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Fas-ligand system functions in hepatocytes in the early stage of fulminant hepatic failure in rats
Hepatology Research 11 (1998) 103 – 114
The Fas/Fas-ligand system functions in hepatocytes in the early stage of fulminant hepatic failure in rats Susumu Eguchi a, Sumihiro Matsuzaki a, Hikaru Fujioka a, Takehiko Koji b, Yoshikazu Higami c, Takashi Kanematsu a,* a
Department of Surgery II, Nagasaki Uni6ersity School of Medicine, 1 -7 -1 Sakamoto, Nagasaki 852 -8501, Japan b Department of Anatomy III, Nagasaki Uni6ersity School of Medicine, 1 -7 -1 Sakamoto, Nagasaki 852 -8102, Japan c Department of Pathology I, Nagasaki Uni6ersity School of Medicine, 1 -7 -1 Sakamoto, Nagasaki 852 -8102, Japan Received 18 December 1997; received in revised form 5 February 1998; accepted 10 February 1998
Abstract Apoptosis is involved in various liver diseases, but in the case of fulminant hepatic failure (FHF) much remains to be determined. We investigated apoptosis in the remnant liver lobes of FHF rats regarding the Fas/Fas ligand and the TGF-b system. Rats (n= 30) with FHF were euthanized at postoperative 3, 6, 12, 24 and 36 h. Changes in the Fas/Fas-ligand and TGF-b were studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining. Apoptosis was explored using terminal deoxynucleotidyl transferase mediated dUDP-nick end labeling method (TUNEL). Within 6 h, a relatively large number of hepatocytes, mainly in zone 3, were apoptotic. During the same period, the expression of Fas and Fas-ligand mRNA and protein, but not that of TGF-b, were observed and decreased with time. Both Fas and Fas-ligand proteins were present in the hepatocytes in zone 3. These results suggest that the Fas/Fas-ligand system, rather than TGF-b, has a role in apoptosis during the early stages of FHF, possibly through an autocrine loop. © 1998 Elsevier Science Ireland Ltd. All rights reserved. * Corresponding author. Tel.: [email protected]
+ 81 95 8497312; fax:
+81 95 8497319; e-mail: kane-
1386-6346/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S1386-6346(98)00020-5
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Keywords: Fas (Fas/APO-1/CD95); Fulminant hepatic failure; Apoptosis
1. Introduction Fulminant hepatic failure (FHF) is defined as the appearance of encephalopathy less than 8 weeks after the onset of symptoms in a patient without any previous liver disease [1]. The majority of patients will rapidly deteriorate and die, unless they undergo immediate liver transplantation; only a small percentage of these patients can recover and subsequently have normal liver function [2]. Despite improvement in the determination of etiology in the majority of cases, it is still unclear what specific abnormalities are pathognomonic for FHF. A novel experimental model of FHF in the rat was reported [3]. Although we have investigated and characterized pathological events in the remnant liver lobes in FHF, the role of apoptosis which is highlighted in FHF, has not been thoroughly explored. Apoptosis is thought to play a homeostatic role after mild tissue injury, leaving intact the overall cytoarchitecture of the tissue [4]. The first description of apoptosis in the liver dates back to the late 1960s, but there had been no documentation on the role of this phenomenon as a mechanism of acute liver failure. Hirata et al. [5] reported that apoptosis of fat-storing cells could be the main cause of microcirculatory failure in drug-induced liver injury. Apoptosis also occurs in liver diseases such as viral hepatitis B [6], hepatitis C [7] and ischemia-reperfusion injury [8]. Inflammatory infiltrate is characteristic of each of these liver pathologies, consistent with the possibility that infiltrating cells such as a CTL (cytotoxic T-lymphocytes) may be involved in the apoptotic process. [9] In addition, several cytokines and chemical agents were reported to induce apoptosis in hepatocytes, including tumor necrosis factor-a [10], interferon-g [10], oxygen radicals [11], TGF-b [12], and Fas (Fas/APO-1/CD95)/ Fas-ligand [13]. Fas (Fas/APO-1/CD95) is a cell surface protein that induces apoptosis in hepatocytes, under physiological and pathological conditions. The intraperitoneal administration of anti-Fas antibody induced FHF-like massive liver damage with apoptosis through Fas antigen on their hepatocytes. [14] It was concluded that accumulation of Fas ligand expressing cells around the target cells was critical for effectiveness of the Fas system. On the other hand, TGF-b has been reported as a strong inducer of apoptosis as well. [12] We examined the Fas/Fas-ligand and TGF-b systems as possible inducers of apoptosis in the liver of the rats with FHF. The transition and localization of these systems in the liver were investigated using RT-PCR and immunohistochemical staining.
2. Materials and methods
2.1. Animals Healthy adult male Sprague Dawley rats weighing 250–300 g obtained from
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Charles River Japan (Atsugi, Japan) were acclimatized to our laboratory conditions for 1 week prior to use in the experiments. They were housed in a climate controlled 21°C room under a 12 h light/dark cycle. Tap water and commercial rat chow (Oriental Kobo Kogyo, Tokyo, Japan) were provided ad libitum. All procedures were in accordance with University of Nagasaki Research Animal Resources guidelines. At the completion of the surgical procedure, each animal was given 20 ml of 5% glucose in Lactate Ringer’s solution in a subcutaneous bolus injection (s.c.).
2.2. Surgical animal models FHF was induced as described [3]. Briefly, the abdomen was entered through a midline incision. The common pedicle to the right liver lobes (24% of the liver) was ligated to induce necrosis, while the two anterior liver lobes (68% of the liver) were removed using the standard Higgins and Anderson technique. [15] The two omental liver lobes (8% of the liver) were left intact. The anterior liver lobes were fully mobilized and a ligature was placed high around their common pedicle, so that there would be no interference with the arterial blood supply to the liver remnant or impairment of venous outflow.
