with Bonferroni's
post hoc test.
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Effect of estriol treatment on gut permeability and portal endotoxin
levels.
Two hours after estriol treatment, gut permeability was increased
dramatically (Fig.
3A).
Levels were about threefold higher than values from control rats;
however, permeability was not affected by treatment with antibiotics.
Levels of endotoxin in peripheral blood plasma from rats in this study
were below the limits of detection. Interestingly, portal endotoxin
levels of 6 ± 3 pg/ml in normal rats were increased by estriol
administration to 61 ± 19 pg/ml (Fig.
3B), an effect blocked by
antibiotics.

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Fig. 3.
Effect of estriol on gut permeability and endotoxin.
A: some rats were treated with
antibiotics before experiments for 4 days. Two hours after
administration of estriol (20 mg/kg ip), segments of ileum were
isolated and permeability to horseradish peroxidase (HRP) was detected.
Values are means ± SE; n = 4. * P < 0.05 vs. control.
B: portal plasma endotoxin was
determined by Limulus amoebocyte
lysate pyrogen test. Values are means ± SE;
n = 4. * P < 0.05 vs. control.
# P < 0.05 vs. estriol by ANOVA with Bonferroni's post hoc test.
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Effect of estriol on LPS-induced increases in
[Ca2+]i
in Kupffer cells.
As reported previously, LPS increases
[Ca2+]i
transiently in isolated Kupffer cells. Here, Kupffer cells from control
female rats exhibited small increases in
[Ca2+]i
with 100 ng/ml LPS to 83 ± 6 nmol/l (Figs.
4A and
5A).
[Ca2+]i in Kupffer cells from
estriol-treated female rats, however, was increased to levels about
threefold higher, confirming previous work (16) (Figs.
4B and
5A). This phenomenon was also
blocked by treatment with antibiotics (Figs.
4C and
5A), indicating that the effect of
estriol is dependent on gut-derived endotoxin. In contrast, addition of
estriol in vitro did not alter LPS-induced increases in
[Ca2+]i,
indicating that the effect of estriol on Kupffer cells is not direct.
Although Kupffer cells from male control rats exhibited similar
increases in
[Ca2+]i
with 100 ng/ml LPS to 114 ± 33 nmol/l, estriol had no
effect on this phenomenon (Table 2). In
castrated female rats, control Kupffer cells also showed similar
increases in
[Ca2+]i
with LPS.
[Ca2+]i
in Kupffer cells from estriol-treated castrated female rats, however,
was increased only ~1.5-fold. Briefly, estriol was not effective in
male rats, and its effect was markedly diminished in castrated female
rats compared with normal female rats (Table 2). These data suggest
that estriol sensitivity depends on several factors, but estrogen and
estrogen receptors are probably pivotal.

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Fig. 4.
Effect of estrogen on LPS-induced increases in intracellular
Ca2+ concentration
([Ca2+]i)
in Kupffer cells.
[Ca2+]i
in isolated Kupffer cells was measured fluorometrically using fura 2. Changes in
[Ca2+]i
after addition of LPS (supplemented with 5% rat serum) are plotted.
A: LPS (100 ng/ml) was added to
Kupffer cells from control rats. B:
LPS (100 ng/ml) was added to Kupffer cells from estriol-treated rats.
C: LPS (100 ng/ml) was added to
Kupffer cells from rats treated with antibiotics and estriol. Data are
representative traces of experiments repeated 5 times.
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Fig. 5.
Influence of antibiotics on LPS-induced increases in
[Ca2+]i
and tumor necrosis factor- (TNF- ) production in isolated Kupffer
cells from estriol-treated rats. Some rats were treated with
antibiotics before experiments for 4 days (150 mg · kg 1 · day 1
of polymyxin B and 450 mg · kg 1 · day 1
of neomycin). Twenty-four hours after administration of estriol (20 mg/kg ip), Kupffer cells were isolated and cultured in 6-cm culture
dishes and 24-well culture plates.
