INTRODUCTION

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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

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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.

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 = K_d[(R  R_min)/(R_max  R)](F₀ /F_s) where F₀ /F_s 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 R_max and R_min 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 (K_d) 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

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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.

View larger version (K): [in this window] [in a new window]   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.

View larger version (K): [in this window] [in a new window]   View larger version (K): [in this window] [in a new window]   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.

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.

View larger version (K): [in this window] [in a new window]   View larger version (K): [in this window] [in a new window]   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.

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 cells¹ · 4 h¹, 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 cells¹ · 24 h¹, control and estrogen-treated groups, respectively).

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