Desensitization to LPS after ethanol involves the effect of endotoxin on voltage-dependent calcium channels

Nobuyuki Enomoto, Shunhei Yamashina, Moritaka Goto, Peter Schemmer, and Ronald G. Thurman

Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7365


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hepatic macrophages are sensitized to alcohol in 24 h due to increases in the endotoxin receptor, CD14; however, desensitization to lipopolysaccharide (LPS), which occurred earlier, could not be explained by changes in CD14. Therefore, the purpose of this work was to attempt to understand factors responsible for ethanol-induced desensitization to LPS in hepatic macrophages. Rats were given ethanol (5 g/kg body wt) intragastrically, and hepatic macrophages were isolated 2 h later. After addition of endotoxin, intracellular Ca2+ concentration ([Ca2+]i) was measured using fura 2 and tumor necrosis factor (TNF)-alpha was measured by ELISA. Ethanol given 2 h before injection of LPS totally prevented liver injury and blunted LPS-induced increases in [Ca2+]i and TNF-alpha in hepatic macrophages. Furthermore, the protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate and acute ethanol treatment both activated PKC and largely prevented the influx of [Ca2+]i caused by LPS. Sterilization of the gut with antibiotics completely blocked all effects of ethanol on [Ca2+]i and TNF-alpha release. Thus ethanol-induced desensitization of hepatic macrophages correlates with gut-derived endotoxin after ethanol and involves the effect of PKC on voltage-dependent Ca2+ channels.

intracellular calcium; tumor necrosis factor-alpha ; protein kinase C; lipopolysaccharide


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RECENTLY, IT WAS SHOWN that ethanol causes sensitization of rat hepatic macrophages via mechanisms dependent on endotoxin and increases in CD14 (11); however, desensitization after alcohol could not be explained by decreases in CD14. Ethanol attenuates the lipopolysaccharide (LPS)-induced production of various biologically active substances such as proteases, toxic radicals, eicosanoids, and cytokines from hepatic macrophages (4, 9, 19, 21, 29). Moreover, it was previously demonstrated that desensitization of hepatic macrophages was caused by gut-derived endotoxin (11). Reasons for this desensitization to ethanol are not understood.

Membrane depolarization opens voltage-dependent Ca2+ channels, promoting Ca2+ entry and leading to subsequent Ca2+-dependent signaling events in various cell types (5). Intracellular Ca2+ concentration ([Ca2+]i) most likely plays an important role in activation of hepatic macrophages (7, 8). For example, a transient increase of [Ca2+]i is required for LPS-induced expression of tumor necrosis factor (TNF)-alpha by a macrophage cell line (34). Furthermore, hepatic macrophages contain voltage-dependent Ca2+ channels(15), which most likely play an important role in modulating hepatic macrophage function by regulating [Ca2+]i. Paradoxically, voltage-dependent Ca2+ channels were inactivated by acute exposure to ethanol (i.e., desensitization) (14) but were easier to open after chronic alcohol treatment (i.e., sensitization) (12). However, the mechanisms by which voltage-dependent Ca2+ channels are regulated in hepatic macrophages remain unknown and whether or not they participate in desensitization to ethanol in vivo and in vitro is not clear.

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) are exogenous compounds that activate protein kinase C (PKC) in a manner analogous to diacylglycerol, an endogenous product of phosphatidylinositol breakdown (22). PKC has many regulatory roles, including cell proliferation and differentiation (17), and has been shown to modulate ion channels, including Ca2+ channels (27), by phosphorylating the beta -subunit of the channel. Although hepatic macrophages are known to have PKC activity (10), whether PKC participates in the mechanism of the regulation of voltage-dependent Ca2+ channels is not clear. Therefore, the present study was designed to determine if cellular desensitization in hepatic macrophages due to acute ethanol treatment involves voltage-dependent Ca2+ channels and PKC.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Animals and treatments. Female Sprague-Dawley rats weighing between 200 and 250 g were used in this study. All animals were given humane care in compliance with institutional guidelines. Rats were given ethanol (5 g/kg body wt po) before experiments (31, 36). Some ethanol-treated rats were also treated for 4 days with polymyxin B and neomycin (26) to prevent growth of intestinal bacteria, the primary source of endotoxin in the gastrointestinal tract. On the basis of the 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). Some rats were treated with microcystin (50 µg/kg ip) 1 h before intragastric injection of ethanol to inhibit dephosphorylation.

