TY - JOUR
AU - SERGENT,, O.
AB - Abstract The aim of this study was to examine how macrophages could act on ethanol-induced oxidative stress in rat hepatocytes during inflammatory conditions, well-known to induce nitric oxide (NO) synthase. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocyte cultures. Co-cultures were then supplemented with lipopolysaccharide (LPS) and interferon γ (IFN) for 18 h, in order to induce NO synthase before the addition of 50 mM ethanol. In cultures of hepatocytes alone, the addition of LPS and IFN protected from ethanol-induced oxidative stress. It has been shown previously that NO generated in hepatocytes was responsible for this effect. When macrophages were added to primary rat hepatocyte cultures supplemented with LPS and IFN, protection provided by NO against ethanol-induced oxidative stress in hepatocytes ceased. Using a pretreatment of macrophages with Ng-monomethyl-l-arginine, a NO synthase inhibitor, it was concluded that NO generated by macrophages was responsible for macrophage toxicity. Taken together, our observations suggest that NO biosynthesis in hepatocytes protects them from ethanol-induced oxidative stress, whereas NO production in macrophages deprives hepatocytes of this NO protection. INTRODUCTION It is well-known that Kupffer cells, resident macrophages of the liver, and other inflammatory macrophages recruited to the liver, can aggravate the hepatotoxicity of ethanol. Indeed, following chronic alcohol intoxication of rats, an increase in macrophage and neutrophil amounts is observed in the portal tract (Karakucuk et al., 1989) and the liver (Bautista and Spitzer, 1992). Moreover, ethanol intoxication stimulates some activities of macrophages and neutrophils, such as the release of superoxide anion and cytokines (Williams and Barry, 1987; Yamada et al., 1991; Bautista and Spitzer, 1992; Goto et al., 1994), mediators involved in the pathogenesis of alcohol-induced liver disease. Thus, macrophages and neutrophils are directly involved in the hepatotoxicity of alcohol. For instance, the extent of macrophage and neutrophil infiltration of the liver is correlated with the severity of hepatocellular injury (Takahashi et al., 1987; Ishii et al., 1994) and gadolinium chloride, a selective inhibitor of Kupffer cells, reduces alcohol-induced liver injury (Adachi et al., 1994; Koop et al., 1997). In the liver, many cell types, including Kupffer cells (Billiar et al., 1989) and hepatocytes (Curran et al., 1989) have the ability to synthesize nitric oxide (NO) during endotoxaemia and inflammation. Moreover, an increase in NO production has been reported in monocytes of patients with chronic liver diseases (Hunt and Goldin, 1992) and in livers of rats chronically fed with ethanol (Wang et al., 1995; Chamulitrat and Spitzer, 1996). Previously, we have shown that RAW 264.7 macrophages and peritoneal macrophages are able to downregulate the ability of hepatocytes to generate nitric oxide, even when these cells were stimulated by supplementation with lipopolysaccharide (LPS) and interferon γ (IFN), well-known to induce NO synthase (Griffon et al., 1998). We also reported that NO biosynthesis in hepatocytes protects them from ethanol-induced oxidative stress (Sergent et al., 1997); this prompted us to consider another mechanism for macrophage cytotoxicity, whereby macrophages could make hepatocytes more susceptible to ethanol injury by decreasing NO production in these cells. For this purpose, cytotoxicity was estimated by the evaluation of oxidative stress in rat hepatocytes co-cultured with RAW 264.7 macrophages and supplemented with LPS and IFN to induce NO synthesis before the addition of ethanol. In the present study, we report that activated macrophages, by producing NO, abolished the protective effect of LPS and IFN against ethanol-induced oxidative stress. MATERIALS AND METHODS Materials IFN and LPS from Escherichia coli serotype 055:B5, Ng-monomethyl-l-arginine (l-NMMA), and indomethacin were purchased from Sigma (Saint Quentin