Modulation of endotoxin stimulated interleukin-6 production in monocytes and Kupffer cells by S-adenosylmethionine (SAMe)

www.elsevier.com/locate/issn/10434666 Cytokine 28 (2004) 214e223

Modulation of endotoxin stimulated interleukin-6 production in monocytes and Kupffer cells by S-adenosylmethionine (SAMe) Zhenyuan Songa, Theresa Chenb, Ion V. Deaciuca, Silvia Uriartea, Daniell Hilla, Shirish Barvea,b, Craig J. McClaina,b,c,* a Department of Medicine, University of Louisville School of Medicine, Louisville, KY 40292, United States Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, United States c Department of Veterans Affairs Medical Center, Louisville, KY 40292, United States

b

Received 22 August 2003; received in revised form 22 June 2004; accepted 2 August 2004

Abstract Interleukin-6 (IL-6) is a multifunctional cytokine having primarily anti-apoptotic and anti-inflammatory effects. Recent reports have documented that IL-6 plays a key role in liver regeneration. Intracellular deficiency of S-adenosylmethionine (SAMe) is a hallmark of toxin-induced liver injury. Although the administration of exogenous SAMe attenuates liver injury, its mechanisms of action are not fully understood. Here we investigated the effects of exogenous SAMe on IL-6 production in monocytes and Kupffer cells. RAW 264.7 cells, a murine monocyte cell line, and isolated rat Kupffer cells were stimulated with lipopolysaccharide (LPS) in the absence or presence of exogenous SAMe. IL-6 production was assayed by ELISA and intracellular SAMe concentrations were measured by HPLC. We have found that exogenous SAMe administration enhanced both IL-6 protein production and gene expression in LPS-stimulated monocytes and Kupffer cells. Cycloleucine (CL), an inhibitor for extrahepatic methionine adenosyltransferases (MAT), inhibited LPS-stimulated IL-6 production. The enhancement of LPS-stimulated IL-6 production by SAMe was inhibited by ZM241385, a specific antagonist of adenosine (A2) receptor. Our results demonstrate that SAMe administration may exert its anti-inflammatory and hepatoprotective effects, at least in part, by enhancing LPS-stimulated IL-6 production. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Adenosine; S-adenosylmethionine; Glutathione; IL-6; Liver disease

1. Introduction S-adenosylmethionine (SAM, SAMe, or AdoMet) is produced from methionine and ATP by methionine adenosyltransferase (MAT). SAMe is a key intermediate in the hepatic transsulfuration pathway and serves as a precursor for glutathione (GSH) as well as a methyl * Corresponding author. Department of Internal Medicine, University of Louisville Medical Center, 550 South Jackson Street, ACB 3rd Floor, Louisville, KY 40292, United States. Tel.: C1 502 562 3899; fax: C1 502 852 0846. E-mail address: [email protected] (C.J. McClain). 1043-4666/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2004.08.004

donor in most transmethylation reactions [1]. Clinical studies have reported that administration of stable salts of SAMe has beneficial effects on many hepatic disorders ranging from cholestasis to alcoholic liver disease [2e7]. Although the mechanisms of its action are not fully established, one possibility is its ability to regulate functions of the immune system by modulating production of cytokines by endotoxin (LPS) stimulated monocytes/macrophages. It has recently been shown that SAMe supplementation to RAW 264.7, a murine monocyte cell line, decreases the amount of TNFa released in the conditioned medium, and steady-state mRNA concentrations following LPS stimulation [8].

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2. Results 2.1. Effects of SAMe and cycloleucine (CL) treatment on intracellular SAMe concentration The effects of exogenous SAMe and cycloleucine treatments on intracellular concentrations of SAMe are shown in Fig. 1. The basal concentration of SAMe in RAW cells was 90 pmol/mg protein and remained