Analytical methods. Rats were forced to breathe into a closed, heated chamber (37°C) for 20 s, and 1 ml of breath was collected using a gas-tight syringe to measure ethanol by gas chromatography (31, 36). 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 using the Limulus amebocyte lysate assay (Whitaker Bioproducts, Walkerville, MD). Levels of endotoxin in plasma from normal rats were below the limits of detection. Serum was stored at -20°C in microtubes, and aspartate transaminase and alanine aminotransferase were measured by standard enzymatic procedures (6). Livers were formalin fixed, embedded in paraffin, and stained with hematoxylin and eosin to assess inflammation and necrosis.

Hepatic macrophage preparation and culture. Hepatic macrophages were isolated by collagenase digestion and differential centrifugation using Percoll (Pharmacia, Uppsala, Sweden) as described elsewhere, with slight modifications (24). Briefly, the liver was perfused through the portal vein with Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS) at 37°C for 5 min at a flow rate of 26 ml/min. Subsequently, perfusion was performed with HBSS containing 0.025% collagenase IV (Sigma Chemical, St. Louis, MO) 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 hepatic macrophage fraction was collected and washed again with buffer. Viability of cells, determined by trypan blue exclusion, was >90%, and purity, which was assessed histologically, was also >90%. Neutrophils and lymphocytes could be easily differentiated from hepatic macrophages based on incorporation of latex beads and nuclear shape. These preparations were virtually free of neutrophils and lymphocytes. Moreover, there were no differences between control, ethanol, and antibiotics plus ethanol groups with respect to viability and purity (Table 1). Cells were seeded onto 25-mm glass coverslips and cultured in DMEM (GIBCO Life Technologies, 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 buffer, and cells were cultured for 24 h before experiments.

                              
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Table 1.   Effect of ethanol and antibiotics on cell preparations

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). Hepatic macrophages were incubated in modified Hanks' buffer (in mmol/l: 115 NaCl, 5 KCl, 0.3 Na2HPO4, 0.4 KH2PO4, 5.6 glucose, 0.8 MgSO4, 1.26 CaCl2, and 15 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 hepatic macrophages 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 hepatic macrophages. Each value was corrected by subtracting the system dark noise and autofluorescence, assessed by quenching fura 2 fluorescence with Mn2+, as described previously (15). [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 using a buffer containing 3 mmol/l EGTA and 1 µmol/l ionomycin ([Ca2+]min) or 10 mmol/l Ca2+ and 1 µmol/l ionomycin ([Ca2+]max). R is the ratio of fluorescent intensities at excitation wavelengths of 340 nm 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 nmol/l was used (13).

TNF-alpha detection. Hepatic macrophages were seeded onto 24-well plates and cultured in DMEM supplemented with 10% FBS and antibiotics at 37°C in the presence of 5% CO2. Cells were incubated with fresh medium containing LPS (0 and 1 µg/ml supplemented with 5% rat serum) for an additional 4 h. Samples of medium were collected and kept at -80°C until assay. TNF-alpha in the culture medium was measured using an ELISA kit (Genzyme, Cambridge, MA), and data were corrected for dilution.

[3H]phorbol 12,13-dibutyrate binding to subcellular fractions from hepatic macrophages. Hepatic macrophages from rats treated acutely with ethanol for 2 h were collected and resuspended in homogenizing buffer at a density of ~30 × 106 cells/ml. Cells were then incubated in the dark with either PMA (100 nmol/l, Sigma Chemical) or DMSO vehicle. After 15 min, the reaction was stopped by rinsing cells three times with ice-cold PBS. Cells were then collected using a rubber policeman, homogenized (30-40 times) on ice in a 2-ml homogenizing tube, and centrifuged in an airfuge (50,000 rpm, 1 h, 4°C). The pellet was resuspended in 250 µl of buffer, and the supernatant was concentrated to a final volume of 250 µl (33).

