Dexamethasone Inhibits the Proliferation of Hepatocytes and Oval Cells But Not Bile Duct Cells in Rat Liver

Transcription

1 Dexamethasone Inhibits the Proliferation of Hepatocytes and Oval Cells But Not Bile Duct Cells in Rat Liver PETER NAGY, 1 ANDRAS KISS, 2 JANOS SCHNUR, 1 AND SNORRI S. THORGEIRSSON 2 Recent advances have implicated the importance of tumor necrosis factor (TNF) and interleukin 6 (IL-6) in the regulation of liver growth. Therefore, we studied how dexamethasone, a well-known inhibitor of these cytokines, influences the proliferation of different hepatic cell populations. As we expected, dexamethasone pretreatment suppressed the expression of both TNF and IL-6 after partial hepatectomy and significantly reduced the proliferative response of the hepatocytes. Furthermore, the proliferative response of hepatocytes could be rescued by IL-6 administration. Dexamethasone also severely diminished the induction and expansion of oval cells induced by the 2-acetylaminofluorene/partial hepatectomy (AAF/PH) protocol but did not have any effect on the proliferation of the bile duct cells stimulated by bile duct ligation. The differential inhibition of these two morphologically very similar cell types may be used to characterize divergent regulatory mechanisms responsible for the proliferative response of oval cells and adult bile epithelial cells. (HEPATOLOGY 1998;28: ) Although the adult liver is mitotically silent, the proliferation of different hepatic cell populations can be triggered by appropriate stimulation. An increasing number of compounds have been described recently that are able to induce liver hyperplasia without preceding necrosis or cell loss. 1-3 Hepatocyte proliferation is traditionally studied by surgical partial hepatectomy (PH). 4 Similar mitotic activity is seen in these experimental models, but biologically they are not equal. For example, although compensatory hyperplasia promotes tumorigenesis in the liver, direct mitogens do not exert any promoting effect. 5 Regulation of the two proliferative processes also is not identical. Transforming growth factor (TGF) and hepatocyte growth factor (HGF) are thought to be the major mitogenic growth factors driving proliferation after PH, but these cytokines are not upregulated during chemically induced primary hyperplasia of the liver. 6 When PH is performed and the hepatocytic response is inhibited, the liver regenerates via the proliferation and differentiation of facultative stem cells traditionally referred to as oval cells. 7 Ligation of the common bile duct and consequent bile congestion is an established model for induction of the proliferation of bile duct cells. 8 Although the different proliferative models must be regulated differently, there may be common regulatory mechanisms. In fact, dexamethasone has been shown to abrogate the proliferative response of primary mitogens effectively by inhibiting tumor necrosis factor (TNF) production. 9,10 TNF is also required for compensatory liver growth after PH. 11 However, little is known about the effect of dexamethasone on the proliferation of different cell types in other models of hepatic proliferation. Therefore, we compared the effects of dexamethasone on the proliferative capacity of hepatocytes, bile duct cells, and oval cells in vivo. The results show that dexamethasone effectively inhibits regenerative liver growth after PH and oval cell induction/ proliferation. However, dexamethasone does not influence the proliferation of bile duct cells after bile duct ligation. An analysis of this differential inhibition may provide further understanding of hepatic growth control. MATERIALS AND METHODS Animal Models Male Fischer F344 rats (range, g) were used for the experiments. At least three animals were killed in each experiment at every time point studied. The animals were treated with 2 mg/kg dexamethasone administered intraperitoneally (IP) 1 hour before surgery unless otherwise noted. The dexamethasone (Sigma Chemical Co., St. Louis, MO) was dissolved in a small volume of ethanol and suspended in water. PH 4 and double bile duct ligation 8 were performed under metophane anesthesia. The 2-acetylaminofluorene (AAF)/PH protocol has been described previously. 