[Ca2+]i
was measured using a microspectrofluorometer with fluorescent indicator
fura 2, and TNF- was measured by ELISA. Basal levels of TNF-
production in control rats was 26 ± 2 pg · 5 × 105
cells 1 · 4 h 1 (24 ± 4 and 25 ± 4 pg · 5 × 105
cells 1 · 4 h 1 in control and
antibiotics + estriol groups, respectively). Values are means ± SE;
n = 4. * P < 0.05 vs. control.
# P < 0.05 vs. estriol by ANOVA with Bonferroni's post hoc test.
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Table 2.
Effect of estriol on LPS-induced increases in
[Ca2+]i in isolated Kupffer cells
from male and female rats
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Effect of estriol treatment in vivo on LPS-induced production of
TNF-
in isolated Kupffer cells.
Because TNF-
is a pivotal cytokine involved in the development of
endotoxin shock, LPS-induced TNF-
production by isolated Kupffer
cells was measured (Fig. 5B).
Kupffer cells from control rats produced TNF-
in response to LPS
(100 ng/ml); however, isolated cells from estriol-treated animals
produced about three times more TNF-
than controls. This effect was
blocked by treatment with antibiotics. Interestingly, addition of
estriol (100 nM) directly to the culture medium for 24 h before LPS did
not alter TNF-
production due to LPS (100 ng/ml) by isolated Kupffer
cells (260 ± 27 and 312 ± 16 pg · 106
cells
1 · 4 h
1 in control and
estriol-treated groups, respectively).
Effect of estriol and antibiotics on CD14 expression in Kupffer
cells.
To determine whether the LPS/LBP receptor (CD14) was altered in Kupffer
cells by estriol treatment in vivo, Western blotting with ED-9
antibody, which recognizes rat CD14, was performed (Fig. 6). As expected, Kupffer cells from control
rats expressed the 55-kDa CD14; however, the band was about twofold
more intense in Kupffer cells from estriol-treated rats. Furthermore,
the effect of estriol on CD14 protein levels was blunted by
antibiotics.

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Fig. 6.
Effect of estriol on CD14 expression in Kupffer cells. Protein extracts
from cultured Kupffer cells 24 h after estriol treatment were analyzed
by Western blotting with use of mouse anti-rat ED-9 antibody. Specific
bands for CD14 (55 kDa) are shown: lane
1, Kupffer cells from control rats;
lane 2, Kupffer cells from rats
treated with estriol for 24 h before isolation; lane
3, Kupffer cells from rats treated with antibiotics for
4 days and estriol for 24 h before isolation. Data are representative
of 3 individual experiments.
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 |
DISCUSSION |
Possible mechanism of greater susceptibility of women to alcoholic
liver disease.
It is still unclear why alcoholic liver injury is more rapid and more
extensive in women than in men; however, hormonal effects are an
obvious possibility. Estriol has profound effects on lipid metabolism,
and women have lower levels of hepatic triglyceride lipase and higher
levels of plasma lipoprotein lipase than men (3). Also, estriol therapy
in postmenopausal women has been shown to increase plasma triglyceride
(3, 27) and decrease hepatic triglyceride lipase activity (2). Indeed,
hepatic triglycerides were increased about 3-fold in women but only
1.5-fold in men 6 h after a single dose of ethanol (18). Therefore,
estriol may have an additive effect on alcohol-induced fat accumulation in the liver.
On the other hand, Iimuro et al. (14) showed that plasma endotoxin
levels were significantly higher in women than in men after exposure to
ethanol. In the present study, estriol increased portal endotoxin via
mechanisms most likely dependent on gut permeability (Fig.
3A), consistent with the hypothesis
that higher plasma endotoxin levels lead to more extensive Kupffer cell
activation in women than in men.
Recently, it was shown that ethanol-induced sensitization of Kupffer
cells was caused by gut-derived endotoxin and involved increases in
CD14 (9). Farhat et al. (10) demonstrated that estrogen promoted
vasodilatation and stimulated microvascular permeability (6). It is
also possible that treatment with estriol increases gram-negative
bacterial species, a major source of endotoxin in the gut microflora,
in the portal vein via increased gut permeability. In this study it was
demonstrated that estriol indeed increased permeability of the isolated
small intestine (Fig. 3A). As
expected, permeability was not affected by treatment with antibiotics.