Fallavier, France). Ethanol was obtained from Prolabo (Paris, France). Cell culture and treatment Adult rat hepatocytes were isolated and cultured as described previously (Guguen et al., 1975). Briefly, adult rat hepatocytes were isolated from 2-month-old Sprague–Dawley animals by the two-step collagenase perfusion method. The cells were collected in Leibovitz medium containing 1 mg of bovine serum albumin and 5 mg of bovine insulin/ml. Cell suspensions were filtered on gauze and allowed to sediment for 20 min to eliminate cell debris, blood, and sinusoidal cells. The cells were washed three times by centrifugation at 50 g, tested for viability (≥85%) and counted. Typically, 18 × 106 hepatocytes were plated in 175 cm2 Nunclon® flasks in a medium (Ref. 015650, Eurobio, Les Ulis, France) consisting of 75% Eagle minimum essential medium and 25% medium 199 with Hanks' salts and containing streptomycin (50 μg), penicillin (5 μg), bovine insulin (5 μg), bovine serum albumin (1 mg), and sodium bicarbonate (2.2 mg)/ml. Mouse monocytes/macrophages from cell line RAW 264.7 [European Collection of Cell Cultures (ECACC)] were grown on 100-mm2 plastic tissue-culture dishes in Dulbecco's modified essential medium (DMEM) supplemented with 10% (v/v) fetal calf serum, 4 mM glutamine, 5% (v/v) sodium bicarbonate, 50 μg/ml streptomycin, and 5 μg/ml penicillin. Cells were diluted by a fifth in fresh medium every 7 days. Hepatocyte:macrophage co-cultures were performed as follows. Confluent RAW 264.7 macrophages were washed by phosphate buffered saline (PBS) and removed from culture dishes by scraping according to the recommendations of ECACC. Cells were then centrifuged and resuspended in hepatocyte medium to a concentration of 1 × 106 cells/ml. Cells were then added directly to adherent hepatocytes with hepatocyte:macrophage ratios of 6:1, 2:1, and 1:1. These ratios were respectively similar to those found in normal liver with only resident Kupffer cells (Kuiper et al., 1994), in pathological livers with a weak inflammation (Laskin and Pilaro, 1986) or a severe inflammation for which monocytes/macrophages were recruited to the liver (Laskin and Pilaro, 1986). Co-cultures were then supplemented with IFN (500 IU/ml) and LPS (20 μg/ml) or not at all. After an 18-h incubation period at 37°C, some cultures were supplemented with 50 mM ethanol for 5 h. In some experiments, 9 × 106 RAW 264.7 macrophages were preincubated for 3 h with 500 μM l-NMMA (an NO synthase inhibitor), washed three times with PBS and added to 18 × 106 hepatocytes. These co-cultures were then treated as above. With this protocol, no NO synthesis, estimated by nitrite levels in the medium of the pure macrophage cultures was found during a 23-h incubation time with LPS and IFN (data not shown). In a further set of experiments, co-cultures with a 2:1 hepatocyte:macrophage ratio were treated for 23 h simultaneously with LPS and IFN, with 10 μM indomethacin, a cyclooxygenase inhibitor. Whatever the type of supplement, addition of macrophages did not lead to loss of viability or leakage of cell enzymes. Evaluation of oxidative stress Oxidative stress was analysed by lipid peroxidation measurement using extracellular free malondialdehyde (MDA) as marker. According to the method previously described (Morel et al., 1990), extracellular free MDA was estimated in the ultrafiltrate of culture medium by size-exclusion chromatography. Previous work has shown that 95% of total free MDA was released into the culture medium after 5 h of incubation with ethanol (Sergent et al., 1995). As MDA has a high affinity for the primary amino group of proteins (Janero, 1990), experiments were performed to find out whether a decrease of free MDA levels could be due to this binding. For this purpose, the pH of the culture medium was adjusted to 13 and then the culture medium was incubated in a water bath at 60°C for 30 min before ultrafiltration. This pretreatment is known to release MDA from the bound form of MDA– biomolecule complexes (Lee et al., 1987). Determination of ethanol