Introduction

Summary Introduction Materials & Methods Results Discussion References

NO, the smallest known biologic mediator produced by mammalian cells, is involved in a diverse array of activities, including vasodilation, neurotransmission, and antimicrobial functions. NO is derived from L-arginine and can be produced by constitutively expressed NO synthase in cells such as endothelial cells (1), neurons (2), and cardiac myocytes (3) or by iNOS in cells such as macrophages (4), hepatocytes (5), and vascular smooth muscle cells (6). Because iNOS can produce a large amount of NO, its induction plays an influential role in cell death, tissue damage, and inflammation (7, 8). Maximal induction of iNOS depends on synergistically combining stimuli; the most effective stimuli vary with cell type. These stimulatory compounds include TNF-, IL-1, interferon-, and LPS. In hepatocytes, LPS, TNF-, or IL-1 alone did not significantly stimulate NO formation, but double or triple combinations of LPS with the cytokines induced a significantly higher NO production than any of the agents alone (9). Evidence, although inconclusive, suggests that the induction mechanism by LPS and combined cytokines might include protein kinase C, phospholipase A₂, or protein tyrosine kinases in hepatocytes (9). A previous study postulated that reactive oxygen species participate in transducing the signal, thereby leading to activation of iNOS by TNF- in rat hepatocytes (10). The mechanism underlying multiple cytokines or other agents inducing iNOS expression appears to be quite complicated and varies with different cell types.

Chronic inflammation has long been recognized as a risk factor for a variety of human cancers. Increasing evidence suggests that NO produced in inflamed tissues may contribute to the multistage carcinogenesis process (11). Epidemiological studies indicate that long term alcohol consumption can lead to hepatic injuries such as fatty liver, necrosis, inflammation, and fibrosis (12). However, whether NO plays a prominent role in initiating such alcoholic liver lesions remains unclear. In contrast, a previous study demonstrated that in vivo administration of NOS inhibitor increased the severity of liver injury, indicating that NO plays a protective role in alcohol liver disease (13). Taken together, although these results seem to be controversial, it implicates that NO is likely to be involved in ethanol-mediated modulation of the function of hepatic cells during alcohol intake. Another possible cause for increased NO production could be high levels of circulating endotoxin and TNF, which are markedly raised in acute alcoholic hepatitis (14). The aim of this study was to examine whether ethanol can stimulate NO production in primary rat hepatocytes in the presence of LPS and TNF- and the signal transduction pathway responsible for the response.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Reagents. Recombinant human TNF- (specific activity, 2.86 × 10⁷ units/mg) was supplied by R and D Systems (Minneapolis, MN). Recombinant rat interferon- was purchased from GIBCO BRL (Gaithersburg, MD). LPS, herbimycin A, staurosporine, NAC, and genistein were obtained from Sigma Chemical (St. Louis, MO). Ethanol was purchased from Merck (Darmstadt, Germany).

Animals and cell culture. Study animals were 1-month-old male Wistar rats that were fed ad libitum. Hepatocytes were isolated from Wistar rats by collagenase perfusion (15) and purified by differential centrifugation to produce cultures of 90% viability and 95% purity. Hepatocytes were plated onto rat tail collagen-coated culture plates (Corning, Palo Alto, CA) in Weymouth's medium supplemented with 0.5 mM L-arginine, 10⁶ M insulin, L-glutamine, penicillin, streptomycin, and 10% fetal calf serum. BNL Cl.2 mouse liver cell line was obtained from American Type Culture Collection (Rockville, MD). Cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 10 units/mL penicillin.

Measurement of NO₂. Accumulation of NO₂ in the medium was determined via the Greiss reagent (16) and was taken as an index of NO production. The measurement of NO₂ accumulation in the medium was determined by mixing 0.5 ml of medium with an equal volume of Greiss reagent (1% sulfanilamide/0.1% naphthylethylene diamine dihydrochloride/2% H₃PO₄) and incubation at room temperature for 15 min. Absorbance at 550 nm was measured in a spectrophotometer, and NO₂ was determined with NaNO₂ as a standard.

