Estrogen increases sensitivity of hepatic Kupffer cells to
endotoxin
Kenichi
Ikejima1,2,
Nobuyuki
Enomoto1,
Yuji
Iimuro1,
Ayako
Ikejima2,
Dawn
Fang1,
Juliana
Xu1,
Donald T.
Forman3,
David A.
Brenner2, and
Ronald G.
Thurman1
1 Laboratory of Hepatobiology
and Toxicology, Department of Pharmacology,
2 Division of Digestive Diseases and Nutrition,
Department of Medicine, and
3 Department of Pathology,
University of North Carolina at Chapel Hill, Chapel Hill, North
Carolina 27599-7365
 |
ABSTRACT |
The relationship
among gender, lipopolysaccharide (LPS), and liver disease is complex.
Accordingly, the effect of estrogen on activation of Kupffer cells by
endotoxin was studied. All rats given estrogen 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 pretreatment with gadolinium chloride, a Kupffer
cell toxicant. Peak serum tumor necrosis factor-
(TNF-
) values as
well as TNF-
mRNA in the liver after LPS were twice as high in the
estrogen-treated group as in the untreated controls. Plasma nitrite
levels and inducible nitric oxide synthase in the liver were also
elevated significantly in estrogen-treated rats 6 h after LPS.
Furthermore, Kupffer cells isolated from estrogen-treated rats produced
about twice as much TNF-
and nitrite as controls did in response to LPS. In addition, Kupffer cells from estrogen-treated rats required 15-fold lower amounts of LPS to increase intracellular
Ca2+ than controls did, and
Kupffer cells from estrogen-treated animals expressed more CD14, the
receptor for LPS/LPS binding protein, than controls. Moreover, estrogen
treatment increased LPS binding protein mRNA dramatically in liver in
6-24 h. It is concluded that estrogen treatment in vivo sensitizes
Kupffer cells to LPS, leading to increased toxic mediator production by
the liver.
lipopolysaccharide; tumor necrosis factor-
; nitric oxide; intracellular calcium; CD14
 |
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 (21) to produce chemical mediators [i.e., eicosanoids, interleukin-1
(IL-1), IL-6, tumor necrosis factor-
(TNF-
), superoxide, and
nitric oxide (NO)]. Kupffer cells contain voltage-dependent
Ca2+ channels (11), and
intracellular Ca2+ is an important
second messenger in the production and release of chemical mediators
(5, 15). Indeed, Ca2+ channel
blockers increased graft survival after transplantation (24) and
reduced liver injury due to alcohol (12), presumably by preventing
activation of Kupffer cells.
It is well known that sensitivity to endotoxin in vivo is increased
during pregnancy, when estrogen levels are high. In 1935, Apitz (1)
demonstrated that pregnant animals are more susceptible than
nonpregnant animals to a generalized Shwartzman reaction induced by
endotoxin. Furthermore, after a single injection of endotoxin, pregnant
rats develop more severe inflammation and necrosis in the liver than
nonpregnant rats (28). In addition, the syndrome of hemolysis, elevated
liver enzymes, and low platelet count (HELLP syndrome), a serious
complication of some preeclamptic and eclamptic patients (27), is
mimicked by LPS treatment in pregnant animals (20).
However, it is unclear how estrogen increases liver injury in
pregnancy. One possibility is that female hormones alter susceptibility
of the liver to endotoxin. The purpose of this study, therefore, was to
evaluate the hypothesis that estrogen enhances the sensitivity of
Kupffer cells to endotoxin.
 |
MATERIALS AND METHODS |
Estrogen treatment in vivo.
Female Sprague-Dawley rats weighing between 200 and 250 g were used for
all experiments. All animals were given humane care in compliance with
institutional guidelines. Rats were given an intraperitoneal injection
of estrogen (20 mg/kg estriol; Sigma Chemical, St. Louis, MO) 24 h
before experiments. All control rats received saline vehicle without
estrogen. A sublethal dose of LPS (5 mg/kg,
Escherichia coli 0111:B4; Sigma
Chemical) was injected intravenously via the tail vein, and survival
was assessed after 24 h. Some rats were given gadolinium chloride
(GdCl3; 10 mg/kg in saline)
intravenously 24 h before estrogen treatment. While rats were under
pentobarbital anesthesia, serum and liver samples were collected at
1.5, 3, 6, 12, and 24 h after estrogen treatment and 1, 3, and 6 h
after LPS injection and kept frozen at
80°C until assay.