PKC activity for both particulate and soluble fractions was estimated by measuring the binding of [3H]phorbol 12,13-dibutyrate ([3H]PDBu) to the active form of the PKC molecule (37). The reaction mixture contained 46 mmol/l Tris · HCl (pH 7.4), 1 mmol/l CaCl2, 0.1 mg/ml BSA, 0.02 mol/l MgCl2, 2 mmol/l dithiothreitol, 0.04 mg/ml phosphatidylserine, and [3H]PDBu (15.6 mCi · nmol-1 · l-1). Sample protein (50 µl) was incubated with an equal volume of reaction mixture for 30 min at room temperature. Nonspecific binding was determined by displacement of label by incubation with excess PMA (1 µmol/l). The binding reaction was terminated by addition of 1 ml of ice-cold 10 mmol/l Tris · HCl (pH 7.4) to each tube. Particulate fractions were collected on 1.2-µm Millipore filters and rinsed twice with ice-cold Tris · HCl. Dried filters were placed in scintillation vials containing 5 ml of liquid scintillation cocktail (Ecolume, Biomedical, Irvine, CA), and radioactivity was measured.

Gel filtration chromatography using a Sephadex G-25 column (Amersham Pharmacia Biotech, Piscataway, NJ) was employed to separate free [3H]PDBu from bound radioligand in the soluble fraction. The column was previously equilibrated with 50 mmol/l Tris · HCl, pH 7.5, at 4°C, and samples were eluted with 1-ml aliquots of ice-cold buffer. The void volume, determined by elution of dextran blue, was analyzed for receptor-bound ligand. Each sample was assayed in duplicate, and values from reaction mixtures containing PMA were subtracted from those without ligand to determine specific binding. Protein concentration for each sample was determined by the method of Lowry et al. (20) using BSA as the standard.

Statistical analysis. All results are expressed as 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Blood ethanol and endotoxin levels. After oral administration of ethanol (5 g/kg) to untreated, normal rats, blood ethanol levels increased gradually and reached a value of 268 ± 38 mg/dl after 90 min. Similar results were obtained in rats treated with antibiotics (245 ± 34 mg/dl). Subsequently, levels declined toward basal values over the next 6 h. Endotoxin was not detectable in peripheral blood; however, levels were increased in portal blood by ethanol treatment to values of 77 ± 6 pg/ml at 90 min and declined subsequently to baseline. Levels were three- to fourfold significantly higher than values from rats treated with antibiotics (23 ± 1 pg/ml, P < 0.05).

Effect of ethanol on survival after LPS injection. LPS (10 mg/kg) was injected via the tail vein, and 24-h survival rates were assessed (Fig. 1). Mortality rates were ~50% with 10 mg/kg of LPS in control rats, with most rats dying between 8 and 24 h. However, mortality was prevented totally by ethanol administration (5 g/kg) 2 h before LPS. This phenomenon was abolished by treatment with antibiotics, confirming that endotoxin is involved in mechanisms of desensitization to endotoxin due to ethanol in the animal (11).


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Fig. 1.   Effect of ethanol and antibiotics on mortality after lipopolysaccharide (LPS) injection. LPS (10 mg/kg, E. coli serotype 0111:B4, Sigma) was injected via the tail vein, and mortality rates were monitored for 24 h. Data represent percentage of dead animals (numbers listed above bars) after 24 h. EtOH, ethanol. * P < 0.05 vs. control. # P < 0.05 vs. ethanol + LPS by chi 2 test.

Effect of ethanol and LPS on liver histology and serum transaminases. Liver specimens were collected for histology 24 h after administration of LPS (10 mg/kg). Histology was normal in control rats (Fig. 2A), whereas 24 h after LPS administration focal necrosis and neutrophil infiltration were observed in the liver as expected (Fig. 2B). Two hours after ethanol treatment, however, necrosis and infiltration caused by LPS were decreased (Fig. 2C). Moreover, antibiotics prevented the improvement in histology observed with ethanol (Fig. 2D). Similar results were obtained with serum transaminases (Fig. 3). Mean values for aspartate transaminase and alanine aminotransferase in the control group were 26 ± 3 and 29 ± 4 IU/l, respectively. LPS increased transaminases dramatically to 823 ± 84 and 730 ± 128 IU/l, respectively. However, in ethanol-treated rats, this effect was blunted, and protection was prevented with antibiotics (Fig. 3).