Western blot analysis. Treated hepatocytes were washed and pelleted in PBS. Cellular lysates were prepared as previously described (17). A 50-µg sample of each lysate was subjected to electrophoresis on 8% and 15% SDS-polyacrylamide gels for detecting iNOS and MAPK, respectively. The samples were then electroblotted onto nitrocellulose paper. After blocking, blots were incubated with anti-iNOS (Transduction Laboratories, Lexington, KY) or anti-MAPK (Santa Cruz Biochemicals, Santa Cruz, CA) antibody in PBS/Tween 20 for 1 hr followed by two washes (15 min each) in PBS/Tween 20 and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham, Arlington Heights, IL) for 30 min. After washing, the blots were incubated for 1 min with Western blotting reagent ECL, and chemiluminescence was detected by exposure of the filters to Kodak X-Omat films for 10 sec to 10 min. The specificity of this antibody to the 130-kDa iNOS was ensured by comparison with a standard macrophage iNOS.

RNA isolation and Northern blotting. The cDNA probe, derived from the mouse macrophage iNOS gene and having a length of ~1.8 kb, was purchased from Cayman Chemical (Ann Arbor, MI). The probe was random-primer labeled and used in a Northern blot analysis. Total RNA from the cells grown in cell culture dishes was isolated using 4 M guanidine isothiocyanate. Fifteen micrograms of RNA was used for Northern blotting as previously described (18).

pp60^c-src kinase reaction. Kinase assays were performed essentially as described by Gould and Hunter (19). After treatment, hepatocytes were rinsed with cold PBS, lysed in 1 ml of cold RIPA buffer, and clarified at 15,000 × g for 1 hr at 4°. Next, pp60^c-src was immunoprecipitated by incubating the lysate (300 µg) on ice for 1 hr with 1 µg of anti-c-Src antibody (Santa Cruz Biochemicals), followed by the addition of 40 µl of protein A-Sepharose beads (Santa Cruz Biochemicals) for an additional 4 hr, and immunocomplexes were collected by centrifugation at 4°. The immunocomplexes were washed three times with RIPA buffer and once with kinase buffer (20 mM HEPES, pH 7.0, 6 mM MgCl₂, 20 mM sodium orthovanadate) and suspended in 20 ml of kinase buffer containing 10 µCi of [-³²P]ATP (3000 Ci/mmol; Amersham) followed by the addition of 10 µg of acid-denatured enolase (Sigma). The reaction was incubated for 10 min at 30° and terminated by the addition of 2× SDS sample buffer (0.5 M Tris·Cl, pH 8.8, 0.4% SDS, 20% glycerol; 2% mercaptoethanol, 1% Bromphenol Blue). The proteins were resolved on SDS-10% polyacrylamide gels. Wet gels were exposed for 30-60 min at room temperature. Later, the gels were stained, and bands were excised and quantified by scintillation counting.