2.3. Experimental design FHF rats (n =30) were euthanized in batches of six rats at 3, 6, 12, 24 and 36 h, respectively. At death, the omental liver lobes were removed, frozen in liquid nitrogen for RNA extraction, or fixed in formalin. The tissue was sectioned and stained with hematoxylin and eosin and immunostained for Fas, Fas-ligand and TGF-b.
2.4. Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling After fixing with 10% buffered formalin, sections (4 mm) were deparaffinized and hydrated. Sections were reacted with 20 mg/ml proteinase K (Sigma, St. Louis, MO) at 37°C for 20 min and rinsed in distilled water. After immersion in terminal deoxynucleotidyl transferase (TdT) buffer (Gibco BRL, Gaithersburg, MO), sections were incubated overnight with TdT in TdT buffer, rinsed in buffer solution and the non-specific reaction was blocked with 10% normal rabbit serum. For visualization of incorporated biotin-16-dUTP (Boehringer Mannheim Biochemica, Mannheim, Germany), the sections were incubated with streptavidin-alkaline phosphatase conjugate for 30 min at room temperature. Sections were washed in Tris-buffered saline and alkaline phosphate substrate buffer (ASB; 0.1 mol/l Tris – HCl pH 9.5, 0.1 mol/l NaCl and 0.05 mol/l MgCl2) and incubated with the substrate solution containing 4-nitroblue tetrazolium chloride (NBT, Gibco BRL, Gaithersburg, MO) and 5-bromo-4-chloro3-indolylphosphate (BCIP, Gibco BRL, Gaithersburg, MO) in ASB.
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2.5. Apoptotic index For an apoptotic index in the remnant liver, we calculated as the number of terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL)-positive hepatocytes per 1000 hepatocytes. For each liver, hepatocytes in 100 consecutive high power fields (hpfs) were counted.
2.6. Fas, Fas-ligand and TGF-b mRNA expression RNA expression of Fas and TGF-b in the liver tissue was detected using RT-PCR. From the livers kept frozen in liquid nitrogen until assay, total RNA was extracted according to the RNAzol B (TEL-TEST, Friendswood, TX) protocol. Single strand complementary DNA (cDNA) was synthesized by RT using the random primer and 1 mg of mRNA in 10 ml of reaction mixture. PCR primers were synthesized for rat Fas [16], rat Fas-ligand [17], rat TGF-b [18] and rat b-actin [16] cDNA. The 5%-primer of the rat Fas was 5%CAAGGGACTGATAGCATCTTTGAGG-3% and the 3%-primer of the rat Fas was 5%-GTCCTTAACTTTTCGTTCACCCAGG-3% (PCR product size= 141 bp). The 5%-primer of the rat Fas-ligand was 5%-TCCACCACCACCTCCATCAC-3% and the 3%-primer of the rat Fas-ligand was 5%-CCAACCTTACCCCAATCCTT3% (PCR product size=152 bp). The 5%-primer of the rat TGF-b was 5%GGACTCTCCACCTGCAAGAC-3% and the 3%-primer of the rat TGF-b was 5%-CTCTGCAGGCGCAGCTCTG-3% (PCR product size = 390 bp). The 5%primer of the rat b-actin was 5%-CACTGCCGCATCCTCTTCCT-3% and the 3%primer of the rat b-actin was 5%-AGCCACCAATCCACACAGAG-3% (PCR product size= 347 bp). Relative levels of mRNA were detected using equal amounts of input single-stranded DNA (2 ml per 30 ml) and tested in parallel using GeneAmp RNA PCR kits (Perkin Elmer, Norwalk, CT). PCR consisted of denaturation at 94°C, annealing at 60°C and extension at 72°C using a Thermal Cycler (Perkin Elmer, Norwalk, CT). After staining with ethidium bromide, gels were photographed using Polaroid film (Fuji Film, Tokyo, Japan). In preliminary studies, the parallel amplification efficiency of PCR for different targets was determined after several different amounts of initial DNA template, as reported [16]. The optimal number of thermal cycles for Fas, Fas-ligand, TGF-b and b-actin was determined to be 28, 32, 25 and 22 cycles, respectively. Gel images showing ethidium bromide fluorescence of each band in stored images were analyzed using a computer software system (NIH image, Version 1.61).
2.7. Immunohistochemical staining for Fas, Fas-ligand and TGF-b1 Immunohistochemical staining was performed using an indirect, two-step labeling technique with peroxidase-conjugated IgG. Polyclonal anti-mouse Fas (P4; dilution, 1:200)/anti-rat Fas-ligand (P5; dilution, 1:100) antibodies from
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rabbit were used [13,16]. Sections (4 mm thick) were deparaffinized in toluene and rehydrated by passing through a graded series of ethanol. Endogenous peroxidase was blocked by incubation of the slides with 0.3% H2O2 in methanol for 15 min at room temperature. After washing in phosphate-buffered saline (PBS), 500 mg/ml of normal goat IgG in 1% bovine serum albumin (BSA)/PBS was applied for 60 min at room temperature for blocking. Slides were incubated overnight with anti-Fas and anti-Fas ligand antibodies for 2 h, and subsequently exposed to prediluted goat antirabbit peroxidase-conjugated IgG (1:400) for 40 min at room temperature. The peroxidase reaction was developed by incubating in H2O2 and 3,3%-diaminobenzidine tetrachloride. As a control, the adjacent sections were reacted with normal rabbit IgG in the presence of the specific antibody. Counterstaining was performed with methylgreen. All washing procedure was done using 0.075% Brij 35 (Polyoxyethylene Lauryl Ether, Nacarai Tesque, Kyoto, Japan) in PBS. For immunodetection of TGFb1 protein, the liver sections were exposed to microwaves to retrieve antigenicity. Chicken polyclonal anti-human TGF-b1 antibody (R&D Systems, Minneapolis, MN) was used followed by detection using secondary anti-chicken IgY alkaline phosphatase conjugate (Promega, Madison, WI) with our modification. [19] Sections were washed in Tris-buffered saline and ASB and incubated with the substrate solution containing NBT and BCIP in ASB.