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In addition, we recently reported that exogenous SAMe supplementation to RAW 264.7 cells increased LPS-stimulated protein production and gene expression of IL-10, an important anti-inflammatory cytokine [9]. Interleukin-6 (IL-6) is a multifunctional cytokine produced by many different cell types, but the main sources in vivo are stimulated monocytes/macrophages, fibroblasts and vascular endothelial cells [10]. IL-6 has been classified as both a pro- and anti-inflammatory cytokine. However, the current view is that IL-6 has primarily anti-apoptotic and anti-inflammatory effects. IL-6 has been shown to render protection in animal models of fulminant hepatic failure through mechanisms involving hepatocyte apoptosis [11]. Pretreatment with IL-6 protected the livers of both normal rats and IL-6deficient mice from warm ischemia/reperfusion injury [12]. IL-6 induced hepatoprotection of steatotic liver isografts via preventing sinusoidal endothelial cell necroapoptosis and consequent amelioration of hepatic microcirculation, and protecting against hepatocyte death [13]. IL-6 pretreatment also protected mice from endotoxin shock induced by either Staphylococcus aureus endotoxin B following D-galactosamine sensitization [14], or by challenge with a lethal dose of LPS [15]. Recent studies using IL-6 null mice demonstrated that IL-6ÿ/ÿ mice are significantly more sensitive than IL-6C/C mice to apoptotic liver injury induced by the Fas agonist, Jo-2 mAb. Pretreatment of both wild-type and IL-6ÿ/ÿ mice with recombinant IL-6 significantly reduced the level of Fas agonist-induced apoptosis and increased survival rates compared to untreated controls [16]. In addition to being an anti-apoptotic factor, recent reports have documented that IL-6 is a key factor in liver regeneration. IL-6 knockout mice exhibit impaired liver regeneration, which is reversed by IL-6. The impaired liver regeneration in TNF receptor1 knockout mice can also be reversed by exogenous IL-6 [17]. Although the functions of SAMe in liver protection and immune modulation have been widely investigated, the effect(s) of exogenous administration of SAMe on IL-6 production and gene expression have not been examined. The purpose of the current study is to evaluate the effects of SAMe on both IL-6 protein synthesis and gene expression in LPS-stimulated monocytes and Kupffer cells.

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Fig. 1. Changes in intracellular SAMe concentrations in SAMe or CLtreated cells. RAW cells were plated at a density of 0.5 ! 106 cells/ml and incubated overnight, then cells were treated with medium alone (UT), 1 mM SAMe or 40 mM cycloleucine (CL) dissolved in the media. Cells were harvested at the indicated time points and intracellular SAMe concentrations were quantified by HPLC as described in Section 4. Values represent means G S.D. from three separate experiments. aP ! 0.05 compared to corresponding control cells.

unchanged during the culture conditions used in these experiments. Treatment with 1 mM exogenous SAMe resulted in a 3-fold elevation of intracellular SAMe concentrations within 2 h of exposure. These levels remained elevated over 8 h. Cycloleucine is an inhibitor of MAT2A, which is expressed in all extrahepatic tissues including cells of the immune system. Treatment with 40 mM exogenous CL depleted intracellular SAMe concentrations within 2 h of exposure. 2.2. Effects of SAMe treatment on the release of IL-6 by LPS-stimulated RAW cells The effect of exogenous SAMe on IL-6 production was determined by pretreating RAW cells with SAMe for 2 h and then stimulating with LPS. After a number of preliminary studies in which the pretreatment period with SAMe was varied from a few minutes to overnight, we designed this protocol to optimize the changes in LPS-stimulated IL-6 release by RAW cells without affecting their viability. Fig. 2A summarizes the doseeresponse of SAMe-enhanced IL-6 release into the conditioned medium. Cells treated with 250 mM SAMe demonstrated a significant increase in IL-6 production following stimulation by LPS. When the cells were pretreated with 1 mM SAMe for 2 h, IL-6 production was increased to almost 2.8 times of that of cells treated by LPS alone by 18 h after LPS stimulation. IL-6 production was very low in either untreated cells or those treated with 1 mM SAMe without LPS. The viability of these cells, measured by the conversion of MTT to formazan, was unaffected by concentrations of

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pendent increase during the 24 h culture period (from 54 G 7 pg/ml in 2 h to 1611.9 G 115.4 pg/ml in 24 h) after LPS stimulation. Compared to control cells, SAMe-treated cells produced higher level of IL-6 by 4 h after LPS stimulation. By 24 h after LPS stimulation, IL-6 production by SAMe-treated cells reached almost 1.5-fold of that in control cells (1611.9 G 115.4 pg/ml in control cells versus 2527.7 G 289.7 pg/ml in SAMetreated cells).

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To determine the effect of SAMe on IL-6 production from Kupffer cells stimulated by LPS, Kupffer cells were isolated from the livers of normal rats and incubated with different doses of SAMe for 2 h followed by stimulation with LPS (300 ng/ml) for 20 h. The medium was analyzed by the rat IL-6 ELISA kit revealing that pretreatment of Kupffer cells with SAMe significantly induced the LPS-stimulated IL-6 production (Fig. 3).