	Results

Summary Introduction Materials & Methods Results Discussion References

Ethanol potentiates TNF-- and LPS-stimulated NO₂ production. Primary rat hepatocytes and mouse BNL Cl.2 liver cell lines were treated with cytokines and LPS in combination to determine their production of NO₂ (i.e., a stable oxidation product of NO). The 24-hr treatment of both cell systems with cytokines such as IL-1 (5 ng/ml), TNF- (1 ng/ml), interferon- (10 ng/ml), or LPS (1 µg/ml) alone did not stimulate increased NO₂ level. Although double combinations of LPS with the cytokines induced a higher NO₂ production than any of the agents alone, we found the amount of NO₂ was still difficult to detect (0.02-0.03 absorbance units by the Greiss reaction) (data not shown). Interestingly, the simultaneous addition of ethanol (0.02-2 mM) to both cell systems significantly increased LPS/TNF--induced NO₂ production in a concentration-dependent manner (Fig. 1, A and B); in addition, the potentiating effect of ethanol in primary rat hepatocytes seemed more remarkable than that in mouse BNL Cl.2 cells. However, if the ethanol concentration exceeded 2 mM, enhancement of NO₂ production by ethanol would be decreased in both cells. A time course of NO₂ accumulation in hepatocytes after 1 mM ethanol plus LPS and TNF- is showed in Fig. 1C. The result clearly shows that the induction of NO₂ by ethanol/LPS/TNF- appeared to be time dependent. The ethanol-mediated enhancement of this effect was significantly blocked by the concomitant addition of 100 µM N-nitro-L-arginine to both cells (Table 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Enhancing effect of ethanol on TNF-/LPS-induced NO production in (A) primary rat hepatocytes or (B) mouse BNL Cl.2 liver cells. C, A time course of NO2 accumulation in rat hepatocytes after treatment with ethanol (1 mM)/TNF- (1 ng/ml)/LPS (1 µg/ml). Cells were incubated with TNF- (1 ng/ml)/LPS (1 µg/ml) and varying concentrations of ethanol (0-3 mM) for 24 hr or treated with ethanol (1 mM)/TNF- (1 ng/ml)/LPS (1 µg/ml) for 0-24 hr; then, the NO2 level was determined by Greiss reagent as described in Materials and Methods. Results are mean ± standard error of five separate experiments. *, p < 0.05 versus control group at the same time point. **, p < 0.01 versus control group.

View this table: [in this window] [in a new window]   TABLE 1 Effects of antioxidants and protein kinase inhibitors on TNF/LPS/ethanol-stimulated nitrite production in rat hepatocytes

Characterization of iNOS expression. In the experiment, Northern and Western blots were used to determine the amount of iNOS in primary rat hepatocytes treated with ethanol (1 mM)/TNF- (1 ng/ml)/LPS (1 µg/ml) or with any of three agents alone. Fig. 2 (top) reveals that exposure of hepatocytes to ethanol/TNF-/LPS for 4 hr resulted in an increase of iNOS mRNA, which was subsequently slightly increased in a time-dependent manner. Corroborating with the observation on mRNA increment, Western blotting analysis indicated that the level of iNOS protein was also initially detected after treatment for 4 hr and gradually increased thereafter (Fig. 2, bottom). No detectable signal of iNOS mRNA or protein appeared during treatment with TNF-, LPS, or ethanol alone (data not shown).

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Characterization of TNF-/LPS/ethanol-induced iNOS gene expression. Top, kinetics of iNOS mRNA induction by TNF- (1 ng/ml)/LPS (1 µg/ml)/ethanol (1 mM). Total RNA was extracted from 107 hepatocytes at different time points after the combined agents treatment as indicated in the figure. The signal of glyceraldehyde-3-phosphate dehydrogenase as an internal control to normalize the level of iNOS in respective sample. The number indicates the fold-change of iNOS transcripts of treated cells from control cells. Bottom, kinetics of immunoreactive iNOS protein expression by TNF-/LPS/ethanol. Total protein was collected from treated or untreated rat hepatocytes and assayed for iNOS protein by Western blotting with a specific anti-iNOS antibody as described in Materials and Methods. Similar findings were observed in three separate experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Top, effect of age on the level of iNOS protein in rat hepatocytes treated with TNF-/LPS/ethanol. Hepatocytes isolated from 1-month-old (lane 1), 2-month-old (lane 2), 3- month-old (lane 3), or 4-month-old (lane 4) rats were treated with TNF- (1 ng/ml)/LPS(1 µg/ml)/ethanol (1 mM) for 24 hr; Then, iNOS proteins were determined by Western blotting as described in Materials and Methods. Bottom, effect of age on the accumulation of NO2 in rat hepatocytes treated with TNF-/LPS/ethanol. The NO2 production from different ages of rat hepatocytes treated with TNF- (1 ng/ml)/LPS (1 µg/ml)/ethanol (1 mM) was determined by Greiss reagent as described in Material and Methods. Experimental conditions were identical to those in Fig. 3A. Values are mean ± standard error of triplicate samples from two to four experiments. *, p < 0.05 versus hepatocytes from young rats (1 and 2 months old).