concentration in culture media Alcohol concentration in the culture media was determined by gas chromatographic analysis as described previously (Sergent et al., 1995). For experimental purposes, ethanol at a final concentration of 50 mM was added to hepatocyte cultures and to hepatocyte:macrophage co-cultures (1:1) pretreated for 18 h with LPS and IFN. Cultures were then maintained at 37°C for 1 h in closed flasks. After the first hour of incubation, it has been shown that there is no further change in ethanol concentration in the culture media (Sergent et al., 1995). Measurement of protein levels in hepatocytes The results obtained for the whole indices were corrected for cellular protein concentration which was determined according to Bradford's reaction by using Bio-Rad (Bio-Rad, Ivry, France) reagent (Bradford, 1976). Statistical analysis Values are expressed as means ± SD from four independent experiments. ANOVA and Newman–Keuls tests were used to identify statistical significance for multiple comparisons. Differences were considered significant when P was < 0.05. RESULTS Macrophage inhibition of the antioxidant effect of LPS and IFN on ethanol-induced lipid peroxidation in hepatocytes In rat hepatocytes cultured alone, ethanol or the combination of LPS and IFN increased MDA levels when compared to control cultures. However, when these cells were treated with ethanol after a preincubation with LPS and IFN, lipid peroxidation was reduced significantly (Fig. 1). Previously, both effects of supplementing with LPS and IFN, i.e. pro-oxidant properties, when added alone, and antioxidant properties against ethanol-induced oxidative stress were demonstrated to be linked to NO production (Sergent et al., 1997). When macrophages were added to rat hepatocytes, preincubation with LPS and IFN before addition of ethanol did not reduce lipid peroxidation, contrary to hepatocyte cultures supplemented in the same conditions but without macrophages (Fig. 1). An elevation of lipid peroxidation, which increased with the number of macrophages, could be observed (Fig. 1). For the hepatocyte:macrophage ratios 2:1 and 1:1, lipid peroxidation levels were higher than in hepatocyte cultures incubated with ethanol alone. However, it should be noted that macrophages did not have any significant influence on ethanol-induced lipid peroxidation if they were not stimulated with LPS and IFN (Fig. 1). Moreover, macrophage addition led to a decrease in lipid peroxidation induced by LPS and IFN when compared to hepatocytes cultured alone in the same conditions. This decrease in lipid peroxidation became stronger as the number of macrophages increased (Fig. 1). It should be noted that the various supplements previously used in hepatocyte:macrophage co-cultures did not lead to any increase of free MDA in cultures of macrophages alone (data not shown). In a set of experiments, RAW 264.7 macrophages were replaced by primary rat peritoneal macrophages to evaluate the consequences of using cell lines. As RAW 264.7 macrophages, peritoneal macrophages also inhibited the antioxidant effect of LPS and IFN on ethanol-induced lipid peroxidation in hepatocytes (data not shown). Because RAW 264.7 macrophages gave the same results and were much easier to obtain in large numbers, these were used for further experiments. In order to establish whether activated macrophages could decrease free MDA levels by increasing protein release into the culture medium and thereby promoting binding of MDA to proteins, culture media were pretreated as described in Materials and methods to release free MDA from its bound form. No significant difference could be found when compared with results obtained without this pretreatment (data not shown). Involvement of NO generated by macrophages in the macrophage inhibition of the antioxidant effect of LPS and IFN on ethanol-induced lipid peroxidation in hepatocytes Since macrophages were previously shown to decrease NO levels in hepatocytes through their NO production (Griffon et al., 1998), they were preincubated for 3 h with a structural analogue of l-arginine, l-NMMA, which inhibits NO synthase. When NO production was inhibited in macrophages before adding to hepatocytes, pretreatment of co-cultures with LPS and IFN again led to inhibition of ethanol-induced oxidative stress (Fig. 2). Conversely, it should be noted that, in cultures of hepatocytes co-cultured with l-NMMA-pretreated macrophages and supplemented with LPS and IFN, an increase in MDA levels was again observed, when compared to control co-cultures (Fig. 2). Beneficial effect of indomethacin, an inhibitor of prostaglandin synthesis, on ethanol-induced lipid peroxidation in hepatocyte:macrophage co-cultures NO generated by macrophages was previously shown to decrease NO production in hepatocytes via prostaglandin release (Griffon et al., 1998). Therefore, the effect of indomethacin, an inhibitor of prostaglandin synthesis through cyclo-oxygenase inhibition, was tested in co-cultures, in order to re-establish NO levels and thereby to protect from ethanol toxicity. In co-cultures incubated with LPS and IFN, treatment with indomethacin led to recovery of the inhibition by LPS and IFN of ethanol-induced lipid peroxidation (Fig. 3). It should be noted that indomethacin had no effect on lipid peroxidation measured in control hepatocytes culture alone or on ethanol-induced lipid peroxidation. Lack of effect of macrophages on ethanol concentration in culture media Addition of macrophages, whether or not stimulated with LPS and IFN, to hepatocyte cultures did not modify ethanol concentration in the culture medium (Table 1). Moreover, no detectable changes in ethanol concentrations were found in culture media of macrophages cultured alone. DISCUSSION In this paper, macrophages have been shown to be able to suppress the protective effect of LPS and IFN against ethanol-induced oxidative stress and even to enhance ethanol toxicity. These data support previous observations that reported involvement of macrophages in alcohol-induced liver injury (Adachi et al., 1994; Takeyamo et al., 1996; Koop et al., 1997). However, to our knowledge, this is the first study that has taken into account changes of macrophage amount and the state of liver inflammation. Activation of macrophages in large amounts, corresponding to a possible infiltration of the liver by monocytes/macrophages, contributed much more to ethanol-induced toxicity in hepatocytes than stimulation of macrophages with an amount equal to that of a normal liver. Our data are in agreement with some studies performed in vitro on the toxicity of galactosamine, each using different amounts of macrophages (Kmiec et al., 1993; McMillan and Jollow, 1995). With a hepatocyte:macrophage ratio corresponding to normal liver, Kmiec et al. (1993) did not observe an enhancement of galactosamine toxicity in hepatocytes, whereas McMillan and Jollow (1995) obtained an increase in toxicity for a hepatocyte:macrophage ratio of 1:4, which was in accord with a high recruitment of macrophages. As the protective effect of LPS and IFN against ethanol-induced oxidative stress was previously shown to be linked to NO production in hepatocytes (Sergent et al., 1997), our present results have shown that macrophages were able to abolish NO protection and even to induce toxicity. This could partly explain the discrepancies which still exist in vivo about the protective or toxic effect of NO toward toxicant-induced liver injuries (Chamulitrat et al., 1994; Nagase et al., 1995; Gardner et al., 1998), which would depend on the degree of macrophage liver infiltration. The aim of this study was to elucidate the mechanism whereby macrophages are able to inhibit the protection provided by NO synthesized in hepatocytes toward ethanol-induced oxidative stress. Previous studies prompted us to consider the effect of NO generated by macrophages on oxidative stress induced in hepatocytes, since we had reported that NO biosynthesis in macrophages decreased NO production in rat hepatocytes (Griffon et al., 1998) (Fig. 4). In our present study, inhibition of macrophage NO synthase, by pretreatment of these cells with l-NMMA, demonstrated that NO generated by macrophages was responsible for the ability of these cells to restore ethanol-induced oxidative stress in rat hepatocytes supplemented with LPS and IFN (Fig. 4). According to our previous results (Griffon et al., 1998), it is likely that the mechanism of toxicity for macrophages was inhibition of NO formation in hepatocytes. More precisely, NO generated in macrophages was previously shown to inhibit NO production in hepatocytes via prostaglandin release (Griffon et al., 1998). When cyclo-oxygenase, which catalyses prostaglandin production in macrophages, was inhibited by indomethacin, the antioxidant effect of LPS and IFN on ethanol-induced oxidative stress was re-established in hepatocytes co-cultured with macrophages. Thus, prostaglandin released from macrophages by inducing downregulation of endogenous NO production in hepatocytes, caused an increase in vulnerability of hepatocytes to ethanol (Fig. 4). This toxicity of macrophages toward the beneficial production of NO in hepatocytes could be added to other detrimental effects previously reported with Kupffer cells, such as inhibition of mitochondrial function (Stadler et al., 1991; Kurose et al., 1993). It should be noted that macrophages did not increase lipid peroxidation levels by activation of their own reactive oxygen species production, either by ethanol or by LPS and IFN, as no elevation of MDA levels was obtained in co-cultures incubated under these conditions, when compared to pure hepatocyte cultures. Moreover, macrophage addition did not modify ethanol concentration in the medium, even though Wickramasinghe (1989) reported that macrophages could oxidize ethanol to acetaldehyde extracellularly. An increase in acetaldehyde is known to cause lipid peroxidation (Muller and Sies, 1982) and would have therefore increased MDA levels. It should be noted that, in hepatocyte cultures incubated only with LPS and IFN, macrophage addition led to no toxicity and even in high ratios, resulted in a decrease in oxidative stress. This could be explained easily by the ability of NO generated in hepatocytes to induce oxidative stress (Sergent et al., 1997), whereas NO produced in macrophages can downregulate NO formation in hepatocytes (Griffon et al., 1998). Furthermore, when NO biosynthesis was inhibited in macrophages, LPS- and IFN-elicited cytotoxicity was again observed. At first sight, the lack of toxicity of macrophages in cultures incubated with LPS and IFN alone seemed to disagree with many studies performed in vivo, which pointed to the toxic effect of Kupffer cells during endotoxaemia (Limuro et al., 1994; Jaeschke et al., 1994; Suzuki et al., 1996). This could, however, be explained by the ability of Kupffer cells to induce neutrophil infiltration (Adachi et al., 1994; Suzuki et al., 1996; Mawet et al., 1996). Neutrophils have been described to be inflammatory cells which are much more harmful than macrophages because they are able to release reactive oxygen species more rapidly, producing hypochlorous acid, a powerful oxidant, and to synthesize more active proteolytic enzymes (Adams et al., 1980). Taken together, our results lead to the conclusions that, in cultures supplemented with LPS and IFN, macrophages induced enhancement of ethanol toxicity in hepatocytes and that NO generated in macrophages was involved in this toxicity. Therefore, NO plays a double role in the liver: NO biosynthesis in hepatocytes protects against ethanol-induced oxidative stress whereas NO production in macrophages prevents the NO protection of hepatocytes through prostaglandin release. From these observations, it may be postulated that clinical use of NO donors or NO synthase inhibitors in patients with inflammatory disease and monocyte/macrophage infiltration of the liver, such as alcoholic hepatitis, could be ineffective and even harmful, and that inhibition of prostaglandin production by non-steroidal anti-inflammatory drugs should be the preferred method of treatment. Table 1. Ethanol concentration in culture medium of primary rat hepatocyte cultures, hepatocyte:macrophage co-cultures and RAW 264.7 macrophage cultures . Ethanol concentration (mM) . Treatment . Hepatocytes . Hepatocytes:macrophages . Macrophages . Cultures were supplemented with lipopolysaccharide (LPS) and interferon γ (IFN) for 18 h, and then with 50 mM of ethanol (EtOH) for 1 h. Control cultures were not supplemented. EtOH 40.20 ± 1.71 40.79 ± 1.57 49.60 ± 1.16 EtOH + LPS and IFN 39.20 ± 2.76 39.73 ± 1.67 48.78 ± 1.33 . Ethanol concentration (mM) . Treatment . Hepatocytes . Hepatocytes:macrophages . Macrophages . Cultures were supplemented with lipopolysaccharide (LPS) and interferon γ (IFN) for 18 h, and then with 50 mM of ethanol (EtOH) for 1 h. Control cultures were not supplemented. EtOH 40.20 ± 1.71 40.79 ± 1.57 49.60 ± 1.16 EtOH + LPS and IFN 39.20 ± 2.76 39.73 ± 1.67 48.78 ± 1.33 Open in new tab Table 1. Ethanol concentration in culture medium of primary rat hepatocyte cultures, hepatocyte:macrophage co-cultures and RAW 264.7 macrophage cultures . Ethanol concentration (mM) . Treatment . Hepatocytes . Hepatocytes:macrophages . Macrophages . Cultures were supplemented with lipopolysaccharide (LPS) and interferon γ (IFN) for 18 h, and then with 50 mM of ethanol (EtOH) for 1 h. Control cultures were not supplemented. EtOH 40.20 ± 1.71 40.79 ± 1.57 49.60 ± 1.16 EtOH + LPS and IFN 39.20 ± 2.76 39.73 ± 1.67 48.78 ± 1.33 . Ethanol concentration (mM) . Treatment . Hepatocytes . Hepatocytes:macrophages . Macrophages . Cultures were supplemented with lipopolysaccharide (LPS) and interferon γ (IFN) for 18 h, and then with 50 mM of ethanol (EtOH) for 1 h. Control cultures were not supplemented. EtOH 40.20 ± 1.71 40.79 ± 1.57 49.60 ± 1.16 EtOH + LPS and IFN 39.20 ± 2.76 39.73 ± 1.67 48.78 ± 1.33 Open in new tab Fig. 1. Open in new tabDownload slide Effect of addition of various amounts of RAW 264.7 macrophages on lipid peroxidation in rat hepatocyte cultures supplemented with ethanol. Lipid peroxidation was estimated by the measurement of MDA. Co-cultures contained 18 × 106 hepatocytes and 3 × 106 macrophages (6:1 ratio), 9 × 106 macrophages (2:1 ratio) or 18 × 106 macrophages (1:1 ratio). Cultures were incubated without any supplement for 23 h (Control), with LPS and IFN for 23 h (LPS–IFN), with 50 mM ethanol for 5 h (ETOH), or with LPS and IFN for 18 h, and then ethanol for the next 5 h (ETOH + LPS–IFN).*P < 0.05 compared with control hepatocyte cultures. †P < 0.05 compared with hepatocyte cultures supplemented with ethanol alone. Fig. 1. Open in new tabDownload slide Effect of addition of various amounts of RAW 264.7 macrophages on lipid peroxidation in rat hepatocyte cultures supplemented with ethanol. Lipid peroxidation was estimated by the measurement of MDA. Co-cultures contained 18 × 106 hepatocytes and 3 × 106 macrophages (6:1 ratio), 9 × 106 macrophages (2:1 ratio) or 18 × 106 macrophages (1:1 ratio). Cultures were incubated without any supplement for 23 h (Control), with LPS and IFN for 23 h (LPS–IFN), with 50 mM ethanol for 5 h (ETOH), or with LPS and IFN for 18 h, and then ethanol for the next 5 h (ETOH + LPS–IFN).*P < 0.05 compared with control hepatocyte cultures. †P < 0.05 compared with hepatocyte cultures supplemented with ethanol alone. Fig. 2. Open in new tabDownload slide Effect of RAW 264.7 macrophage preincubation with l-NMMA on lipid peroxidation in hepatocyte:macrophage co-cultures supplemented with ethanol. Lipid peroxidation was estimated by the measurement of MDA. Co-cultures contained 18 × 106 hepatocytes and 9 × 106 macrophages (2:1 ratio). Cultures were incubated without any supplement for 23 h (Control), with LPS and IFN for 23 h (LPS–IFN), with 50 mM ethanol for 5 h (ETOH), or with LPS and IFN for 18 h, and then ethanol for the next 5 h (ETOH + LPS–IFN). *P 14;< 0.05 compared with control hepatocyte cultures. Fig. 2. Open in new tabDownload slide Effect of RAW 264.7 macrophage preincubation with