2.8. Statistical analysis Data were analyzed using one-way ANOVA. P values equal or less than 0.05 were considered significant. Data are presented as means9 S.D.
3. Results Our FHF model made evident many aspects of clinical FHF in a highly reproducible manner and liver regeneration in remnant intact lobes was completely halted as reported. Mean survival time was 399 11 h [3].
3.1. Apoptotic index Apoptotic index, expressed as the number of TUNEL-positive hepatocytes within 1000 hepatocytes in the remnant omental lobe peaked as early as 3 h following induction of FHF, as shown in Fig. 1 (PB0.05), a significant finding compared to the subsequent period. It then decreased with time and almost no TUNEL-positive cell was detected at 36 h like baseline. Apoptotic cells were hepatocytes mainly around central vein (zone 3).
3.2. Expression of Fas, Fas-ligand and TGF-b mRNA in remnant li6er lobes In FHF rats, mRNA of Fas was upregulated at 3 h and subsequently decreased
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Fig. 1. Apoptotic index in the remnant liver of FHF rats. For the indicated times, data are plotted as means9 S.D. Apoptotic indices in the liver remnant was calculated as the number of TUNEL-positive hepatocytes per 1000 hepatocytes. In each liver, hepatocytes in 100 consecutive high power fields (hpfs) were counted. *P 5 0.05.
to baseline level. Fas-ligand mRNA was detected for up to 6 h post-induction, then was undetectable. TGF-b mRNA was detectable after 6 h and upregulated until expiration (Fig. 2). The baseline expression of Fas mRNA was minimal and that of Fas-ligand was undetectable. TGF-b mRNA was undetectable at baseline, while b-actin mRNA was constantly expressed at all time point.
Fig. 2. Fas, Fas-ligand, TGF-b and b-actin gene expression in the remnant liver of FHF rats. Animals were killed at 0, 3, 6, 12, 24 and 36 h following surgery. After extraction of RNA, 1 mg of total RNA was used for RT-PCR analysis, as described.
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Fig. 3. Immunohistochemical staining of FHF livers using anti-Fas antibody. (A) Sham liver stained with anti-Fas antibody ( × 100). (B) FHF liver at 3 h stained with anti-Fas antibody ( × 100). (C) FHF liver at 36 h stained with anti-Fas antibody ( ×100). (D) Positive staining was seen in hepatocytes at 3 h (×400).
3.3. Histological changes in remnant li6er tissue The omental lobes in FHF rats had an interstitial edema and extensive lipid vacuolization of the hepatocytes. The derangement was most evident in specimens of the 36-h group. Congestion was present in the whole sample thereby reflecting portal hypertension. In the 3- and 6-h groups, apoptotic cells, defined as nuclei displaying chromatin condensation, were relatively more as compared to the group in late stage (36 h).
3.4. Immunohistochemical staining of the Fas and Fas-ligand Fas protein was minimal in sham liver. Staining for Fas protein was present in hepatocytes around central vein (zone 3) at 3–6 h following induction of FHF (Fig. 3). At this same period, Fas-ligand was expressed in hepatocytes in a zone identical with that for Fas protein (Fig. 4). Subsequently, expression of Fas and Fas-ligand protein decreased with time and the general condition of the rats deteriorated and they died. Location of expression of Fas and Fas-ligand was in the plasma membrane and cytoplasm of hepatocytes (Fig. 3D and Fig. 4D).
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3.5. Immunohistochemical staining of the TGF-b TGF-b protein was negative for up to 12 h following the induction of FHF. It began to be expressed at 24 h and was most evident at 36 h when rats were in coma and about to die. TGF-b protein was evident in the cytoplasm of hepatocytes rather than non-parenchymal cells (Fig. 5).
4. Discussion We observed apoptosis in hepatocytes in the early stages of FHF, an event which coincided with expression of the Fas/Fas-ligand but not the TGF-b system. Expression of Fas protein and Fas-ligand protein was evident in hepatocytes, not infiltrating lymphocytes, thereby indicating a possible autocrine loop of apoptotic process. As far as Fas (Fas/APO-1/CD95) in liver disease is concerned, Ogasawara et al. [14] a reported lethal effect of anti-Fas antibody on mice to induce FHF through apoptosis. Aside from FHF, in liver tissue of patients with chronic hepatitis B [20] and hepatitis C [21], Fas was involved. Galle et al. [22] reported the localization of
Fig. 4. Immunohistochemical staining of FHF livers using anti-Fas-ligand antibody. (A) Sham liver stained with anti-Fas-ligand antibody ( ×200). (B) FHF liver at 3 h stained with anti-Fas-ligand antibody ( × 200). Brown stained hepatocytes represent positive staining. (C) FHF liver at 36 h stained with anti-Fas-ligand antibody ( ×200). (D) Positive staining was seen in hepatocytes at 3 h ( ×400).
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Fig. 5. Immunohistochemical staining of FHF livers, using anti-TGF-b antibodies. (A) Sham liver stained with anti-TGF-b antibody ( × 100). (B) FHF liver at 3 h stained with anti-TGF-b antibody (× 100). (C) FHF liver at 36 h stained with anti-TGF-b antibody ( ×100). (D) Positive staining was seen in hepatocytes rather than non-parenchymal cells at 36 h ( × 400).