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Time after LPS addition (h) Fig. 2. Effects of SAMe treatment on the release of IL-6 by LPSstimulated RAW cells. (A) Dose-dependent effect of SAMe on LPSstimulated IL-6 release. Cells were plated as described in the caption of Fig. 1. SAMe was added to the culture medium at various concentrations; after 2 h incubation, LPS was added to the culture medium to a final concentration of 100 ng/ml. Cells were incubated in the continued presence of SAMe and LPS for a further 20 h. Conditioned media were collected and IL-6 was quantified by ELISA as described in Section 4. Values are expressed as folds of LPS alone treatment and represent means G S.D. from three separate experiments. (B) Time course of IL-6 release by LPS-stimulated RAW cells in the presence and absence of SAMe. Cells were plated as described in the caption of Fig. 1, then pretreated with 1 mM SAMe dissolved in medium, or an equal volume of medium alone for 2 h; they were then stimulated with 100 ng/ml LPS. Conditioned media were collected at the indicated times and analyzed for IL-6 concentration by ELISA. Values represent means G S.D. from three separate experiments. a P ! 0.05 compared to corresponding untreated cells. bP ! 0.05 compared to cells treated with LPS alone.

SAMe up to 1 mM (more than 90% viability). Treatment of cells with higher concentrations of SAMe (e.g. R3 mM), however, significantly lowered cellular viability to approximately 70% (data not shown). Fig. 2B shows the the time course changes of LPSstimulated IL-6 release in the absence or presence of 1 mM SAMe. In the control cells (no SAMe), IL-6 levels in the conditioned medium demonstrated a time-de-

The effect of CL on LPS-stimulated IL-6 production is shown in Fig. 4. In comparison to control cells, CL pretreatment for 2 h significantly decreased IL-6 pro-

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+ LPS (300ng/ml) Fig. 3. Effects of SAMe treatment on IL-6 production by LPSstimulated Kupffer cells. Kupffer cells were isolated from rats as described in Section 4 and plated at a density of 1.2 ! 106 cells/ml and incubated overnight, then SAMe was added to the culture medium at various concentrations; after 2 h incubation, LPS was added to the culture medium to a final concentration of 300 ng/ml. Cells were incubated in the continued presence of SAMe and LPS for a further 20 h. Conditioned media were collected and IL-6 was quantified by ELISA as described in Section 4. Values are expressed as folds of LPS alone treatment and represent means G S.D. from three separate experiments.

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duction 18 h after LPS stimulation. SAMe addition prior to CL treatment not only caused a recovery of the IL-6 level, but also resulted in a higher level of IL-6 production when compared to cells treated with LPS alone.

2.5. Effects of exogenous SAMe on LPS-Induced IL-6 gene expression RT-PCR was performed to determine whether the effects of SAMe on IL-6 expression could be attributed to changes in IL-6 mRNA levels following exposure to LPS. IL-6 mRNA could not be detected in either control or SAMe treatment alone; however, it was detected 4 h after stimulation in both LPS alone and SAMe C LPS cells (Fig. 5A). Cells treated with SAMe C LPS expressed higher levels of IL-6 mRNA than cells treated with LPS alone at all time points after LPS stimulation (4e8 h) (Fig. 5B).

2.7. Effects of N-acetylcysteine (NAC) on LPSstimulated IL-6 production Previous studies have reported that alteration of intracellular redox status, particularly changes in GSH levels, may modulate LPS-stimulated cytokine production from monocytes and Kupffer cells [18]. To determine whether intracellular GSH elevation induced by SAMe treatment contributes to increased IL-6 production in our study, we pretreated RAW cells with different concentrations of NAC, a widely used GSH precursor, for 2 h followed by LPS stimulation. Fig. 7 showed the results of IL-6 levels 20 h after LPS stimulation. NAC lowered LPS-stimulated IL-6 production in a dose-dependent manner. Pretreatment of RAW cells with 10 mM NAC caused about 30% decrease in IL-6 production in comparison to cells treated with LPS alone and 50 mM NAC almost abolished IL-6 production by 20 h after LPS stimulation. 2.8. Effects of anti-TNF antibody on LPSstimulated IL-6 production