l-NMMA on lipid peroxidation in hepatocyte:macrophage co-cultures supplemented with ethanol. Lipid peroxidation was estimated by the measurement of MDA. Co-cultures contained 18 × 106 hepatocytes and 9 × 106 macrophages (2:1 ratio). Cultures were incubated without any supplement for 23 h (Control), with LPS and IFN for 23 h (LPS–IFN), with 50 mM ethanol for 5 h (ETOH), or with LPS and IFN for 18 h, and then ethanol for the next 5 h (ETOH + LPS–IFN). *P 14;< 0.05 compared with control hepatocyte cultures. Fig. 3. Open in new tabDownload slide Effect of indomethacin on lipid peroxidation in hepatocyte:macrophage co-cultures supplemented with ethanol. Lipid peroxidation was estimated by the measurement of MDA. Co-cultures contained 18 × 106 hepatocytes and 9 × 106 macrophages (2:1 ratio). Rat hepatocyte cultures (Hep) and hepatocyte–macrophage cocultures (Hep-MΦ) were incubated without any supplement for 23 h (Control), with LPS and IFN for 23 h (LPS–IFN), and with 50 mM of ethanol for 5 h (ETOH) or with LPS–IFN for 18 h, and then with ethanol for the next 5 h (LPS–IFN + ETOH). *P0.05 compared with control hepatocyte cultures. Fig. 3. Open in new tabDownload slide Effect of indomethacin on lipid peroxidation in hepatocyte:macrophage co-cultures supplemented with ethanol. Lipid peroxidation was estimated by the measurement of MDA. Co-cultures contained 18 × 106 hepatocytes and 9 × 106 macrophages (2:1 ratio). Rat hepatocyte cultures (Hep) and hepatocyte–macrophage cocultures (Hep-MΦ) were incubated without any supplement for 23 h (Control), with LPS and IFN for 23 h (LPS–IFN), and with 50 mM of ethanol for 5 h (ETOH) or with LPS–IFN for 18 h, and then with ethanol for the next 5 h (LPS–IFN + ETOH). *P0.05 compared with control hepatocyte cultures. Fig. 4. Open in new tabDownload slide Proposed mechanism of enhancement by macrophages of ethanol-induced oxidative stress in rat hepatocytes supplemented with LPS and IFN. Activated macrophages, by generating NO and prostaglandin, decrease hepatocyte production of NO, which protected them from ethanol-elicited toxicity. PG = prostaglandin; iNOS = inducible NO synthase; COX = cyclo-oxygenase. Fig. 4. Open in new tabDownload slide Proposed mechanism of enhancement by macrophages of ethanol-induced oxidative stress in rat hepatocytes supplemented with LPS and IFN. Activated macrophages, by generating NO and prostaglandin, decrease hepatocyte production of NO, which protected them from ethanol-elicited toxicity. PG = prostaglandin; iNOS = inducible NO synthase; COX = cyclo-oxygenase. * Author to whom correspondence should be addressed. This work was supported by IREB (Institut de Recherches Scientifiques sur les Boissons, Paris, France) (Contract number 97/22), by FRM (Fondation pour la Recherche Médicale, Paris, France) and Langlois fondation (Rennes, France). The authors also wish to thank Professor Jean-Pierre Anger for his technical assistance. REFERENCES Adachi, Y., Bradford, B. U., Gao, W., Bojes, H. K. and Thurman, R. G. ( 1994 ) Inactivation of Kupffer cells prevents early alcohol-induced liver injury. Hepatology 20 , 453 –460. Adams, D. O., Kao, K., Farb, R. and Pizzo, S. U. ( 1980 ) Effector mechanisms of cytolytically activated macrophages. Secretion of cytolytic factor by activated macrophages and its relation to secreted neutral protease. Journal of Immunology 124 , 293 –300. Bautista, A. P. and Spitzer, J. J. ( 1992 ) Acute ethanol intoxication stimulates superoxide anion production by in situ perfused rat liver. Hepatology 15 , 892 –898. Billiar, T. R., Curran, R. D., Stuehr, D. J., West, M. 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TI - ACTIVATED MACROPHAGES INCREASE THE SUSCEPTIBILITY OF RAT HEPATOCYTES TO ETHANOL-INDUCED OXIDATIVE STRESS: CONFLICTING EFFECTS OF NITRIC OXIDE
JF - Alcohol and Alcoholism
DO - 10.1093/alcalc/35.3.230
DA - 2000-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/activated-macrophages-increase-the-susceptibility-of-rat-hepatocytes-jbStwCT5H4
SP - 230
EP - 235
VL - 35
IS - 3
DP - DeepDyve
ER -