mRNA of Fas ligand in liver cirrhosis developed from hepatitis B and alcohol-related liver damage. The signal of the Fas-ligand was found in infiltrated lymphocytes in the presence of viral disease, while in alcohol-related disease, the Fas ligand was found in hepatocytes. On the other hand, TGF-b is a multifunctional cytokine with diverse effects on cell growth, differentiation and function. Apoptosis can be induced by TGF-b and accounts for the inadequate hepatic regeneration that occurs with liver disease [23]. It has recently been reported that TGF-b was produced in not only non-parenchymal cells but also in hepatocytes during liver regeneration and has some role through an autocrine loop. [24] In our model of FHF, TGF-b did not play a role in inducing apoptosis. There was no evidence of liver regeneration in our expired animals used as models of FHF. [3] No relationship was found between TUNEL positive hepatocytes and proliferation with PCNA. [25] It would thus appear that apoptosis has little effect on subsequent liver regeneration. In the present study, TGF-b was upregulated from 6 h on. Therefore, TGF-b may have the role on insufficient liver regeneration, since the HGF/c-met system as stimulator was upregulated after 18 h following induction of FHF. [3] We found that apoptotic death of hepatocytes was evident within 6 h following the induction of FHF, which means suggesting that apoptosis may be the pathognomosis in the early stage of FHF. The expression of mRNA and protein of the
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Fas/Fas-ligand in hepatocytes coincided with the occurrence of apoptosis of hepatocytes, while that of TGF-b was observed from 6 h, thus preceding apoptosis by 6 h. In addition, the localization of Fas/Fas-ligand proteins was concomitant with that of apoptosis. These results suggest that the Fas/Fas-ligand system rather than TGF-b might give rise to apoptosis in hepatocytes in remnant hepatocytes in FHF. With regard to apoptosis in FHF, there have been reports, including a transgenic mouse model of fulminant hepatitis [26], liver injury induced by endotoxin [27] and thioacetamide. [26] In clinical fulminant hepatitis, TGF-b was expressed in both metaplastic hepatocytes and adjacent metaplastic bile duct forming cells, while Fas antigen was positive only on the cell membrane of bile duct forming cells, but not on hepatocytes. [28] In addition, expression of Fas antigen was not detectable at the sites where TUNEL was positive, thus suggesting that induction of apoptosis was not mediated by Fas antigen in their clinical samples with various liver diseases. However, the clinical stage in which they obtained specimens was not standardized. As we demonstrated in the present study, apoptosis seems to be involved in the early stage of FHF and it would be very difficult to obtain such specimens in a clinical setting. Our finding is in keeping with the report of Ledda-Columbano et al. [29] in which apoptosis was evident within 6 h, while necrosis was observed at 24 h accompanied by massive inflammatory reactions. In hepatocytes under oxidative stress or the influence of cytokines, the signal of the Fas/Fas-ligand might be up-regulated so that injured hapatocytes are eliminated through apoptotic cell death to maintain homeostasis. It is intriguing that interruption of the Fas/Fas-ligand system using a neutralizing anti-Fas antibody can prevent the advance of disease. In addition, the presence of necrosis in our model may play some role in enhancing the vicious circle of cytokines and toxic substances specific in FHF. [30] It is concluded that, in the liver of rats with FHF, apoptosis of remnant hepatocytes occurs in the early stage of disease. In particular, the Fas/Fas-ligand, but not TGF-b, seems to play an important role in such processes, since increase in number of apoptotic cells paralleled the expression of Fas and Fas-ligand at both mRNA and protein levels.
Acknowledgements We thank M. Ohara for reading the manuscript. We also wish to acknowledge T. Terao and Y. Tsuji for technical assistance, and all members of the Laboratory Animal Center of Nagasaki University for animal care.
References [1] Trey C. The fulminant hepatic failure surveillance study. Can Med Assoc J 1972;106:525 – 7. [2] Lidofsky S. Liver transplantation for fulminant hepatic failure. Gastroenterol Clin North Am 1993;22:257–69.
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[3] Eguchi S, Kamlot A, Ljubimova J, Lebow LT, Demetriou AA, Rozga J. Fulminant hepatic failure in rats: survival and effect on blood chemistry and liver regeneration. Hepatology 1996;24:1452 – 9. [4] Raff MC. Social controls on cell survival and cell death. Nature 1992;356:397. [5] Hirata K, Ogata I, Ohta Y, Fujiwara K. Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats. J Pathol 1989;158:157 – 69. [6] Ando K, Guidotti LG, Wirth S, et al. Class I-restricted cytotoxic T-lymphocytes are directly cytopathic for their target cells. J Immunol 1994;152:3245 – 53. [7] Mita E, Hayashi N, Iio S, et al. Role of Fas ligand in apoptosis induced by hepatitis C virus infection. Biochem Biophys Res Commun 1994;204:468 – 74. [8] Sasaki H, Matsuno T, Tanaka N, Orita K. Activation of apoptosis during the reperfusion phase after rat liver ischemia. Transplant Proc 1996;28:1908 – 9. [9] Krams SM, Egawa H, Quinn MB, Martinez OM. Apoptosis as a mechanism of cell death in a rat model of liver allograft rejection. Transplant Proc 1995;27:466 – 7. [10] Shinagawa T, Yoshioka K, Kakumu S, et al. Apoptosis in cultured rat hepatocytes the effects of tumor necrosis factor alpha and interferon gamma. J Pathol 1991;165:247 – 53. [11] Suematsu M, Suzuki H, Ishii H, et al. Early midzonal oxidative stress preceding cell death in hypoperfused rat liver. Gastroenterology 1994;103:994 – 1000. [12] Oberhammer F, Bursch W, Tiefenbacher R, et al. Apoptosis is induced by transforming growth factor-b1 within 5 hours in regressing liver without significant fragmentation of the DNA. Hepatology 1993;18:1238–46. [13] Hakuno N, Koji T, Yano T, et al. Fas/APO-1/CD95 system as a mediator of granulosa cell apoptosis in ovarian follicle atresia. Endocrinology 1996;137:1938– 48. [14] Ogasawara J, Watanabe-Fukunaga R, Adachi M, et al. Lethal effect of the anti-Fas antibody in mice. Nature 1993;364:806–9. [15] Higgins GM, Anderson RM. Restoration of the liver of the white rat following partial surgical removal. AMA Arch Pathol 1931;12:186 – 202. [16] Higami Y, Shimokawa I, Tomita M, et al. Aging accelerates but life-long dietary restriction suppresses apoptosis-related Fas expression on hepatocytes. Am J Pathol 1997;151:659 – 63. [17] Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993;75:1169 – 78. [18] Gao C, Gressner G, Zoremba M, Gressner AM. Transforming growth factor b (TGF-b) expression in isolated