2.6. Effects of SAMe and CL on intracellular GSH levels Time-dependent changes of intracellular GSH concentrations following SAMe and CL treatments were monitored and results were shown in Fig. 6. The basal concentration of GSH in RAW cells was approximately 7.5 G 0.8 nmol/mg protein and remained unchanged during the culture conditions used in these experiments. Treatment with 1 mM exogenous SAMe resulted in a minor, but significant elevation of intracellular GSH concentrations within 4 h of exposure and remained elevated over 24 h. Treatment with 40 mM CL did not significantly lower intracellular GSH levels until 8 h; although GSH level in CL treatment rebounded by 24 h, it did not return to the normal level.

The effects of anti-TNF on IL-6 production by LPSstimulated RAW cells are shown in Fig. 8. Although anti-TNF antibody at a dose of 10 mg/ml neutralized TNF produced by LPS-stimulated RAW cells pretreated with 1 mM SAMe (Fig. 8A), it did not affect SAMeinduced enhancement of IL-6 production by LPS (Fig. 8B). 2.9. Effects of selective antagonist of adenosine receptor A2 on LPS-stimulated IL-6 production SAMe is precursor of adenosine. To determine whether or not the adenosine (A2) receptor (AR), a subtype of ARs mainly expressed on monocytes and macrophages, was involved in the enhancement of

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Fig. 5. RT-PCR for IL-6 mRNA in the presence and absence of SAMe. (A) After 2 h of pretreatment with 1 mM SAMe dissolved in the media, RAW cells were stimulated with 100 ng/ml LPS in the continued presence of treatments. Cells were collected at the indicated times after exposure to LPS, the RNA was isolated for RT-PCR assay as described in Section 4. (B) Arbitrary densitometric units. This is a representative example of three separate experiments.

antagonism on A2 receptor, RAW cells were pretreated with adenosine (1 mM) for 2 h, then stimulated with LPS in the absence or presence of ZM241385. Our results show that pretreatment with adenosine resulted in enhanced IL-6 production from LPS-stimulated monocytes, which was similar to that with SAMe pretreatment. Moreover, ZM241385 completely inhibited this enhancement.

SAMe on LPS-stimulated IL-6 production, ZM241385, a selective antagonist of A2 receptor, was added to the media at a dose of 30 mM before LPS stimulation and results are shown in Fig. 9. ZM241385 itself had no effect on LPS-stimulated IL-6 production from monocytes; however, it inhibited enhancement of IL-6 production resulting from SAMe pretreatment. In order to verify that this inhibition by ZM241385 was due to its

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3. Discussion S-adenosylmethionine (SAMe) is the first product in methionine metabolism pathway in the liver via methionine adenosyltransferase (MAT)-catalyzed con-

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Fig. 9. Effects of selective antagonist of adenosine (A2) receptor on LPS-stimulated IL-6 production. Cells were plated as described in the caption of Fig. 1, then pretreated with medium alone, 1 mM SAMe, or 1 mM adenosine (ADO); ZM241385 (ZM) was added to the media at a dose of 30 mM at 30 min before LPS (100 ng/ml) stimulation. Conditioned media were collected 20 h later and analyzed for IL-6 by ELISA. Values in bars that do not share a letter differ (P ! 0.05).

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Fig. 8. Effects of anti-TNF-a antibody on LPS-stimulated IL-6 production. Cells were plated as described in the caption of Fig. 1, then pretreated with medium alone, 1 mM SAMe, or 1 mM SAMe C 10 mg/ml anti-TNF-a antibody for 2 h; they were then stimulated with 100 ng/ml LPS. Conditioned media were collected 20 h later and analyzed for both TNF (A) and IL-6 (B) by ELISA. Values in bars that do not share a letter differ (P ! 0.05).