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effect of passage number on iNOS expression in mouse BNL Cl.2 liver cell line by TNF-/LPS/ethanol. Top, Western blot analysis of iNOS. Total proteins were collected after 24-hr treatment of TNF- (1 ng/ml)/LPS (1 µg/ml)/ethanol (1 mM) and assayed for iNOS protein by immunoblotting. Lane 1, passages 12. Lane 2, passages 13. Lane 3, passages 14. Lane 4, passages 15. Lane 5, passages 16. Bottom, nitrite production in different passages of BNL Cl.2 liver cell lines treated with TNF-/LPS/ethanol. Mouse BNL Cl.2 cells were treated as described above. Media samples were collected and assayed for total NO2 and nitrate accumulation. Values are mean ± standard error of duplicate samples in two experiments. *, p < 0.05 versus early passages of BNL Cl.2 cells (passages 12 and 13).

Effects of signal transduction modulators on ethanol/TNF-/LPS-induced iNOS expression. To gain further insight into the mechanism by which ethanol potentiates TNF-/LPS-stimulated iNOS expression, we used various kinds of signal transduction modulators to examine such an event. Based on the hypothesis that the oxidation/reduction status may regulate TNF-- or ethanol-caused effects (10), hepatocyte cultures were treated with antioxidants NAC, ascorbic acid, and -tocopherol to examine their effects on NO₂ production induced by ethanol/TNF-/LPS. According to the results presented in Table 1, none of these antioxidants significantly inhibited ethanol/TNF-/LPS-stimulated NO₂ production. Consequently, several kinase inhibitors, including staurosporine (protein kinase C inhibitor), genistein (tyrosine kinase inhibitor), curcumin (nonspecific kinase inhibitor), and herbimycin A (Src-related kinase inhibitor), were used to examine their effects on the stimulation. Among them, only herbimycin A completely inhibited the NO₂ production induced by the combination of agents. The concentrations of modulators used herein were noncytotoxic to rat hepatocytes.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Top, effect of protein kinase inhibitors and antioxidants on iNOS protein expression by TNF-/LPS/ethanol in rat hepatocytes. Hepatocytes (5 × 106) were incubated in the absence (C) or presence of TNF- (1 ng/ml)/LPS (1 µg/ml)/ethanol (1 mM) () or plus various inhibitors such as herbimycin A (HER, 5 µM), NAC (5 mM), staurosporine (SAT, 1 µM), or curcumin (CCM, 10 µM) for 24 hr; then, the iNOS protein level of total cell lysates was determined by Western blotting. Bottom, inhibition of herbimycin A on TNF-/LPS/ethanol-induced iNOS mRNA expression. Hepatocytes were treated as follows. Lane 1, untreated. Lane 2, TNF- (1 ng/ml)/LPS (1 µg/ml)/ethanol (1 mM) for 6 hr. Lane 3, TNF-/LPS/ethanol plus herbimycin A (5 µM) for 6 hr. Lane 4, herbimycin A (5 µM) for 6 hr. Methods for total RNA isolation and Northern blotting are described in Materials and Methods. Number, fold-change of iNOS transcripts of treated cells from control cells.