and cultured rat hepatocytes. J Cell Physiol 1996;167:394 – 405. [19] Su HC, Ishikawa R, Biron A. Transforming growth factor-beta expression and natural killer cell responses during virus infection of normal, nude and SCID mice. J Immunol 1993;151:4874– 90. [20] Mochizuki K, Hayashi N, Hiramatsu N, et al. Fas antigen expression in liver tissues of patients with chronic hepatitis B. J Hepatol 1996;24:1 – 7. [21] Hiramatsu N, Hayashi N, Katayama K, et al. Immunohistochemical detection of Fas antigen in liver tissue of patients with chronic hepatitis C. Hepatology 1994;19:1354 – 9. [22] Galle PR, Hofmann WJ, Walczak H, et al. Involvement of the CD95 (APO-1/Fas) receptor and ligand in liver damage. J Exp Med 1995;182:1223– 30. [23] Russell WE, Coffey RJ, Ouellette AJ, Moses HL. Type b transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proc Natl Acad Sci USA 1988;85:5126–30. [24] Bissel DM, Wang SS, Jarnagin WR, Roll FJ. Cell-specific expression of transforming growth factor-b in rat liver. J Clin Invest 1995;96:447 – 55. [25] Yamada T, Nakayama T, Saito M. Apoptosis in liver of the autopsy cases with acute hepatic failure. Int Pathol Commun 1995;3:76. [26] Ando K, Moriyama T, Guidotti LG, et al. Mechanism of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med 1993;178:1541– 54. [27] Tsutsui H, Matsui K, Nakanishi K, Higashino K, Osward IP, Inoue M. Fas – Fas ligand interactions underlie the pathogenesis of endotoxin-induced apoptotic liver injury. Hepatology 1994;20:183A.
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[28] Takiya S, Tagaya T, Takahashi K, et al. Role of transforming growth factor b1 on hepatic regeneration and apoptosis in liver diseases. J Clin Pathol 1995;43:1093 – 7. [29] Ledda-Columbano GM, Coni P, Curto M, et al. Rapid induction of apoptosis in rat liver by cycloheximide. Am J Pathol 1991;139:1099– 109. [30] Chen SC, Watanabe FD, Wang C-C, Eguchi S, Demetriou AA, Rozga J. Presence of liver necrosis is needed for for development of intracranial hypertension in acute liver failure. Hepatology 1995;22:381A.
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The Fas/Fas-ligand system functions in hepatocytes in the early stage of fulminant hepatic failure in rats Susumu Eguchi a, Sumihiro Matsuzaki a, Hikaru Fujioka a, Takehiko Koji b, Yoshikazu Higami c, Takashi Kanematsu a,* a
Department of Surgery II, Nagasaki Uni6ersity School of Medicine, 1 -7 -1 Sakamoto, Nagasaki 852 -8501, Japan b Department of Anatomy III, Nagasaki Uni6ersity School of Medicine, 1 -7 -1 Sakamoto, Nagasaki 852 -8102, Japan c Department of Pathology I, Nagasaki Uni6ersity School of Medicine, 1 -7 -1 Sakamoto, Nagasaki 852 -8102, Japan Received 18 December 1997; received in revised form 5 February 1998; accepted 10 February 1998
Abstract Apoptosis is involved in various liver diseases, but in the case of fulminant hepatic failure (FHF) much remains to be determined. We investigated apoptosis in the remnant liver lobes of FHF rats regarding the Fas/Fas ligand and the TGF-b system. Rats (n= 30) with FHF were euthanized at postoperative 3, 6, 12, 24 and 36 h. Changes in the Fas/Fas-ligand and TGF-b were studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining. Apoptosis was explored using terminal deoxynucleotidyl transferase mediated dUDP-nick end labeling method (TUNEL). Within 6 h, a relatively large number of hepatocytes, mainly in zone 3, were apoptotic. During the same period, the expression of Fas and Fas-ligand mRNA and protein, but not that of TGF-b, were observed and decreased with time. Both Fas and Fas-ligand proteins were present in the hepatocytes in zone 3. These results suggest that the Fas/Fas-ligand system, rather than TGF-b, has a role in apoptosis during the early stages of FHF, possibly through an autocrine loop. © 1998 Elsevier Science Ireland Ltd. All rights reserved. * Corresponding author. Tel.: [email protected]
+ 81 95 8497312; fax:
+81 95 8497319; e-mail: kane-
1386-6346/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S1386-6346(98)00020-5
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S. Eguchi et al. / Hepatology Research 11 (1998) 103–114
Keywords: Fas (Fas/APO-1/CD95); Fulminant hepatic failure; Apoptosis
1. Introduction Fulminant hepatic failure (FHF) is defined as the appearance of encephalopathy less than 8 weeks after the onset of symptoms in a patient without any previous liver disease [1]. The majority of patients will rapidly deteriorate and die, unless they undergo immediate liver transplantation; only a small percentage of these patients can recover and subsequently have normal liver function [2]. Despite improvement in the determination of etiology in the majority of cases, it is still unclear what specific abnormalities are pathognomonic for FHF. A novel experimental model of FHF in the rat was reported [3]. Although we have investigated and characterized pathological events in the remnant liver lobes in FHF, the role of apoptosis which is highlighted in FHF, has not been thoroughly explored. Apoptosis is thought to play a homeostatic role after mild tissue injury, leaving intact the overall cytoarchitecture of the tissue [4]. The first description of apoptosis in the liver dates back to the late 1960s, but there had been no documentation on the role of this phenomenon as a mechanism of acute liver failure. Hirata et al. [5] reported that apoptosis of fat-storing cells could be the main cause of microcirculatory failure in drug-induced liver injury. Apoptosis also occurs in liver diseases such as viral hepatitis B [6], hepatitis C [7] and ischemia-reperfusion injury [8]. Inflammatory infiltrate is characteristic of each of these liver pathologies, consistent with the possibility that infiltrating cells such as a CTL (cytotoxic T-lymphocytes) may be involved in the apoptotic process. [9] In addition, several cytokines and chemical agents were reported to induce apoptosis in hepatocytes, including tumor necrosis factor-a [10], interferon-g [10], oxygen radicals [11], TGF-b [12], and Fas (Fas/APO-1/CD95)/ Fas-ligand [13]. Fas (Fas/APO-1/CD95) is a cell surface protein that induces apoptosis in hepatocytes, under physiological and pathological conditions. The intraperitoneal administration of anti-Fas antibody induced FHF-like massive liver damage with apoptosis through Fas antigen on their hepatocytes. [14] It was concluded that accumulation of Fas ligand expressing cells around the target cells was critical for effectiveness of the Fas system. On the other hand, TGF-b has been reported as a strong inducer of apoptosis as well. [12] We examined the Fas/Fas-ligand and TGF-b systems as possible inducers of apoptosis in the liver of the rats with FHF. The transition and localization of these systems in the liver were investigated using RT-PCR and immunohistochemical staining.