version (Fig. 10). Although its mechanism(s) of action remain unclear, SAMe has been increasingly utilized for the treatment of liver diseases. In animal models of alcoholic liver disease and carbon tetrachloride hepatotoxicity, exogenous administration of SAMe prevented the depletion of SAMe and GSH levels and significantly ameliorated liver injury, including fibrosis [3e7]. SAMe has also been used to prevent hepatic carcinogenesis [19,20]. It has been proposed that SAMe’s protective effect involves several pathways, including increasing GSH levels, changing DNA methylation status and maintaining mitochondrial GSH stores [21e23]. Increasing evidence suggests that SAMe also plays a critical role in the regulation of the immune function by modulating cytokine production/secretion. Our previous studies have shown that exogenous SAMe administration can down-regulate release of TNF-a [8] and up-regulate IL-10 production [9] from RAW cells. In the present study, we investigated the effects of SAMe on another critical cytokine, interleukin-6, in LPSstimulated RAW 264.7 cells and isolated rat Kupffer cells. Our results showed that exogenous SAMeenhanced LPS-induced IL-6 production and gene expression. Initially, IL-6 was primarily considered to be a proinflammatory cytokine. However, IL-6 more recently has been shown to exhibit anti-inflammatory and antiapoptotic properties. Unlike IL-1b and TNF, IL-6 does not up-regulate major inflammatory mediators such as nitric oxide or matrix metalloproteinases. Rather, IL-6

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Fig. 10. Simplified hepatic methionine metabolism. 1. Methionine adenosyltransferase (MAT); 2. Transmethylation reactions; 3. Sadenosylhomocysteine hydrolase; 4. Cystathionine b-synthase (CBS); 5. g-Cystathionase; 6. g-Glutamylcysteine synthetase; 7. GSH synthetase.

appears to be the primary inducer of the hepatocytederived acute-phase proteins, many of which have antiinflammatory properties. IL-6 directly inhibits the expression of TNF and IL-1b and is a potent inducer of IL-1ra, which exerts anti-inflammatory activity by blocking IL-1b receptors [24]. Knockout of Gp-130, the common signal transducer of IL-6 family cytokines, in nonparenchymal liver cells and not hepatocytes resulted in fibrosis progression [25]. Interleukin-6ÿ/ÿ mice express lower levels of Bcl-2, Bcl-xL and FLIP and are more sensitive than IL-6C/C mice to FasL-induced apoptosis. Pretreatment with IL-6 significantly reduces the level of Fas agonist-induced apoptosis and increases survival rates [16]. Moreover, IL-6 is a key factor in liver regeneration. Taub’s laboratory showed that IL-6 knockout mice exhibit impaired liver regeneration, which could be reversed by IL-6. Furthermore, the impaired liver regeneration in TNFR1 knockout mice could also be reversed by IL-6 [17]. The effects of SAMe on protein production and gene expression of IL-6 in vitro has not been evaluated. Our study showed for the first time that SAMe increased IL6 production in LPS-stimulated RAW cells and Kupffer cells. To establish the link between the intracellular level of SAMe and IL-6 production, time course changes of intracellular SAMe concentration after administration of exogenous SAMe were monitored by a well-established HPLC assay. Our results showed that treatment with 1 mM exogenous SAMe resulted in a 3-fold elevation of intracellular SAMe concentrations within 2 h of exposure. These levels remained elevated over 8 h. Furthermore, our results showed that cycloleucine, an inhibitor of MAT2A, which completely depleted in-

tracellular SAMe levels at a dose of 40 mM, decreased LPS-stimulated IL-6 production, and this decrease was blocked by the addition of SAMe. Overall, our study suggests that there is a direct link between intracellular SAMe level and IL-6 production in LPS-stimulated RAW cells and isolated rats Kupffer cells. At present, the mechanisms by which SAMe modulates protein production and gene expression of cytokines in monocytes are not fully understood. In the current study, we explored possible mechanisms involving the modulation of SAMe to IL-6 production in LPS-stimulated RAW cells (Fig. 10). Based on our finding that SAMe administration caused a modest but significant increase in the intracellular GSH level within 4 h and cycloleucine decreased GSH within 8 h, we initially investigated whether or not the changes in intracellular GSH level resulted in increased IL-6 production. By treating RAW cells with increasing doses of N-acetylcysteine (NAC), a GSH prodrug, we excluded that possibility because NAC actually decreased IL-6 production. Additionally, it is well documented that besides LPS, TNF alone can induce IL-6 production from monocytes/macrophages [26]. Therefore, we investigated if the increased IL-6 production by SAMe administration was secondary to LPS-stimulated TNF production. By using anti-TNF antibody in the medium, our results demonstrate that neutralization of TNF to the basal level does not affect enhancement of IL-6 production by SAMe in LPSstimulated RAW cells. Taken together, the findings of present study indicate that the enhancement of IL-6 production in LPS-stimulated monocytes by SAMe pretreatment is independent of observed increased intracellular GSH level and LPS-induced TNF production. Both in vivo and in vitro studies have demonstrated that adenosine exhibits potent anti-inflammatory properties that are mediated through A2 receptor, a subtype of adenosine receptors expressed on the cells of immune system [27]. Adenosine and adenosine (A2) receptor agonists inhibited LPS-induced TNF-a [28] production and enhanced IL-10 [29] secretion by human monocytes. Our study showed that adenosine increased LPSstimulated IL-6 production from RAW 264.7 cells and the enhancement was inhibited by ZM241385, a selective antagonist of A2 adenosine receptor. Moreover, the enhancement of LPS-stimulated IL-6 production by SAMe was also completely inhibited by ZM241385, suggesting that modulation of IL-6 production by SAMe treatment in monocytes was A2 receptor-mediated. Considering the fact that ZM241385 was added to the media at 90 min after SAMe treatment in our in vitro model, it seems unlikely that SAMe binds to the A2 receptor directly as a receptor agonist. SAMe could act as a precursor of adenosine, whose intracellular level is increased by exogenous SAMe. However, the