Measurement of serum estrogen levels.
Serum samples were collected 1.5 and 24 h after intraperitoneal
injection of estriol and were stored frozen at
20°C until assay. Serum estriol levels were determined by RIA (31). The amount of
125I-labeled estriol bound to
antibody is inversely proportional to the concentration of the
unlabeled estriol present. Separation of free and bound antigen is
rapidly achieved using a double antibody system (19). An ultrasensitive
unconjugated estriol procedure was employed (DSL-1400, Diagnostic
Systems Laboratories, Webster, TX).
Blood sampling and measurement of TNF-
.
Serial blood samples were collected for TNF-
determination as
reported previously (14). Briefly, an intravenous catheter was placed
into the femoral vein under methoxyflurane anesthesia (Metofane,
Pittman-Moore, Mundelein, IL), and blood was drawn from a catheter
before and 60, 150, 210, and 300 min after LPS injection (5 mg/kg). We
collected 200 µl of whole blood and then injected the same volume of
lactated Ringer solution at each time point. Serum was mixed with the
protease inhibitor aprotinin (Sigma Chemical) immediately, and samples
were stored at
80°C until assay. Serum TNF-
levels were
measured using an ELISA kit (Genzyme, Cambridge, MA), and data were
corrected for dilution.
Measurement of plasma nitrite levels.
Some animals were killed before and 6 h after injection of LPS (5 mg/kg) to obtain plasma samples for the measurement of nitrite, which
was determined colorimetrically using the Griess reaction (8). Briefly,
500 µl of plasma were mixed with an equal volume of Griess reagent
(1% sulfanilamide, 0.1% naphthalene-ethylenediamine dihydrochloride
in 15%
H3PO4)
and incubated for 5 min at room temperature. The resulting
product,
N-(1-naphthyl)ethylenediamine, was
quantitated spectrophotometrically at 550 nm. Nitrite levels were
calculated using a standard curve generated with known concentrations of sodium nitrite.
Western blotting for inducible NO synthase and CD14.
Total protein extracts of the liver or cultured Kupffer cells were
obtained by homogenizing samples in a buffer containing 10 mM HEPES, pH
7.6, 25% glycerol, 420 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM
dithiothreitol, 40 µg/ml Bestatin, 20 mM
-glycerophosphate, 10 mM 4-nitrophenyl phosphate, 0.5 mM Pefabloc,
0.7 µg/ml pepstatin A, 2 µg/ml aprotinin, 50 µM
Na3VO4,
and 0.5 µg/ml leupeptin. Protein concentration was
determined using the Bradford assay kit (Bio-Rad Laboratories,
Hercules, CA). Extracted protein was separated by 7.5% and 10%
SDS-PAGE for inducible NO synthase (iNOS) and CD14, respectively, and
transferred to polyvinylidene fluoride membranes. Membranes were
blocked by Tris-buffered saline-Tween 20 containing 5% skim milk,
probed with rabbit anti-mouse iNOS polyclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) or mouse anti-rat ED9 monoclonal
antibody (Serotec, Oxford, United Kingdom), followed by horseradish
peroxidase-conjugated secondary antibody as appropriate. Membranes were
incubated with a chemiluminescence substrate (ECL reagent, Amersham
Life Science, Buckinghamshire, United Kingdom) and exposed to X-OMAT
films.
RNA preparation, RT-PCR, and Northern blotting.
Total liver RNA was prepared by guanidium-CsCl centrifugation as
described previously (2). The integrity and concentration of RNA was
determined by measuring absorbance at 260 nm followed by
electrophoresis on agarose gels.