2. Materials and methods
2.1. Animals Healthy adult male Sprague Dawley rats weighing 250–300 g obtained from
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Charles River Japan (Atsugi, Japan) were acclimatized to our laboratory conditions for 1 week prior to use in the experiments. They were housed in a climate controlled 21°C room under a 12 h light/dark cycle. Tap water and commercial rat chow (Oriental Kobo Kogyo, Tokyo, Japan) were provided ad libitum. All procedures were in accordance with University of Nagasaki Research Animal Resources guidelines. At the completion of the surgical procedure, each animal was given 20 ml of 5% glucose in Lactate Ringer’s solution in a subcutaneous bolus injection (s.c.).
2.2. Surgical animal models FHF was induced as described [3]. Briefly, the abdomen was entered through a midline incision. The common pedicle to the right liver lobes (24% of the liver) was ligated to induce necrosis, while the two anterior liver lobes (68% of the liver) were removed using the standard Higgins and Anderson technique. [15] The two omental liver lobes (8% of the liver) were left intact. The anterior liver lobes were fully mobilized and a ligature was placed high around their common pedicle, so that there would be no interference with the arterial blood supply to the liver remnant or impairment of venous outflow.
2.3. Experimental design FHF rats (n =30) were euthanized in batches of six rats at 3, 6, 12, 24 and 36 h, respectively. At death, the omental liver lobes were removed, frozen in liquid nitrogen for RNA extraction, or fixed in formalin. The tissue was sectioned and stained with hematoxylin and eosin and immunostained for Fas, Fas-ligand and TGF-b.
2.4. Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling After fixing with 10% buffered formalin, sections (4 mm) were deparaffinized and hydrated. Sections were reacted with 20 mg/ml proteinase K (Sigma, St. Louis, MO) at 37°C for 20 min and rinsed in distilled water. After immersion in terminal deoxynucleotidyl transferase (TdT) buffer (Gibco BRL, Gaithersburg, MO), sections were incubated overnight with TdT in TdT buffer, rinsed in buffer solution and the non-specific reaction was blocked with 10% normal rabbit serum. For visualization of incorporated biotin-16-dUTP (Boehringer Mannheim Biochemica, Mannheim, Germany), the sections were incubated with streptavidin-alkaline phosphatase conjugate for 30 min at room temperature. Sections were washed in Tris-buffered saline and alkaline phosphate substrate buffer (ASB; 0.1 mol/l Tris – HCl pH 9.5, 0.1 mol/l NaCl and 0.05 mol/l MgCl2) and incubated with the substrate solution containing 4-nitroblue tetrazolium chloride (NBT, Gibco BRL, Gaithersburg, MO) and 5-bromo-4-chloro3-indolylphosphate (BCIP, Gibco BRL, Gaithersburg, MO) in ASB.
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2.5. Apoptotic index For an apoptotic index in the remnant liver, we calculated as the number of terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL)-positive hepatocytes per 1000 hepatocytes. For each liver, hepatocytes in 100 consecutive high power fields (hpfs) were counted.
2.6. Fas, Fas-ligand and TGF-b mRNA expression RNA expression of Fas and TGF-b in the liver tissue was detected using RT-PCR. From the livers kept frozen in liquid nitrogen until assay, total RNA was extracted according to the RNAzol B (TEL-TEST, Friendswood, TX) protocol. Single strand complementary DNA (cDNA) was synthesized by RT using the random primer and 1 mg of mRNA in 10 ml of reaction mixture. PCR primers were synthesized for rat Fas [16], rat Fas-ligand [17], rat TGF-b [18] and rat b-actin [16] cDNA. The 5%-primer of the rat Fas was 5%CAAGGGACTGATAGCATCTTTGAGG-3% and the 3%-primer of the rat Fas was 5%-GTCCTTAACTTTTCGTTCACCCAGG-3% (PCR product size= 141 bp). The 5%-primer of the rat Fas-ligand was 5%-TCCACCACCACCTCCATCAC-3% and the 3%-primer of the rat Fas-ligand was 5%-CCAACCTTACCCCAATCCTT3% (PCR product size=152 bp). The 5%-primer of the rat TGF-b was 5%GGACTCTCCACCTGCAAGAC-3% and the 3%-primer of the rat TGF-b was 5%-CTCTGCAGGCGCAGCTCTG-3% (PCR product size = 390 bp). The 5%primer of the rat b-actin was 5%-CACTGCCGCATCCTCTTCCT-3% and the 3%primer of the rat b-actin was 5%-AGCCACCAATCCACACAGAG-3% (PCR product size= 347 bp). Relative levels of mRNA were detected using equal amounts of input single-stranded DNA (2 ml per 30 ml) and tested in parallel using GeneAmp RNA PCR kits (Perkin Elmer, Norwalk, CT). PCR consisted of denaturation at 94°C, annealing at 60°C and extension at 72°C using a Thermal Cycler (Perkin Elmer, Norwalk, CT). After staining with ethidium bromide, gels were photographed using Polaroid film (Fuji Film, Tokyo, Japan). In preliminary studies, the parallel amplification efficiency of PCR for different targets was determined after several different amounts of initial DNA template, as reported [16]. The optimal number of thermal cycles for Fas, Fas-ligand, TGF-b and b-actin was determined to be 28, 32, 25 and 22 cycles, respectively. Gel images showing ethidium bromide fluorescence of each band in stored images were analyzed using a computer software system (NIH image, Version 1.61).