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exact mechanisms and sequence of events are unclear and further research is required. In conclusion, this research demonstrates that intracellular SAMe concentrations critically regulate LPSstimulated IL-6 production in monocytes/Kupffer cells, and that SAMe elevates protein synthesis and gene expression of IL-6 in LPS-stimulated RAW cells. Considering the anti-apoptotic function of IL-6 and its critical role in liver regeneration, the results of this study suggest that enhanced LPS-stimulated IL-6 production may be one of the mechanisms by which SAMe exerts therapeutic efficiency in liver diseases.

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Difco Laboratories (Detroit, MI). Before use, LPS was suspended in sterile, pyrogen-free water, sonicated and diluted with Hanks’ balanced salt solution. Penicillin, streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), trypsin, fetal bovine serum and Trizol reagent were purchased from Invitrogen Corporation (Grand Island, NY); 24-well and 96-well plates were from Corning Inc. (Corning, NY) and murine IL-6 and TNFa ELISA kits were from Biosource International (Camarillo, CA). Purified hamster anti-mouse/rat TNF antibody, H-89, was from Calbiochem (San Diego, CA). All other reagents were of the highest purity available and unless indicated otherwise were obtained from Sigma (St. Louis, MO, U.S.A.).

4. Materials and methods 4.4. MTT assay 4.1. Cells RAW 264.7 murine monocytes were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured in DMEM containing 10% (v/v) fetal bovine serum, 2 mM glutamine, 5 U/ml penicillin, and 50 mg/ml streptomycin at 37  C in a humidified O2/CO2 (19:1) atmosphere. 4.2. Kupffer cells isolation and culture Kupffer cells were isolated as described earlier [30]. Briefly, the liver was perfused through the portal vein with Ca2C, Mg2C-free Hanks bicarbonate buffer, continuously gassed with O2:CO2, at 37  C for 3e4 min at a flow rate of 25 ml/min. Then the perfusion was switched to a re-circulating system with the perfusion medium (100 ml) as above but containing CaCl2 (4 mM) and 0.05% collagenase (type IV). The perfusion was continued for another 4e6 min. Parenchymal cells were pelleted by centrifugation at 50 ! g for 3 min. Kupffer cells were isolated by centrifugal elutriation from the primary, post-hepatocyte sedimentation supernatant. The latter was centrifuged at 700 ! g for 10 min, the pellet was resuspneded in RPMI medium and used for elutriation. Kupffer cells isolated by elutriation have a viability of no less than 90% and the contamination by other cells is very low (1e2%) if any. Isolated Kupffer cells were cultured in RPMI containing 10% (v/v) fetal bovine serum, 2 mM glutamine, 5 U/ml penicillin, and 50 mg/ml streptomycin at 37  C in a humidified O2/CO2 (19:1) atmosphere. 4.3. Materials SAMe, as its 1,4-butanedisulphonate salt, was provided by Dr. Robert O’Brian (Knoll Pharmaceuticals Co., Piscataway, NJ, U.S.A) and Dr. G. Stramentionoli (Knoll Farmaceutici, Milan, Italy). Lipopolysaccharide (LPS; Escherichia coli O111:B4) was purchased from

Cell viability was assessed by examining cell number with the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treatments, cells were washed twice with PBS; then cell culture medium was removed, and serum-free medium containing 1 mg/ ml MTT was added to the cells. After a 2 h incubation, 100 ml lysis buffer containing 20% SDS and 50% N,Ndimethyformamide (DMF) was added and incubated at 37  C overnight. The OD values were read at 570 nm. 4.5. HPLC assay for intracellular SAMe The intracellular concentrations of S-adenosylmethionine (SAMe) and S-adenosylhomocysteine (SAH) were assayed by reverse-phase HPLC with deprotenized extracts of cells by a modified method of Merali et al. [31]. Cell pellets were mixed with 0.25 ml of 4% metaphosphoric acid (MPA) and centrifuged at 10,000 ! g for 2 min. The supernatants were collected for HPLC analysis. The HPLC system was equipped with a Waters 501 pump, a manual injector, a 5-mm Hypersil C18 reverse-phase column (250 ! 4.6 mm). The mobile phase consisted of 40 mM ammonium phosphate, 8 mM heptane sulfonic acid (ion-pairing reagent) and 6% acetonitrile (pH 5.0) and were run isocratically at a constant rate of 1.0 ml/min. SAMe and SAH were detected using a Waters 740 detector at 254 nm. Standard solutions of SAMe and SAH were prepared in 4% MPA. An internal standard, S-adenosylethionine (SAE), was added to all samples and standard solutions to a concentration of 100 nmol/ml. Protein concentrations were measured by protein assay kit from Bio-Rad in accordance with the manufacturer’s instructions. 4.6. Intracellular GSH assay by HPLC Reduced glutathione (GSH), oxidized glutathione (GSSG) and cysteine in whole blood, cytosol and mitochondria were quantified by HPLC with

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electrochemical detection according to the method of Richie and Lang [32], with slight modifications. In brief, 20-ml samples were injected onto a reversed-phase C18 column (Val-U-Pak HP, fully endcapped ODS, 5 mm, 250 ! 4.6 mm; ChromTech Inc.). The mobile phase, which consisted of a solution of 0.1 M monochloroacetic acid and 2 mM heptanesulfonic acid at pH 2.8 (98%) and acetonitrile (2%), was delivered at a flow rate of 1 ml/min. The compounds were detected in the eluant with a Bioanalytical Systems dual LC4B amperometric detector, using two Au/Hg electrodes in series with potentials of ÿ1.2 V and C0.15 V for the upstream and downstream electrodes, respectively. Standard curves for the analytes were plotted as peak area versus concentration of the analyte. 4.7. ELISA assay for IL-6 and TNF-a Interleukin-6 and TNF-a in conditioned medium were quantified using ELISA kits in accordance with the manufacturer’s instructions. The detection limitation for IL-6 and TNF-a are 4.0, 13.5 pg/ml, respectively. Whereas samples for IL-6 assay were run undiluted, samples for TNF-a were diluted 5-fold. All assays were run in triplicate. 4.8. RNA preparation and RT-PCR for IL-6 Total RNA was isolated from cultured RAW 264.7 cells using Trizol reagent (Gibco BRL) in accordance with the manufacturer’s instruction. RNA was quantitated by measuring absorbance at 260 nm and 280 nm in a SpectronicÒ GenesysÔ spectrophotometer. RNA was stored in small aliquots at ÿ80  C. Aliquots of RNA (5 mg) were used for reverse-transcription by using a cDNA CycleÒ kit from Invitrogen (Carlsbad, CA 92008) according to the manufacturer’s instruction. For each PCR reaction, 5 ml of the resulting cDNA was used as template. All amplifications were performed in 50-ml reaction volumes (1 ! PCR buffer, 0.2 mM dNTP mixture, 2 mM MgSO4, 0.2 mM primer mix, 1.0 unit Taq). Cycling parameters were as follows: initial denaturation at 96  C for 1 min followed by 2 cycles of 1 min at 96  C, 1 min at 57  C, then 33 cycles of 1 min at 94  C, 2.5 min at 57  C; final extension of 10 min at 70  C. Amplification products were electrophoresed on Ready GelÒ precast gels from Bio-Rad (Hercules, CA 94547) according to the manufacturer’s instruction. 4.9. Statistical analyses All data are expressed as mean G S.D. Statistical analysis was performed using one-way ANOVA and further analyzed by NewmaneKeul’s test for statistical difference. Differences between treatments were considered to be statistically significant at P ! 0.05.

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