7 In brief, 10 mg/kg AAF was administered to the rats daily by gavage for 2 weeks. PH was performed in the middle of this period. Ten micrograms of either IL-6 or TNF was administered intravenously to selected rats (described in the text later) 30 minutes after the operation. Animal study protocols were conducted according to National Institutes of Health guidelines for animal care. Abbreviations: PH, partial hepatectomy; TGF, transforming growth factor; HGF, hepatocyte growth factor; TNF, tumor necrosis factor; AAF, acetylaminofluorene; GGT, -glutamyltranspeptidase; NOS, nitric oxide synthase; TNF-RI, tumor necrosis factorreceptor I. From the 1 Ist. Institute of Pathology, Semmelweis Medical University, Budapest, Hungary, and the 2 Laboratory of Experimental Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD. Received January 30, 1998; accepted April 3, Supported by OTKA T , ETT T , and NATO linkage grant Address reprint requests to: Peter Nagy, M.D., First Institute of Pathology, Semmelweis Medical University, Ulloi ut 26, Budapest, Hungary H Fax: Copyright 1998 by the American Association for the Study of Liver Diseases /98/ $3.00/0 423 Histological Techniques BrdU Labeling Index. Animals were killed at the time points described elsewhere. One hour before they were killed, 100 mg/kg BrdU (Boehringer Mannheim, Germany) was given IP. Liver samples were fixed in 70% ethanol, embedded, and cut. Five-micrometer sections were deparaffinized and hydrated through graded ethanol concentrations to water. Endogenous peroxidases were blocked (0.3% hydrogen peroxide, 20 minutes), and the slides were incubated in HCl (3N, room temperature for 15 minutes) to denature DNA. The sections were immunostained for BrdU. A monoclonal anti-brdu antibody was used (Becton Dickinson, San Jose, CA)

2 424 NAGY ET AL. HEPATOLOGY August 1998 diluted 1:100 and developed using the Vector ABC Elite Kit (Vector Laboratories, Burlingame, CA). The number of positively stained nuclei were counted. -Glutamyltranspeptidase (GGT) staining was performed by the method of Rutenberg et al. 12 RNA Isolation and Northern Blot Hybridization RNA was extracted from the tissue with guanidine thiocyanate followed by centrifugation in cesium chloride solution. 13 Poly(A)RNA was selected by oligo(dt)-cellulose chromatography. Five micrograms of poly(a)rna per lane was electrophoresed on 1% agarose gels containing 2.2 mol/l formaldehyde and later transferred to nylon membranes (Nytran plus; Schleicher & Schuell, Keene, NH). After ultraviolet cross-linking, the filters were hybridized with P-32 labeled complementary DNA (cdna) probes. The following cdna fragments were used for labeling: cyclin A, mouse 0.8 kb EcoRI; c-myc, mouse 1.4 kb XhoI; c-fos, rat 2,1 kb EcoRI; P53, human 1.4 kb EcoRI; TGF, 0.31 kb Eco RI; TGF- -I, rat O.9 kb HindIII-XbaI; HGF, human fragment of the coding region from base pair 122 to 969 (kindly provided by R. Zarnegar, University of Pittsburgh, School of Medicine); IL-6, rat 0.9 kb BamHI-PstI; TNF- mouse 2, 1 kb EcoRI; NOS, mouse 4.5 kb NotI. FIG. 1. Height of the columns represents the number of BrdU-positive hepatocytes per 10,000 hepatocytes 24 hours after PH. 1) Control animals; 2) 0, 1 mg; 3) 0, 2 mg; 4) 2 mg/kg dexamethasone pretreatment 1 hour before surgery; 5) 2 mg/kg dexamethasone pretreatment, 10 µg TNF posttreatment; and 6) 2 mg/kg dexamethasone pretreatment, 10 µg IL-6 posttreatment. RESULTS AND DISCUSSION Effect of Dexamethasone on the Proliferation of Different Liver Cell Populations Hepatocytes. Hepatocytes can be induced to proliferate by partial resection of the liver. The proliferative response has been analyzed thoroughly since the experimental protocol was described. 