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Fig. 2.   Photomicrographs of liver tissue. Photomicrographs of livers (hematoxylin and eosin stained) from rats treated as described in MATERIALS AND METHODS. A: no treatment. B: 24 h after LPS (10 mg/kg iv) treatment. C: 2 h after ethanol exposure and 24 h after LPS exposure. D: exposure to antibiotics for 4 days and then 2 h after ethanol exposure and 24 h after LPS exposure. Samples were from rats surviving 24 h after LPS. Typical photomicrographs are shown. Original magnification, ×100.



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Fig. 3.   Effect of acute ethanol treatment on LPS-induced increases in serum transaminases. Rats were treated with ethanol as described in MATERIALS AND METHODS, and blood samples were collected 24 h after LPS exposure (10 mg/kg). At 8 h, aspartate transaminase (AST; A) and alanine aminotransferase (ALT; B) levels were almost the same but were only slightly above the normal range. On the other hand, at 24 h, AST and ALT levels were high but similar. However, at 8 h, there was almost no liver damage. Accordingly, data were collected at 24 h. Under these conditions, ~50% of the rats did not survive. Some rats were given antibiotics (AB) for 4 days before experiments (150 mg · kg-1 · day-1 of polymyxin B and 450 mg · kg-1 · day-1 of neomycin). Samples are from rats surviving 24 h after LPS exposure. Results are means ± SE of 4 rats per group. a P < 0.05 vs. control (cont). b P < 0.05 vs. LPS (10 mg/kg). c P < 0.05 vs. ethanol (2 h) + LPS (10 mg/kg), by ANOVA and Bonferroni's post hoc test.

Effect of ethanol treatment on LPS-induced increases in [Ca2+]i in hepatic macrophages. [Ca2+]i increases both from Ca2+ release from the endoplasmic reticulum as well as from influx from the extracellular space via voltage-dependent Ca2+ channels. Addition of LPS (10 µg/ml) to hepatic macrophages from untreated control rats caused a transient elevation in [Ca2+]i, which consisted of an initial sharp increase followed later by a large increase and a gradual return to the baseline over 3 min. The maximal [Ca2+]i level achieved, including both components, was 304 ± 14 nmol/l (Fig. 4A, Table 2); however, values only reached 116 ± 9 nmol/l and only exhibited the sharp component when extracellular Ca2+ was removed (Fig. 4B). The area under the curve of the [Ca2+]i trace was 315 ± 23 s · nmol-1 · l-1 for cells from control rats. However, area was reduced ~10-fold in cells from rats treated with ethanol. This large component of the [Ca2+]i signal was blocked by a dihydropyridine-type Ca2+ channel blocker (16). Thus the large, slower component was due to Ca2+ entry from the extracellular space via voltage-dependent Ca2+ channels. In cells isolated from rats treated with ethanol 2 h earlier, the extracellular component was also absent (Fig. 4C). Thus these data are consistent with the hypothesis that desensitization due to alcohol involves downregulation or inactivation of voltage-dependent Ca2+ channels in hepatic macrophages. Treatment of rats with antibiotics prevented this desensitization, as the large two-component increase in [Ca2+]i was restored (Fig. 4E). Antibiotics did not affect the small release of Ca2+ from intracellular stores (Fig. 4F). Moreover, the dihydropyridine-type Ca2+ channel blocker, nimodipine, and the phospholipase C inhibitor, 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate, largely prevented the increase in [Ca2+]i in hepatic macrophages after LPS stimulation as expected (Table 2), indicating that the LPS-induced [Ca2+]i response is dependent on voltage-dependent Ca2+ channels via mechanisms involving phospholipase C. However, the response of hepatic macrophages isolated from control rats to LPS was not affected by treatment with antibiotics (data not shown).


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Fig. 4.   Effect of acute ethanol treatment on LPS-induced increases in intracellular Ca2+ in isolated hepatic macrophages. Intracellular Ca2+ in isolated hepatic macrophages was measured fluorometrically using fura 2 as described in MATERIALS AND METHODS. Changes in intracellular Ca2+ after addition of 10 µg/ml LPS, supplemented with 5% rat serum, are plotted. LPS was added to hepatic macrophages from control rats (A and B), to hepatic macrophages from rats treated with ethanol 2 h before isolation (C and D), or to hepatic macrophages from rats treated with antibiotics for 4 days and ethanol 2 h earlier (E and F). LPS was added to hepatic macrophages after removal of extracellular Ca2+ (B, D, and F). Data are representative traces of experiments repeated 4 times.