2.7. Immunohistochemical staining for Fas, Fas-ligand and TGF-b1 Immunohistochemical staining was performed using an indirect, two-step labeling technique with peroxidase-conjugated IgG. Polyclonal anti-mouse Fas (P4; dilution, 1:200)/anti-rat Fas-ligand (P5; dilution, 1:100) antibodies from
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rabbit were used [13,16]. Sections (4 mm thick) were deparaffinized in toluene and rehydrated by passing through a graded series of ethanol. Endogenous peroxidase was blocked by incubation of the slides with 0.3% H2O2 in methanol for 15 min at room temperature. After washing in phosphate-buffered saline (PBS), 500 mg/ml of normal goat IgG in 1% bovine serum albumin (BSA)/PBS was applied for 60 min at room temperature for blocking. Slides were incubated overnight with anti-Fas and anti-Fas ligand antibodies for 2 h, and subsequently exposed to prediluted goat antirabbit peroxidase-conjugated IgG (1:400) for 40 min at room temperature. The peroxidase reaction was developed by incubating in H2O2 and 3,3%-diaminobenzidine tetrachloride. As a control, the adjacent sections were reacted with normal rabbit IgG in the presence of the specific antibody. Counterstaining was performed with methylgreen. All washing procedure was done using 0.075% Brij 35 (Polyoxyethylene Lauryl Ether, Nacarai Tesque, Kyoto, Japan) in PBS. For immunodetection of TGFb1 protein, the liver sections were exposed to microwaves to retrieve antigenicity. Chicken polyclonal anti-human TGF-b1 antibody (R&D Systems, Minneapolis, MN) was used followed by detection using secondary anti-chicken IgY alkaline phosphatase conjugate (Promega, Madison, WI) with our modification. [19] Sections were washed in Tris-buffered saline and ASB and incubated with the substrate solution containing NBT and BCIP in ASB.
2.8. Statistical analysis Data were analyzed using one-way ANOVA. P values equal or less than 0.05 were considered significant. Data are presented as means9 S.D.
3. Results Our FHF model made evident many aspects of clinical FHF in a highly reproducible manner and liver regeneration in remnant intact lobes was completely halted as reported. Mean survival time was 399 11 h [3].
3.1. Apoptotic index Apoptotic index, expressed as the number of TUNEL-positive hepatocytes within 1000 hepatocytes in the remnant omental lobe peaked as early as 3 h following induction of FHF, as shown in Fig. 1 (PB0.05), a significant finding compared to the subsequent period. It then decreased with time and almost no TUNEL-positive cell was detected at 36 h like baseline. Apoptotic cells were hepatocytes mainly around central vein (zone 3).
3.2. Expression of Fas, Fas-ligand and TGF-b mRNA in remnant li6er lobes In FHF rats, mRNA of Fas was upregulated at 3 h and subsequently decreased
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Fig. 1. Apoptotic index in the remnant liver of FHF rats. For the indicated times, data are plotted as means9 S.D. Apoptotic indices in the liver remnant was calculated as the number of TUNEL-positive hepatocytes per 1000 hepatocytes. In each liver, hepatocytes in 100 consecutive high power fields (hpfs) were counted. *P 5 0.05.
to baseline level. Fas-ligand mRNA was detected for up to 6 h post-induction, then was undetectable. TGF-b mRNA was detectable after 6 h and upregulated until expiration (Fig. 2). The baseline expression of Fas mRNA was minimal and that of Fas-ligand was undetectable. TGF-b mRNA was undetectable at baseline, while b-actin mRNA was constantly expressed at all time point.
Fig. 2. Fas, Fas-ligand, TGF-b and b-actin gene expression in the remnant liver of FHF rats. Animals were killed at 0, 3, 6, 12, 24 and 36 h following surgery. After extraction of RNA, 1 mg of total RNA was used for RT-PCR analysis, as described.
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Fig. 3. Immunohistochemical staining of FHF livers using anti-Fas antibody. (A) Sham liver stained with anti-Fas antibody ( × 100). (B) FHF liver at 3 h stained with anti-Fas antibody ( × 100). (C) FHF liver at 36 h stained with anti-Fas antibody ( ×100). (D) Positive staining was seen in hepatocytes at 3 h (×400).
3.3. Histological changes in remnant li6er tissue The omental lobes in FHF rats had an interstitial edema and extensive lipid vacuolization of the hepatocytes. The derangement was most evident in specimens of the 36-h group. Congestion was present in the whole sample thereby reflecting portal hypertension. In the 3- and 6-h groups, apoptotic cells, defined as nuclei displaying chromatin condensation, were relatively more as compared to the group in late stage (36 h).
3.4. Immunohistochemical staining of the Fas and Fas-ligand Fas protein was minimal in sham liver. Staining for Fas protein was present in hepatocytes around central vein (zone 3) at 3–6 h following induction of FHF (Fig. 3). At this same period, Fas-ligand was expressed in hepatocytes in a zone identical with that for Fas protein (Fig. 4). Subsequently, expression of Fas and Fas-ligand protein decreased with time and the general condition of the rats deteriorated and they died. Location of expression of Fas and Fas-ligand was in the plasma membrane and cytoplasm of hepatocytes (Fig. 3D and Fig. 4D).