This led to increases in plasma endotoxin, a phenomenon that was
prevented by treatment with antibiotics (Fig.
3B). Thus it is concluded that
estriol increases portal endotoxin by increasing gut permeability.
Kupffer cells are involved in potentiation of LPS-induced liver
injury by estriol.
In the present study it was demonstrated that pharmacological doses of
estriol similar to levels encountered in late pregnancy increased
mortality due to LPS (Table 1), confirming experiments by Nolan and Ali
(21) and Ikejima et al. (16). This effect of estriol was impressive;
however, precise mechanisms remain unclear. It is well known that
macrophages, including Kupffer cells, contribute to the pathophysiology
of endotoxin shock. Indeed, gadolinium chloride, a Kupffer cell
toxicant, totally prevented mortality due to estrogen plus LPS (16),
indicating that Kupffer cells are necessary for this phenomenon.
Furthermore, antibiotics prevented mortality due to estrogen plus LPS,
indicating that gut-derived endotoxin is also necessary for this
phenomenon (Fig. 3B).
Kupffer cells are activated by endotoxin, leading to rapid increases in
[Ca2+]i
followed by release of inflammatory mediators (e.g., cytokines and
lipid metabolites) as well as reactive oxygen intermediates (20, 26,
30). TNF-
is produced predominantly by the monocyte-macrophage lineage, and the predominant cell type of this lineage is the hepatic
Kupffer cell (8). Moreover,
[Ca2+]i
is required for LPS-induced expression of TNF-
by a macrophage cell
line (31). Increased TNF-
plays a pivotal role in endotoxin shock
and related multiple organ failure (28), and anti-TNF-
antibody
prevents it (29). TNF-
stimulates generation of toxic superoxide
anion from mitochondrial complex III in parenchymal cells, expression
of factors for neutrophil chemotaxis (IL-8/CINC, MIP, MIP-2), and
expression of intracellular adhesion molecule-1, leading to
microcirculatory disturbances (19). However, the effect of estriol on
Kupffer cells has not been studied in much detail. To try to understand
the mechanisms of Kupffer cell sensitivity, here LPS-induced
[Ca2+]i,
TNF-
, and CD14 were monitored in Kupffer cells. LPS-induced production of TNF-
was enhanced in Kupffer cells isolated from rats
treated with estriol (Fig. 5B).
Patterns of LPS-stimulated increases in
[Ca2+]i
(Fig. 5A) and CD14 (Fig. 6) were
similar. Thus it is concluded that estriol sensitizes Kupffer cells to endotoxin.
Estriol increases expression of CD14 in Kupffer cells.
CD14 is a functional LPS/LBP receptor on Kupffer cells. Recently, it
was demonstrated that ethanol-induced sensitization of Kupffer cells
was caused by gut-derived endotoxin via mechanisms dependent on CD14
(9). Here, CD14 was increased by estriol treatment in vivo (Fig. 6), an
effect that was blocked by antibiotics (Fig. 6). This most likely
explains why estriol treatment increased [Ca2+]i
and TNF-
due to LPS in isolated Kupffer cells (Figs. 4 and 5). It is
concluded that CD14 expression on Kupffer cells is increased by
estriol, thereby increasing toxic mediator production by mechanisms involving gut-derived endotoxin. Our working hypothesis is that estriol
increases gut permeability, leading to elevated portal endotoxin.
Fearns and colleagues (11) reported that CD14 is increased by exposure
to LPS, and upregulation of CD14 sensitizes Kupffer cells to LPS (9).
In summary, Kupffer cells isolated from rats treated with estriol
exhibited sensitization to LPS. This phenomenon involves increases in
gut permeability, elevated endotoxin levels, and increased CD14
expression on Kupffer cells.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by grants from the National
Institute of Alcohol Abuse and Alcoholism.
 |
FOOTNOTES |
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: R. G. Thurman,
Laboratory of Hepatobiology and Toxicology, Dept. of Pharmacology, CB
7365, Mary Ellen Jones Bldg., University of North Carolina at Chapel
Hill, Chapel Hill, NC 27599-7365.
Received 20 January 1999; accepted in final form 8 June 1999.
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