14 After PH, the mature hepatocytes quickly enter the cell cycle in a relatively synchronized way. In the rat, the first and highest S-phase peak can be observed approximately 24 hours after surgery. When 2 mg/ kg dexamethasone was administered to the animals 1 hour before surgery, a dramatic decrease was observed in the number of cells entering the S phase at 24 hours. The number of the BrdU-labeled hepatocytes was reduced by 80% compared with control animals (Figs. 1, 2A, and 2B). Lower doses of dexamethasone had a reduced but similar effect. However, if dexamethasone administration was not repeated, a delayed hepatocyte proliferation peak appeared approximately 40 hours after hepatectomy (data not shown). Therefore, inhibition of hepatocyte growth by dexamethasone was dose dependent and reversible. Oval Cell Induction/Proliferation. The AAF/PH model has been studied in detail in our laboratory during the last decade Although AAF very effectively inhibits the proliferation of the hepatocytes and functionally compromises them, it induces the proliferation of small cells in the periportal region. These cells, which we believe to be progeny of the liver stem cells, respond to the PH growth stimulus with strong proliferation. The progeny of the stem cells, the oval cells, invade the liver parenchyme, differentiate into hepatocytes, and regenerate liver mass. 15 Two approaches were used to determine the impact of dexamethasone treatment on this process. First, the rats receiving long-term dexamethasone treatment received 2 mg/kg dexamethasone once every day throughout the experiment 1 hour before AAF feeding and PH. The rats became very weak, especially after PH, and none survived for more than 6 days after PH. No increase in liver weight was observed in this group. Furthermore, no BrdU-labeled cells were identified in the liver at any time in these rats. GGT staining showed oval cells invading the parenchyme in the control rats, but only the pre-existing bile ducts stained positively in the dexamethasone-treated group (Fig. 3A and 3B). The aim of the second approach was to determine how a single dose of dexamethasone would influence oval cell proliferation as well as that of the newly formed basophilic hepatocytes. The rats were treated according to the traditional AAF/PH protocol. Control rats were killed 3, 5, 7, and 9 days after surgery. The experimental animals received 2 mg/kg dexamethasone 24 hours before they were killed at the same 3-, 5-, 7-, and 9-day points. In the control group, BrdU immunohistochemistry showed a high labeling index among the oval cells and at later time points in the newly formed foci of basophilic hepatocytes (Fig. 3C and 3D). However, the number of BrdUpositive cells decreased approximately 10-fold after dexamethasone treatment at all time points, indicating that a single dose of dexamethasone could inhibit proliferation of both oval cells and the newly formed hepatocytes. Furthermore, dexamethasone is capable, at least in this experimental model, of inhibiting activation of the stem cell compartment. Bile Duct Cells. The peak of bile duct cell proliferation occurs 48 hours after ligation of the common bile duct. If dexamethasone was administered 1 hour before surgery and repeated once 24 hours later, the ratio of BrdU-labeled bile duct cells did not differ from that observed in control animals at the peak of proliferation. However, there was a striking difference between the two groups. Whereas probably because of the toxic injury hepatocyte proliferation escorted the bile duct proliferation in the control group, this hepatocytic response was completely abolished by dexamethasone (Fig. 2C and 2D). Because bile duct cells get most of their blood supply from the hepatic artery, dexamethasone was injected into the tail vein of the third group of rats instead of IP administration to avoid preferential exposure of the hepatocytes by the portal blood. No difference in dexamethasone effects was observed between these two forms of treatment. In summary, dexamethasone treatment effectively inhibited the proliferation of hepatocytes and oval cells but had no effect on the replication of bile duct cells. There are contradictory data on how dexamethasone influences the proliferation of hepatocytes. It definitely

3 HEPATOLOGY Vol. 28, No. 2, 1998 NAGY ET AL. 425 FIG. 2. (A and B) BrdU-immunostained liver sections 24 hours after PH: (A) control liver; and (B) after pretreatment with 2 mg/kg dexamethasone. (C and D) BrdU-immunostained liver sections 48 hours after common bile duct ligation: (C) control liver; and (D) after pretreatment with 2 mg/kg dexamethasone.

