Laboratoire de Biologie Cellulaire et Végétale et INSERM U456, UFR des Sciences Pharmaceutiques et Biologiques, 2 av. Pr. Léon Bernard, 35043 Rennes Cedex, France
Received 12 July 1999; in revised form 25 October 1999; accepted 20 November 1999
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ABSTRACT |
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INTRODUCTION |
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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
(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.
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MATERIALS AND METHODS |
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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 SpragueDawley 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 x 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 x 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 x 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 x 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 NewmanKeuls tests were used to identify statistical significance for multiple comparisons. Differences were considered significant when P was < 0.05.
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RESULTS |
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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
).
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DISCUSSION |
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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.
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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.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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