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3.5. Immunohistochemical staining of the TGF-b TGF-b protein was negative for up to 12 h following the induction of FHF. It began to be expressed at 24 h and was most evident at 36 h when rats were in coma and about to die. TGF-b protein was evident in the cytoplasm of hepatocytes rather than non-parenchymal cells (Fig. 5).
4. Discussion We observed apoptosis in hepatocytes in the early stages of FHF, an event which coincided with expression of the Fas/Fas-ligand but not the TGF-b system. Expression of Fas protein and Fas-ligand protein was evident in hepatocytes, not infiltrating lymphocytes, thereby indicating a possible autocrine loop of apoptotic process. As far as Fas (Fas/APO-1/CD95) in liver disease is concerned, Ogasawara et al. [14] a reported lethal effect of anti-Fas antibody on mice to induce FHF through apoptosis. Aside from FHF, in liver tissue of patients with chronic hepatitis B [20] and hepatitis C [21], Fas was involved. Galle et al. [22] reported the localization of
Fig. 4. Immunohistochemical staining of FHF livers using anti-Fas-ligand antibody. (A) Sham liver stained with anti-Fas-ligand antibody ( ×200). (B) FHF liver at 3 h stained with anti-Fas-ligand antibody ( × 200). Brown stained hepatocytes represent positive staining. (C) FHF liver at 36 h stained with anti-Fas-ligand antibody ( ×200). (D) Positive staining was seen in hepatocytes at 3 h ( ×400).
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Fig. 5. Immunohistochemical staining of FHF livers, using anti-TGF-b antibodies. (A) Sham liver stained with anti-TGF-b antibody ( × 100). (B) FHF liver at 3 h stained with anti-TGF-b antibody (× 100). (C) FHF liver at 36 h stained with anti-TGF-b antibody ( ×100). (D) Positive staining was seen in hepatocytes rather than non-parenchymal cells at 36 h ( × 400).
mRNA of Fas ligand in liver cirrhosis developed from hepatitis B and alcohol-related liver damage. The signal of the Fas-ligand was found in infiltrated lymphocytes in the presence of viral disease, while in alcohol-related disease, the Fas ligand was found in hepatocytes. On the other hand, TGF-b is a multifunctional cytokine with diverse effects on cell growth, differentiation and function. Apoptosis can be induced by TGF-b and accounts for the inadequate hepatic regeneration that occurs with liver disease [23]. It has recently been reported that TGF-b was produced in not only non-parenchymal cells but also in hepatocytes during liver regeneration and has some role through an autocrine loop. [24] In our model of FHF, TGF-b did not play a role in inducing apoptosis. There was no evidence of liver regeneration in our expired animals used as models of FHF. [3] No relationship was found between TUNEL positive hepatocytes and proliferation with PCNA. [25] It would thus appear that apoptosis has little effect on subsequent liver regeneration. In the present study, TGF-b was upregulated from 6 h on. Therefore, TGF-b may have the role on insufficient liver regeneration, since the HGF/c-met system as stimulator was upregulated after 18 h following induction of FHF. [3] We found that apoptotic death of hepatocytes was evident within 6 h following the induction of FHF, which means suggesting that apoptosis may be the pathognomosis in the early stage of FHF. The expression of mRNA and protein of the
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Fas/Fas-ligand in hepatocytes coincided with the occurrence of apoptosis of hepatocytes, while that of TGF-b was observed from 6 h, thus preceding apoptosis by 6 h. In addition, the localization of Fas/Fas-ligand proteins was concomitant with that of apoptosis. These results suggest that the Fas/Fas-ligand system rather than TGF-b might give rise to apoptosis in hepatocytes in remnant hepatocytes in FHF. With regard to apoptosis in FHF, there have been reports, including a transgenic mouse model of fulminant hepatitis [26], liver injury induced by endotoxin [27] and thioacetamide. [26] In clinical fulminant hepatitis, TGF-b was expressed in both metaplastic hepatocytes and adjacent metaplastic bile duct forming cells, while Fas antigen was positive only on the cell membrane of bile duct forming cells, but not on hepatocytes. [28] In addition, expression of Fas antigen was not detectable at the sites where TUNEL was positive, thus suggesting that induction of apoptosis was not mediated by Fas antigen in their clinical samples with various liver diseases. However, the clinical stage in which they obtained specimens was not standardized. As we demonstrated in the present study, apoptosis seems to be involved in the early stage of FHF and it would be very difficult to obtain such specimens in a clinical setting. Our finding is in keeping with the report of Ledda-Columbano et al. [29] in which apoptosis was evident within 6 h, while necrosis was observed at 24 h accompanied by massive inflammatory reactions. In hepatocytes under oxidative stress or the influence of cytokines, the signal of the Fas/Fas-ligand might be up-regulated so that injured hapatocytes are eliminated through apoptotic cell death to maintain homeostasis. It is intriguing that interruption of the Fas/Fas-ligand system using a neutralizing anti-Fas antibody can prevent the advance of disease. In addition, the presence of necrosis in our model may play some role in enhancing the vicious circle of cytokines and toxic substances specific in FHF. [30] It is concluded that, in the liver of rats with FHF, apoptosis of remnant hepatocytes occurs in the early stage of disease. In particular, the Fas/Fas-ligand, but not TGF-b, seems to play an important role in such processes, since increase in number of apoptotic cells paralleled the expression of Fas and Fas-ligand at both mRNA and protein levels.
Acknowledgements We thank M. Ohara for reading the manuscript. We also wish to acknowledge T. Terao and Y. Tsuji for technical assistance, and all members of the Laboratory Animal Center of Nagasaki University for animal care.
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