4 426 NAGY ET AL. HEPATOLOGY August 1998 FIG. 3. Effect of dexamethasone treatment on the oval cell induction/proliferation in the AAF/PH model. (A and B) GGT histochemistry 5 days after PH in a control liver section (A) and after long-term dexamethasone treatment (B). (C and D) BrdU immunostaining 9 days after PH: (C) basophilic focus in a control animal; and (D) basophilic focus from a rat treated with 2 mg/kg dexamethasone 24 hours before it was killed.

5 HEPATOLOGY Vol. 28, No. 2, 1998 NAGY ET AL. 427 decreases the proliferative response stimulated by directacting mitogenic compounds such as lead nitrate by supposedly suppressing TNF expression. 9,10 Satoh et al. 19 reported inhibition of the proliferative response after PH in mice. On the contrary, in vitro dexamethasone is a permanent constituent of tissue culture media used for culturing hepatocytes. 20 Block et al. 21 emphasized that dexamethasone is absolutely required to maintain cell proliferation in a newly developed growth medium for long-term culturing of hepatocytes. However, extrapolation from in vitro data to an in vivo situation is at best difficult. This is especially true for dexamethasone, which has pleiotrophic effects and probably influences hepatocytes in vivo directly as well as by extrahepatic and nonparenchymal hepatic means. Dexamethasone is also present in media used for culturing bile duct cells. 22,23 We have not found any previous data on the influence of dexamethasone on oval cell proliferation. The differential growth inhibition of dexamethasone on bile duct and oval cells is an intriguing issue. There is still an ongoing debate about the origin and fate of oval cells. Some authors question their precursor role for the hepatocytic lineage and regard them as nothing more than proliferating bile duct cells. 24 In the present experimental model, we show that at least with respect to response to dexamethasone, the oval cells appear to behave more similarly to hepatocytes than to adult differentiated bile duct cells. Expression of Growth-Related Genes in the Liver After Partial Hepatectomy Although we do not know exactly how the proliferative response is regulated after PH, there is an orchestrated expression of growth-promoting and -inhibiting genes during the compensatory growth. In an attempt to gain insight into the mechanism of dexamethasone-related growth inhibition, we studied the changes in the steady-state messenger RNA (mrna) levels of a number of these growth-associated genes in the liver by Northern blot analysis (Fig. 4). The lower expression of the cyclin A gene in the dexamethasone-treated group supports our observation that significantly fewer cells enter the cell cycle under these conditions. We did not observe any difference in the expression of growth-promoting genes such as TGF-, HGF, and c-jun between the two groups. Therefore, dexamethasone s effect cannot be explained by decreased expression of these genes. Another possible explanation is the up-regulation of a growthinhibitor gene, but there were no signs of that either. In fact, expression of TGF- 1 was lower in the dexamethasonetreated group, probably because the less extensive growth requires less inhibitory action. The implications of increased expression of p53 after PH are not completely understood. The intact p53 protein can execute cell cycle arrest, but the expression of p53 gene was also lower in the dexamethasonetreated group. Increased expression of the inducible form of nitric oxide synthase (NOS) after PH has been reported recently. 