Activation of pp60^c-src but not MAP kinase by ethanol/TNF-/LPS. Due to the potent and specific inhibitory action of herbimycin A on Src tyrosine kinase (20), whether the kinase is involved in the capability of ethanol to enhance TNF-/LPS-mediated iNOS expression in hepatocytes remains unclear. To address this issue, Src immune complex kinase assay was performed in rat hepatocytes exposed to ethanol/TNF-/LPS for varying periods of time. As Fig. 6 (top) depicts, the pp60^c-src kinase activity (phosphorylation of enolase) reached a maximal level at 1 hr after treatment of the agents. The kinase activity then gradually decreased and returned to a control level by 6 hr. As Fig. 6 (bottom) reveals, herbimycin A at the concentration that inhibited iNOS expression also prevented pp60^c-src kinase activation. Similar to the observation on iNOS induction, none of the other kinase modulators were capable of inhibiting pp60^c-src kinase. Table 2 shows that 1-5 µM herbimycin A exerted a dose-dependent inhibition on ethanol/TNF-/LPS-induced Src kinase activity and NO₂ accumulation but that genistein did not affect either event.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Top, kinetics of Src kinase activity induced by TNF-/LPS/ethanol in rat hepatocytes. Hepatocytes (5 × 106) were incubated in the presence of TNF- (1 ng/ml)/LPS (1 µg/ml)/ethanol (1 mM) for different time points as indicated in this figure. Equal amount (100 µg) of cell lysates were immunoprecipitated with anti-c-Src antibody; subsequently, the immunocomplexes were assayed for c-Src kinase activity using enolase as a substrate. The fold increase in c-Src kinase activity was quantified by scintillation counting of the enolase band. The indicated values are the averages of three experiments. Bars, mean ± standard error. Bottom, herbimycin A inhibits TNF-/LPS/ethanol-elicited c-Src activity in rat hepatocytes. Hepatocytes were concomitantly treated with TNF-/LPS/ethanol () and various modulators, such as herbimycin A (HER, 5 µM), NAC (5 mM), staurosporine (SAT, 1 µM), curcumin (CCM, 10 µM), or genistein (GEN, 20 µM) for 1 hr. Methods of immunoprecipitation and c-Src kinase assay are described in Materials and Methods.

View this table: [in this window] [in a new window]   TABLE 2 Effects of herbumycin A and genistein on ethanol/TNF/LPS-induced Src activity and nitrite production

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effect of TNF-/LPS/ethanol on MAPK in rat hepatocytes. Hepatocytes (5 × 106) were treated with TNF-/LPS/ethanol for various time points or treated with hepatocyte growth factor (10 ng/ml) for 1 hr. The cell lysates were subjected to electrophoresis and electrotransferred to nitrocellulose papers. The p44mapk protein on blots was detected as described in Materials and Methods. Tick marks, positions of the hyperphosphorylated (bottom) and hypophosphorylated (top) p44mapk.

	Discussion

Summary Introduction Materials & Methods Results Discussion References

This study clearly demonstrates that a low ethanol concentration potentiates TNF-- plus LPS-stimulated NO₂ production and iNOS expression in both primary rat hepatocytes and mouse liver cell line. Nitrite level in the two hepatic cell systems is not detectable when treating with TNF- alone. In contrast, another study (10) indicated that treatment of rat hepatocytes with TNF- can stimulate a significant increase of NO₂ level. Such a discrepancy may be due to the cytokine concentration (1 ng/ml) used, which is markedly lower than that used in others (1 µg/ml). However, the concentration used in this study is closer to the physiological condition.