First-strand cDNA was transcribed from 1 µg RNA using Moloney murine
leukemia virus RT (Life Technologies, Gaithersburg, MD) and an
oligo(dT)16 primer. PCR was
performed using GeneAmp PCR system 9600 (Perkin Elmer, Foster City,
CA). We amplified 1 µl of cDNA in a 50 µl reaction buffer
containing 10 pmol of forward and reverse primers, 2.5 U
Taq DNA polymerase, 250 mM
2'-deoxynucleoside 5'-triphosphates (dNTPs), and 1×
PCR buffer (Perkin Elmer). The primer sets used in this study are shown
in Table 1. The reaction mixture without
enzyme and dNTPs was heated at 100°C for 4 min, and then a mixture
of Taq polymerase and dNTPs was added
at 80°C. Thereafter, 30 cycles of denaturing at 94°C for 30 s,
annealing at 58°C for 30 s, and extension at 72°C for 30 s
followed by final extension at 72°C for 7 min were carried out. The
size of PCR products was verified by electrophoresis in 1% agarose gel
followed by ethidium bromide staining. Densitometrical analysis using
NIH image software was performed for semiquantification of PCR
products.
For Northern blotting, total RNA (10 µg) was separated in 1% agarose
gel containing formaldehyde followed by capillary transfer to nylon
membranes. Membranes were prehybridized in a buffer containing 50%
formamide, 5× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 100 µg/ml salmon sperm DNA, and 1×
Denhardt's solution and hybridized in the same buffer with 10 × 106 cpm of random prime-labeled
cDNA for LPS binding protein (LBP) overnight. Membranes were then
washed with 2× SSC and 0.1% SDS at 50°C for 30 min and
0.1× SSC and 0.1% SDS at 55°C for 30 min and subjected to
autoradiography. Subsequently, membranes were stripped and
reprobed using cDNA for glyceraldehyde-3-phosphate dehydrogenase.
Kupffer cell isolation and culture.
Kupffer cells from estrogen or vehicle-treated rats were isolated by
collagenase digestion and differential centrifugation, using density
gradients of Percoll (Pharmacia, Uppsala, Sweden) as described
previously with slight modifications (23). Briefly, the liver was
perfused in situ through the portal vein with
Ca2+- and
Mg2+-free Hanks' balanced salt
solution (HBSS) containing 0.5 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic
acid (EGTA) at 37°C for 5 min at a flow rate of 26 ml/min.
Subsequently, perfusion was with HBSS 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, and the filtrate was
centrifuged twice at 50 g at 4°C
for 3 min to remove parenchymal cells. The nonparenchymal cell fraction
was washed with buffer and centrifuged on a density cushion of Percoll
at 1,000 g for 15 min to obtain the
Kupffer cell fraction, followed by washing with buffer again. The
viability of isolated Kupffer cells was determined by trypan blue
exclusion and routinely exceeded 90%. Cells were seeded onto 24-well
culture plates (Corning, Corning, NY) or 25-mm glass coverslips at a
concentration of 5 × 105 and
cultured in DMEM (GIBCO, Grand Island, NY) supplemented with 10% fetal
bovine serum (FBS) 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 replacing the culture medium. All adherent cells
phagocytosed latex beads, indicating that they were Kupffer cells.
Cells were cultured for 24 h before experiments. Cells seeded onto
24-well culture plates were incubated with fresh medium containing LPS
(100 ng/ml, supplemented with 5% rat serum) for an additional 4 or 24 h, and samples were collected for TNF-
and nitrite measurements,
respectively. Samples were kept at
80°C until assay. TNF-
and nitrite in the culture medium were determined by ELISA and the
Griess reaction, respectively, as described above.
Culture of RAW 264.7 cells.
RAW 264.7 cells, a mouse macrophage cell line, were cultured in DMEM
(Gibco) containing 10% FBS and antibiotics at 37°C in 5%
CO2. Total RNA from RAW 264.7 cells was prepared using Trizol reagent (Life Technologies) according
to the manufacturer's suggested protocol.
Measurement of intracellular
Ca2+.
Intracellular Ca2+ in individual
Kupffer cells was measured fluorometrically using the fluorescent
Ca2+ indicator dye fura 2 and a
microspectrofluorometer (Photon Technology International, South
Brunswick, NJ) interfaced with an inverted microscope (Diaphot, Nikon).
Kupffer cells cultured on coverslips were incubated in modified Hanks'
buffer (115 mM NaCl, 5 mM KCl, 0.3 mM
Na2HPO4,
0.4 mM
KH2PO4,
5.6 mM glucose, 0.8 mM MgSO4, 1.26 mM CaCl2, 15 mM HEPES, pH 7.4)
containing 5 µM fura 2-AM (Molecular Probes, Eugene, OR) and 0.03%
Pluronic F127 (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 at 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 (11).
Intracellular Ca2+ concentration
([Ca2+]i)
was determined from the equation
[Ca2+]i = Kd[(R
Rmin)/(Rmax
R)](F0 /Fs)
where
F0 /Fs
is the ratio of fluorescence intensities evoked by 380 nm light from
fura 2-pentapotassium salt loaded in cells using a buffer containing 3 mM EGTA and 1 µM ionomycin
([Ca2+]min)
or 10 mM Ca2+ and 1 µM
ionomycin
([Ca2+]max).
R is the ratio of fluorescence intensities at excitation wavelengths of
340 and 380 nm, and Rmax and
Rmin are values of R at
[Ca2+]max
and
[Ca2+]min,
respectively. The values of these constants were determined at the end
of each experiment, and a dissociation constant
(Kd) of 135 nM
was used (9).
Statistical analysis.
All results except mortality data were expressed as means ± SE.
Mortality was assessed using Fisher's test. Statistical differences between means were determined using analysis of variance (ANOVA) or
ANOVA on ranks as appropriate. P < 0.05 was selected before the study to reflect significance.
 |
RESULTS |
Effect of estrogen on mortality after LPS injection.
To assess the effect of estrogen on endotoxin shock, rats were given an
intraperitoneal injection of estriol 24 h before intravenous injection
of a sublethal dose of LPS via the tail vein. Serum estriol levels 1.5 and 24 h after estriol injection were 27 ± 9 and 6 ± 2 nM,
respectively. Estriol in serum from controls was below detection
limits. Figure 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, 100% mortality was
observed in rats given estriol 24 h previously (20 mg/kg).
Interestingly, mortality due to LPS in estrogen-treated rats was
prevented totally by pretreatment with
GdCl3, a Kupffer cell toxicant,
indicating that Kupffer cells are involved in this phenomenon.

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Fig. 1.
Effect of estrogen on mortality due to lipopolysaccharide (LPS). Rats
were given an intraperitoneal injection of estrogen (20 mg/kg estriol)
24 h before intravenous injection of a sublethal dose of LPS (5 mg/kg)
via the tail vein. Some rats were pretreated with
GdCl3 (10 mg/kg, iv), a Kupffer
cell toxicant, before estrogen treatment. Data represent 24-h mortality
rates. * P < 0.05 vs. control.
** P < 0.05 vs. estriol,
Fisher's test. Nos. above bars represent no. of dead animals per total
no. of animals.
|
|
Effect of estrogen on TNF-
production after LPS
injection.
Because TNF-
is a pivotal cytokine involved in the development of
endotoxin shock, serum TNF-
levels were measured in estrogen-treated rats after LPS injection (Fig.
2A). As
expected, serum TNF-
increased dramatically 150 min after injection
of LPS (5 mg/kg) with a subsequent gradual decrease. Peak levels of
TNF-
were twice as high in the estrogen-treated group as in the
controls. Furthermore, mRNA for TNF-
in the liver was detected by
RT-PCR (Fig. 2B). As expected, TNF-
mRNA was increased 1 h after LPS injection in control livers; however, values were about threefold higher at the same time in the
estrogen-treated group. GdCl3
pretreatment prevented the induction of TNF-
mRNA in the liver from
estrogen-treated animals almost completely.

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Fig. 2.
Effect of estrogen on tumor necrosis factor- (TNF- ) production
after LPS. A: blood samples were
collected before and at 4 time points after an injection of LPS (5 mg/kg), and TNF- was measured by ELISA. ELISA data represent means ± SE from 4 individual preparations.
* P < 0.05 with
Mann-Whitney's rank sum test. B:
total RNA from the liver before and 1 h after LPS injection (5 mg/kg)
was used for detection of TNF- mRNA as described in
MATERIALS AND METHODS. -Actin was
detected as a housekeeping gene, and
X174/Hae III was used to determine
size of PCR products. Data for TNF- mRNA are a representative
picture of 3 individual experiments.
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Effect of estrogen on NO production after LPS injection.
NO produced from macrophages causes lethal hypotension during endotoxin
shock. Therefore, plasma levels of nitrite, which reflect NO production
in vivo, were determined. Figure
3A depicts the effect of estrogen on plasma nitrite levels after LPS
injection. As expected, plasma nitrite increased markedly
6 h after LPS injection (5 mg/kg) in the control group. However,
nitrite levels in estriol-treated rats reached 85 µM, a value 2.5 times higher than in controls, indicating that estrogen treatment in
vivo increases NO production due to LPS. To determine if induction of
NOS in the liver is responsible for this increase, iNOS was detected by
Western blotting (Fig. 3B). iNOS was
increased in the liver 6 h after LPS injection to levels about fivefold
higher in livers from estrogen-treated rats than controls. Furthermore,
mRNA for iNOS was about twofold higher in estrogen-treated animals than
in controls 6 h after LPS injection (Fig.
3C). Because it is known that
interferon-
(IFN-
), which is produced by T lymphocytes and
natural killer cells, is necessary for induction of NOS (4, 30), RT-PCR
for IFN-
mRNA was performed (Fig.
3C). mRNA for IFN-
was increased
3 h after LPS injection in controls; however, estrogen pretreatment
potentiated this increase about threefold. Moreover, since IL-12, which
is produced by macrophages, is known to increase production of IFN-
(18), we also studied IL-12 mRNA in the liver (Fig.
3C). IL-12 mRNA was induced 3 h after LPS injection, and levels were about sevenfold higher in estrogen-treated animals than in controls.

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Fig. 3.
Effect of estrogen on nitric oxide (NO) production due to LPS.
A: estriol-treated rats were injected
with LPS intravenously (5 mg/kg), and plasma samples were
collected before and 6 h later. Plasma nitrite levels were determined
colorimetrically. Nitrite data are means ± SE of 5 individual
samples. * P < 0.05 vs.
control given LPS, Mann-Whitney's rank sum test.
B: protein extracts from whole liver
before and 6 h after LPS injection were separated by 7.5% SDS-PAGE and
immunoblotted using rabbit anti-mouse inducible NO synthase (iNOS)
polyclonal antibody. Specific bands for iNOS (130 kDa) are shown.
C: total liver RNA before and 1, 3, and 6 h after LPS injection (5 mg/ml) was used to detect iNOS,
interferon- (IFN- ), and interleukin-12 (IL-12) mRNA. -Actin
was also detected as a housekeeping gene, and
X174/Hae III was used to determine
size of PCR products. RT-PCR data are a representative picture of 3 individual experiments.
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|
Effect of estrogen treatment in vivo on LPS-induced production of
TNF-
and nitrite in isolated Kupffer cells.
To confirm that Kupffer cells were the source of increased TNF-
in
estrogen-treated animals, TNF-
production by isolated Kupffer cells
was measured (Fig.
4A).
Kupffer cells from control rats produced TNF-
in response to LPS
(100 ng/ml). However, isolated cells from estriol-treated animals
produced about twice as much cytokine. Interestingly, addition of
estriol (100 nM) 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, control and
estrogen-treated groups, respectively).
Furthermore, Kupffer cells isolated from control rats produced small
amounts of nitrite in the presence of LPS (100 ng/ml) (Fig.
4B). However, nitrite levels in the
culture medium of Kupffer cells from estriol-treated rats were about
twofold higher. Interestingly, addition of estriol (1 µM) to the
culture medium for 24 h before LPS also did not alter nitrite
production due to LPS (100 ng/ml) (9.5 ± 1.3 and 9.9 ± 0.5 pmol · 106
cells
1 · 24 h
1, control
and estrogen-treated groups, respectively).