25 NOS expression is down-regulated by corticosteroids, 26 and our results confirm these observations. NOS mrna was detected 12 hours after PH in the control group, but no NOS transcripts were found in the livers of the dexamethasonetreated rats. The function, if any, of NOS in the proliferative response following PH is not known. However, disappearance of the NOS transcripts after dexamethasone treatment is a good indicator of a specific pharmacological effect on the liver. There was a dramatic difference in the expression of TNF and IL-6 as well. Dexamethasone pretreatment almost completely blocked the expression of these two cytokines in the liver at the transcriptional level. It has long been known that TNF and IL-6 induce the so-called acute-phase response in the liver, 27,28 and both are down-regulated by glucocorticoids. What is novel is that the same cytokines are also important for the proliferation of hepatocytes. However, the influence of IL-6 on the proliferative response of hepatocytes is controversial. IL-6 is one of the most pleiotrophic cytokines. 28,29 IL-6 and IL-6 receptor expression are conversely regulated in the liver by glucocorticoids. 20,28,30 These facts may explain, at least partly, the divergent results. Nakamura et al. 31 reported an inhibitory effect of IL-6, and Goss et al. 32 showed that indomethacin up-regulated IL-6 expression in the liver but inhibited liver regeneration. However, most recent reports indicate that IL-6 is required for the compensatory liver growth in vivo. 33 Akerman et al. 11 were able to inhibit liver regeneration after PH by pretreatment with TNF antibody. This antibody treatment also prevented increased serum levels of IL-6 after the operation. Recently, Cressman et al. 33 described a delayed, prolonged response after PH in IL-6 / mice that could be normalized by IL-6 administration before surgery. However, Yamada et al. 34 could reproduce the liver regeneration patterns of IL-6 / by using mice in which the TNF-receptor l (Rl) had been deleted. Furthermore, the proliferative response could be restored in the TNF-RI / mice by administration of IL-6 before surgery. Based on these data, Yamada et al. 34 proposed that TNF signaling through TNF-RI can initiate liver regeneration and that IL-6 is a key target of TNF activation. IL-6 But Not TNF Can Counterbalance Dexamethasone To determine whether the inhibition of TNF and/or IL-6 production is responsible for the observed antiregenerative effect of dexamethasone, we injected 10 µg of either TNF or IL-6 30 minutes after surgery along with the usual dexamethasone pretreatment. The effect was monitored by BrdU incorporation 24 hours after hepatectomy. The TNF was ineffective (Fig. 1) when administered at this dose and schedule. However, the IL-6 administration rescued the proliferative response from dexamethasone inhibition (Fig. 1). The labeling index under these conditions almost reached the level of the control rats. This result strongly supports the recent observation that an intact IL-6 response is required for the liver growth. Therefore, dexamethasone probably inhibits liver regeneration by blocking IL-6 expression. Besides dexamethasone, several other known compounds suppress TNF production. One of these compounds is adenosine, which was recently shown by Kubo et al. 10 to reduce the proliferative response induced by lead nitrate. In our hands, adenosine could also abolish TNF but not IL-6 expression in the liver after PH without significantly influencing the proliferative response (data not shown). CONCLUSIONS Glucocorticoids are important for down-regulating the acute-phase response by suppressing production of TNF and IL-6. 28,30 They can also inhibit liver growth by the same mechanism. However, in the case of acute-phase response, TNF and IL-6 up-regulation does not result in hepatocytic