This study shows that a sustained level of iNOS mRNA was detected at 24 hr after treatment with TNF-/LPS/ethanol. However, previous studies have shown that treatment of rat hepatocytes with various cytokines plus LPS induced iNOS mRNA with a peak at 6-8 hr and then declined slightly by 24 hr (22, 23). This discrepancy is possibly due to the synergizing effect of ethanol to produce prolonged induction of iNOS mRNA. Several agents, such as cAMP, cycloheximide, and 12-O-tetradecanoylphorbol-13-acetate, have been found to affect the stability of iNOS mRNA that led to a synergetic induction of iNOS mRNA (24, 25). This raises a possibility that the potentiating effect of ethanol on iNOS mRNA induction may mediated through an affect on its stability. Although ethanol enhances the expression of iNOS, its potentiating effects on TNF-/LPS-stimulated NO₂ production (~2-4-fold) is lower than that on iNOS mRNA and protein levels (~4-7-fold). A possibility arises that released NO moiety into a culture medium may be neutralized by cellular constituents or nutrients, leading to an underestimation of the actual NO₂ level. Our current finding that ethanol can potentiate iNOS expression in cultured rat hepatocytes contrasts with a recent study in which it was demonstrates that the in vivo administration of ethanol attenuated iNOS expression in a rat liver (26). Reasons for the contradictory results of the two studies remain unknown. However, several possible explanations are available. First, the ethanol concentration used in vivo is higher than that in our in vitro systems. As mentioned earlier, our results indicate that the potentiating effect of ethanol requires a concentration ranging from 0.02 to 2 mM because the addition of an ethanol concentration of >2 mM decreases the NO₂ level. This finding suggests that ethanol, under different concentrations, possibly exerts a complicated effect in regulating NO release. Second, the rats used in in vivo study were older than the rats used in the current study. In addition, according to our results, the potentiating effect of ethanol fails when hepatocytes are obtained from rats >2 months old. This finding clearly suggests that the potentiation of ethanol on iNOS expression is critically determined by the age of the animals. Finally, the in vivo administration of ethanol may result in its biotransformation in the liver to secondary metabolites that can inhibit iNOS induction, which are not formed in our in vitro culture systems.

Our current results reveal that the induction of iNOS mRNA by TNF-/LPS/ethanol in rat hepatocytes is age dependent. It is well documented that many physiological functions or signaling pathways in rat hepatocytes could be affected during the aging process. For example, the expression of heat shock protein 70, epidermal growth factor-stimulated DNA synthesis, or insulin-induced glucosyl-phosphatidyl-inositol signaling could be reduced or attenuated in rat hepatocytes from old rats (27-29). The biological role of NO₂ induced by TNF-/LPS/ethanol in hepatocytes from young rats is unclear. However, previous studies have suggested that adaptive NO synthesis in the hepatic cells is beneficial or protective for the liver in the presence of toxic insults, such as LPS or ethanol (13, 30). In this context, we thus propose that the preferential induction of NO₂ in hepatocytes from young rats may implicate the more actively protective mechanism existing in these cells.

The nonreceptor tyrosine kinase pp60^c-src plays a critical role in modulating cell signals in response to several growth factor, such as platelet-derived growth factor (31), epidermal growth factor (32), and interleukin-3 (33). In addition to growth factors, oxidative stress initiated by ultraviolet irradiation has been found to potentially activate Src tyrosine kinase, thereby leading to trigger of activator protein-1 activity (34). However, this is the first time it has been demonstrated that activation of Src tyrosine kinase is critically involved in inducing iNOS through ethanol/TNF-/LPS treatment. Because treatment of cells with these agents alone did not activate Src kinase, the diverse signals triggered by these single agents are likely a prerequisite to interact and finally converge on Src kinase. As well documented, exposure to ethanol, TNF-, or LPS alone can produce reactive oxygen species in various cell or animal systems (35, 36). Nevertheless, in this study, we failed to decrease both Src kinase activity (Fig. 6B) and iNOS expression (Fig. 5A) by using some well-known antioxidants. This suggests that reactive oxygen species do not contribute to Src-associated iNOS induction by ethanol/TNF-/LPS. A more recent study indicated that iNOS gene expression is regulated differently in response to specific cytokines in each cell type (21). For instance, IL-1 induces iNOS in cardiac myocytes and endothelial cells, whereas interferon- induces iNOS in myocytes but not in endothelial cells.

Activation of MAPK is importantly attributed to iNOS induction by IL-1 or interferon- in its respective cell context. In hepatocytes, we did not find an alteration in MAPK activity after exposure to ethanol/TNF-/LPS (Fig. 7).

In conclusion, this study demonstrates that clinically relevant concentrations of ethanol modulate the expression of iNOS in primary hepatocytes by potentiating the TNF-- and LPS-stimulated elevation of iNOS mRNA and protein. Further evidence suggests that Src tyrosine kinase plays an essential role in regulating iNOS expression by ethanol/TNF-/LPS, but the actual mechanisms by which these combined agents activate Src kinase remain unknown and require further study.