                              
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Table 2.   Effect of ethanol, nimodipine, and NCDC on LPS-induced [Ca2+]i response

Acute exposure to ethanol increases PKC activity in hepatic macrophages. The effect of PMA, the classical PKC agonist, on membrane-bound PKC levels in cultured hepatic macrophages is depicted in Fig. 5A. Treatment of hepatic macrophages with this ligand for 15 min significantly increased PKC activity over control levels around 13-fold. In untreated cells, 62% of the activity was particulate, whereas 38% was in the soluble fraction. After exposure to PMA, however, 96% was particulate and 4% was soluble. Similarly, acute ethanol treatment increased values in the particulate fraction to 100%. The effect of acute treatment with ethanol on membrane-bound PKC activity is depicted in Fig. 5A. PKC activity in hepatic macrophages isolated from rats exposed acutely to ethanol was increased significantly, around 12-fold compared with controls (Fig. 5A).


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Fig. 5.   Effect of phorbol 12-myristate 13-acetate (PMA) and ethanol on protein kinase C (PKC) and intracellular Ca2+ in isolated hepatic macrophages. A: cultured hepatic macrophages were prepared as described in MATERIALS AND METHODS. Cells were treated with 100 nmol/l PMA for 15 min. Some hepatic macrophages were isolated from rats 2 h after intragastric addition of ethanol (5 g/kg). Total PKC activity was measured as described in MATERIALS AND METHODS. Data represent means ± SE; n = 4, *P < 0.05 vs. control by ANOVA and Bonferroni's post hoc test. PDBu, phorbol 12,13-dibutyrate. B: PMA was prepared by dilutions of 1.5 mmol/l stock solution in DMSO. Addition of DMSO to cultured cells had no effect on intracellular Ca2+ concentration ([Ca2+]i) response at final concentrations used in this study. [Ca2+]i in isolated hepatic macrophages was assessed fluorometrically using the Ca2+ indicator, fura 2, as described in MATERIALS AND METHODS. LPS (10 µg/ml) was added at the time indicated by the arrow to control and PMA (1 µmol/l)-treated cells. In cells labeled with PMA, hepatic macrophages were pretreated with PMA 15 min before addition of LPS.

Role of PKC on LPS-induced increases of [Ca2+]i in isolated hepatic macrophages. Representative changes in [Ca2+]i in individual cultured hepatic macrophages caused by LPS are depicted in Fig. 5B. After addition of LPS (10 µg/ml), the maximal [Ca2+]i level in normal hepatic macrophages was 304 ± 14 nmol/l (n > 20). In contrast, when hepatic macrophages were pretreated with PMA (1 µmol/l) 15 min before the addition of LPS, the increase in [Ca2+]i only reached a value of 106 nmol/l in this example (mean = 163 ± 31 nmol/l, P < 0.05). The area under curve of the [Ca2+]i trace was 315 ± 23 s · nmol-1 · l-1 in cells from control rats; however, values only reached 43 ± 9 s · nmol-1 · l-1 in hepatic macrophages pretreated with PMA. PMA alone had no effect on the basal level of [Ca2+]i. Importantly, only the sharp transient peak, similar to what was seen with ethanol (Fig. 4), was observed.

Phosphatase inhibitor microcystin blocks desensitization to ethanol in isolated hepatic macrophages. After addition of LPS (10 µg/ml), the maximal [Ca2+]i level in hepatic macrophages treated with ethanol achieved was 116 ± 9 nmol/l (Fig. 6A). In contrast, when rats were pretreated with 50 µg/kg ip of the phosphatase inhibitor microcystin 1 h before intragastric injection of ethanol, the increase in [Ca2+]i was restored to 300 ± 10 nmol/l. Microcystin alone had no effect on the LPS-induced [Ca2+]i response.


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Fig. 6.   Effect of ethanol, antibiotics, and microcystin on LPS-induced increases in [Ca2+]i and tumor necrosis factor (TNF)-alpha production by cultured hepatic macrophages. 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). Some rats were treated with microcystin (50 µg/kg ip) 1 h before ethanol. Two hours after oral administration of ethanol (5 g/kg body wt po) or saline vehicle, hepatic macrophages were isolated and cultured in 6-cm culture dishes at a density of 5 × 105 cells/dish for [Ca2+]i measurement and in 24-well culture plates at a density of 5 × 105 cells/well for TNF-alpha determination. A: [Ca2+]i was measured using a microspectrofluorometer with the fluorescent indicator, fura 2. B: TNF-alpha was measured by ELISA. TNF-alpha release after 4 h of incubation without LPS was 240 ± 20 pg/ml. Results are means ± SE; n = 4. * P < 0.05 vs. control. # P < 0.05 vs. ethanol, by ANOVA and Bonferroni's post hoc test.

Effect of ethanol treatment in vivo on LPS-induced production of TNF-alpha by isolated hepatic macrophages. To evaluate the effect of ethanol on cytokine production by hepatic macrophages, LPS-induced TNF-alpha production was measured after ethanol treatment (Fig. 6B). As expected, isolated hepatic macrophages produced large amounts of TNF-alpha in the presence of LPS (300% of basal values). However, ethanol completely prevented this effect. Moreover, treatment of rats with antibiotics or microcystin prevented the decrease in TNF-alpha production caused by exposure to ethanol for 2 h.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mechanism of sensitization to endotoxin by ethanol. TNF-alpha levels increase in alcoholics, consistent with the hypothesis that hepatic macrophages of patients with alcoholic liver disease are activated. Shibayama et al. (28) demonstrated that acute administration of ethanol enhances endotoxin hepatotoxicity. Moreover, it was shown recently that hepatic macrophages became sensitized to LPS in cells isolated 24 h after ethanol, reflected by increased [Ca2+]i, TNF-alpha production, and large increases in the endotoxin receptor, CD14 (11). These effects were all blocked by antibiotics, indicating that sensitization of hepatic macrophages by ethanol is also mediated by gut-derived endotoxin. Moreover, hepatic macrophages from rats treated with ethanol 24 h earlier expressed much higher levels of CD14 than rats given antibiotics. This most likely explains why hepatic macrophages ex vivo "remember" that they were treated with ethanol in vivo. Because gut sterilization removes portal endotoxin, hepatic macrophages are not sensitized and liver damage after LPS injection is blunted in vivo (Figs. 2 and 3). Recently, Su et al. (30) showed that CD14 was upregulated in hepatic macrophages from rats exposed to chronic alcohol using the Tsukamoto-French enteral ethanol delivery model.

Endotoxin is involved in desensitization to LPS caused by ethanol. Endotoxin shock causes cardiovascular collapse, lung dysfunction, and altered blood coagulation, and hepatic macrophage-derived cytokines play an important role in this phenomenon (32). Accordingly, the LPS-induced increase of [Ca2+]i and TNF-alpha production was measured in hepatic macrophages. Previous studies have shown that acute alcohol decreases endotoxin-induced increases in cytotoxic mediators such as TNF-alpha (9, 21), superoxide (4), and nitric oxide (29). Furthermore, it was demonstrated that acute ethanol protects against otherwise lethal doses of LPS. Endotoxin is involved in this phenomenon,since sterilization of the gut with antibiotics blocked this effect (Figs. 1-3).

Hepatic macrophages are involved in ethanol-induced desensitization in vivo. Hepatic macrophages represent more than 80% of fixed tissue macrophages (19) and play an important role in removal of gut-derived endotoxin (LPS) (23). Moreover, hepatic macrophages are a rich source of cytokines after LPS treatment (3). LPS elevates [Ca2+]i transiently in isolated hepatic macrophages (Fig. 4). Most likely, Ca2+ enters from the extracellular space via voltage-dependent Ca2+ channels because the LPS-induced increase in [Ca2+]i was largely blocked by the Ca2+ channel blocker, nimodipine (16) (Table 2). Nisoldipine, a related Ca2+ channel blocker, also inhibited the production of TNF-alpha due to LPS in cultured hepatic macrophages (25). Therefore, it is concluded that the transient increase in [Ca2+]i due to LPS is important in triggering the production of TNF-alpha by hepatic macrophages. As expected, ethanol prevented the increase in [Ca2+]i due to LPS (Fig. 4). In this model, increases in TNF-alpha production from hepatic macrophages were also markedly blunted by ethanol (Fig. 6B). Importantly, sterilization of the gut with antibiotics blocked all effects of ethanol on [Ca2+]i and TNF-alpha production completely (Figs. 4 and 6), supporting the hypothesis that desensitization in the hepatic macrophages due to alcohol involves gut-derived endotoxin (4).

Voltage-dependent Ca2+ channels in hepatic macrophages are involved in desensitization to LPS caused by ethanol. Hepatic macrophages isolated from rats exposed to ethanol for 2 h were much less sensitive to LPS, as reflected by blunted [Ca2+]i and TNF-alpha production (Figs. 4 and 6). However, the LPS receptor CD14 was not decreased 2 h after ethanol treatment when the response of the hepatic macrophages to endotoxin was clearly blunted (11). However, Hijioka et al. (14) showed that acute ethanol treatment in vivo reduced the probability that L-type voltage-dependent Ca2+ channels in hepatic macrophages would be open. Increases in intracellular Ca2+ after addition of LPS depend on both Ca2+ release from the endoplasmic reticulum and influx from the extracellular space via voltage-dependent Ca2+ channels. When LPS was added, a sharp increase, followed later by a large increase and a gradual return to the baseline, was observed; however, only the sharp component occurred when extracellular Ca2+ was removed (Fig. 4B). Thus it is concluded that the large, slower component represents Ca2+ entry from the extracellular space via voltage-dependent Ca2+ channels. In cells isolated from rats treated for 2 h with ethanol, the extracellular component was absent (Fig. 4C). Thus it is concluded that desensitization due to alcohol involves inactivation of voltage-dependent Ca2+ channels in hepatic macrophages. Furthermore, it is concluded that endotoxin is involved in this phenomenon, since treatment of rats with antibiotics prevented this desensitization (Figs. 4E and 6B).

Role of PKC. After LPS challenge of murine peritoneal macrophages, production of TNF-alpha was inhibited by the PKC inhibitor H-7 in a dose-dependent manner (18). Furthermore, PKC was upregulated in mouse hepatic macrophages treated with PMA, and more TNF-alpha was produced after LPS stimulation in these cells than in untreated cells (2). However, superoxide production by LPS-treated hepatic macrophages was suppressed by PMA, and PGE2 release by LPS/IFN-gamma -treated hepatic macrophages was increased by treatment with staurosporine (35). Thus the role of PKC in hepatic macrophage activation remains unclear. In this study, PKC activity in hepatic macrophages was increased not only by PMA treatment but by acute ethanol treatment. In both cases, voltage-dependent Ca2+ channels were inactivated (Fig. 5A). The LPS-induced [Ca2+]i response was decreased by treatment with PMA and acute ethanol treatment. The shape of the [Ca2+]i response by treatment with PMA was very similar to acute ethanol treatment. Together, these data strongly support the hypothesis that ethanol-induced increases in endotoxin activate PKC, which inhibits voltage-dependent Ca2+ channels in hepatic macrophages leading to cellular desensitization. Moreover, it is likely that PKC phosphorylates the beta -subunit of voltage-dependent Ca2+ channels. This is based on the finding that inhibition of dephosphorylation by the phosphatase inhibitor, microcystin, reversed the effect of ethanol on [Ca2+]i and TNF-alpha production.

In conclusion, ethanol blunts the elevation of [Ca2+]i in LPS-stimulated hepatic macrophages through inhibition of voltage-dependent Ca2+ channels via mechanisms dependent on PKC. This effect involves endotoxin, since it was blocked by gut sterilization. Moreover, prevention of increases in [Ca2+]i due to LPS by ethanol reduced production of TNF-alpha by hepatic macrophages, leading to improved survival and decreased liver injury due to endotoxin shock (i.e., in vivo desensitization). It is likely that ethanol-induced desensitization in hepatic macrophages was caused by phosphorylation of the beta -subunit of voltage-dependent Ca2+ channels via mechanisms dependent on PKC.


    ACKNOWLEDGEMENTS

This work was supported, in part, by grants from National Institute on 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 Building, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365.

Received 17 July 1998; accepted in final form 31 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(6):G1251-G1258
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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