6 428 NAGY ET AL. HEPATOLOGY August 1998 A B FIG. 4. Northern blot analysis of growth-related genes in the liver after PH without (A) (control) and with (B) dexamethasone pretreatment. The rats killed at 48 hours in the dexamethasone group received a second 2 mg/kg dexamethasone dose 24 hours after surgery. proliferation. In our experimental model, growth-related genes (e.g., c-myc, c-fos, TGF-, HGF) are expressed in the liver, but the cells cannot enter the S phase in the absence of IL-6. Therefore, we propose that the synchronized activation of the TNF/IL-6 pathway and growth-related genes is necessary for effective proliferation after PH. Primary hyperplasia can occur without the expression of traditional growthrelated genes 6 (e.g. HGF, TGF- ), but the TNF/IL-6 pathway is required for both types of proliferation. IL-6 is a mitogen for bile duct epithelial cells. 35 However, in vivo, bile duct cells can proliferate when dexamethasonerelated IL-6 suppression inhibits the proliferation of hepatocytes. Further studies are needed to determine whether the difference in bile duct cells and hepatocytes growth regulation is qualitative or quantitative. Oval cells probably are derived from the bile duct cells. 36 Still, they respond differently to dexamethasone treatment. The key question is whether there are cells in the bile duct tree that are growth-regulated differently from the rest or whether the AAF-related growth stimulus is qualitatively different from that seen in bile statis. Hepatocyte proliferation is inhibited by dexamethasone administration if this response

7 HEPATOLOGY Vol. 28, No. 2, 1998 NAGY ET AL. 429 is triggered by partial hepatectomy or priming mitogens. 9 However, proliferation of the biliary epithelium is not a homogenous response irrespective of the source of the growth stimulus but rather a heterogenous response depending on the nature of the mitogenic stimulus. Further studies on the nature of this heterogenous proliferative response of the bile epithelium may provide important insight into hepatic stem cell biology. REFERENCES 1. Columbano A, Ledda-Columbano GM, Sirigu P, Perra T, Pani P. Liver cell proliferation induced by a single dose of lead nitrate. Am J Pathol 1983;110: Nachtomi E, Farber E. Ethylene dibromide as a mitogen for liver. Lab Invest 1978;38: Levine WG, Ord GM, Stocker LA. Some biochemical changes associated with nafenopine-induced liver growth in rat. Biochem Pharmacol 1977;26: Higgins GM, Anderson RM. Experimental pathology of the liver: restoration of the liver of the white rat following partial surgical removal. Exp Pathol 1931;12: Columbano A, Ledda-Columbano GM, Lee G, Rajalakhsmi S, Sarma DSR. Inability of mitogen-induced liver hyperplasia to support the induction of enzyme-altered islands induced by liver carcinogens. Cancer Res 1987;47: Shinozuka H, Kubo Y, Katyal SL, Coni P, Ledda-Columbano GM, Columbano A, Nakamura T. Roles of growth factors and of tumornecrosis factor-alpha on liver cell proliferation induced in rats by lead-nitrate. Lab Invest 1994;71: Evarts RP, Nagy P, Marsden ER, Thorgeirsson SS. A precursor-product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 1987;8: Cameron GR, Oakley CR. Ligation of the common bile duct. J Pathol Bacteriol 1932;35: Ledda-Columbano GM, Columbano A, Cannas A, Simbula G, Okita K, Kayano K, Kubo Y, Katyal SL, Shinozuka H. Dexamethasone inhibits induction of liver tumor necrosis factor alpha mrna and liver growth induced by lead nitrate and ethylene dibromide. Am J Pathol 1994;145: Kubo Y, Yasunaga M, Masuhara M, Terai S, Nakamura T, Okita K. Hepatocyte proliferation induced in rats by lead nitrate is suppressed by several tumor necrosis alpha inhibitors. HEPATOLOGY 1996;23: Akerman K, Cote P, Yung SQ, McClain G, Nelson S, Bagly GI, Diehl AM. Antibodies to tumor necrosis factor alpha inhibit liver regeneration after partial hepatectomy. Am J Physiol 1992;263:G579-G Rutenberg AM, Kim H, Fischbein IW, Hanker JS, Waserkrug HL, Seligman AM. Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity. J Histochem Cytochem 1969;17: Schweizer G, Goerttler K. Synthesis in vitro of keratin polypeptides directed by mrna isolated from newborn and adult mouse epidermis. Eur J Biochem 1980;112: Michalopoulos GK, DeFrances MC. Liver regeneration. Science 1997;276: Evarts RP, Hu Z, Fujio K, Marsden ER, Thorgeirsson SS. Activation of hepatic stem cell compartment in the rat, role of transforming growth factor-alpha, hepatocyte growth factor, and acidic fibroblast growth factor in early proliferation. Cell Growth Differ 1993;4: Thorgeirsson SS, Evarts RP, Bisgaard HC, Fujio K, Hu Z. Hepatic stem cell compartment: activation and lineage commitment. Proc Soc Exp Biol Med 1993;204: Bisgaard HC, Nagy P, Santoni-Rugiu E, Thorgeirsson SS. Proliferation, apoptosis and induction of hepatic transcription factors are characteristics of the early response of biliary epithelial (oval) cells to chemical carcinogens. HEPATOLOGY 1996;23: Nagy P, Bisgaard HC, Santoni-Rugiu E, Thorgeirsson SS. In vivo infusion of growth factors enhances the mitotic response of rat hepatic ductal (oval) cells after administration of 2-acetylaminofluorene. HEPATOLOGY 1996;23: Satoh M, Adachi K, Suda T, Yamazaki M, Mizuno D. TNF-driven inflammation during mouse liver regeneration after partial hepatectomy and its role in growth regulation in liver. Mol Biother 1991;3: Bauer J, Lengyel G, Bauer MT, Acs G, Gerok W. Regulation of interleukin-6 receptor expression in monocytes and hepatocytes. FEBS Lett 1989;249: Block GD, Locker J, Bowen WC, Petersen BE, Katyal S, Strom SC, Riley T, Howard TA, Michalopoulos GK. Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium. J Cell Biol 1996;132: Demetris AJ, Markus BH, Saidman S, Fung JJ, Makowka L, Graner S, Duquesnoy R. Isolation and primary cultures of human intrahepatic bile ductular epithelium. In Vitro Cell Dev Biol 1988;24: Joplin R, Strain AJ, Neuberger JM. Immunoisolation and culture of biliary epithelial cells from normal human liver. In Vitro Cell Dev Biol 1989;25: Lenzi R, Liu MH, Tarsetti F, Slott PA, Alpini G, Zhai WR, Paronetto F, et al. Histogenesis of bile duct-like cells proliferating during ethionine hepatocarcinogenesis. Evidence for a biliary epithelial nature of oval cells. Lab Invest 1992;66: Hortelano S, Dewez B, Genaro AM, Diaz-Guerra M, Bosca L. Nitric oxide is released in regenerating liver after partial hepatectomy. HEPATOLOGY 1995;21: Geller AD, Nussler AK, DiSilvio M, Lowenstein ChJ, Shapiro RA, Wang SC, Simmons RL, Billiar TR. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthetase in hepatocytes. Proc Natl Acad Sci U S A1993;90: Andus T, Geiger T, Hirano T, Northoff H, Ganter U, Bauer J, Kishimoto T, Heinrich PC. Recombinant human B cell stimulatory factor 2 (BSF-2IFN- B2) regulates beta fibrinogen and albumin mrna levels in Fao-9 cells. FEBS Lett 1987;221: Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute phase response. Biochem J 1990;265: Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6 family of cytokines and gp130. Blood 1995;86: Woloski BMRNJ, Smith EM, Meyer WJ, Fuller GM, Blalock JE. Corticotropin-releasing activity of monokines. Science 1985;230: Nakamura T, Arakaki R, Ichihara A. Interleukin-1 beta is a potent growth inhibitor of adult rat hepatocytes in primary culture. Exp Cell Res 1988;179: Goss AJ, Mangino MJ, Callery MP, Flye W. Prostaglandin E2 downregulates Kupffer cell production of IL-1 and IL-6 during hepatic regeneration. Am J Physiol 1993;264:G601-G6O Cressman DE, Greenbaum LE, DeAngelis RA, Ciliberto G, Furth EE, Poli V, Taub R. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 1996;274: Yamada Y, Kirillova I, Peschon J, Fausto N. Initiation of liver growth by tumor necrosis: Deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proc Natl Acad Sci U S A1997;94: Matsumoto K, Fujii H, Michalopoulos G, Fung JJ, Demetris AJ. Human biliary epithelial cells secrete and respond to cytokines and hepatocyte growth factors in vitro: interleukin-6, hepatocyte growth factor and epidermal growth factor promote DNA synthesis in vitro. HEPATOLOGY 1994;20: Grisham JW, Thorgeirsson SS. Liver Stem Cells. In: Potten CS, ed. Stem Cells. London: Academic, 1997: