Protective effect of hepatocyte growth factor on interferon-gamma-induced cytotoxicity in mouse hepatocytes

Protective Effect of Hepatocyte Growth Factor on InterferonGamma-Induced Cytotoxicity in Mouse Hepatocytes MASAHIKO MORITA, YOSHIFUMI WATANABE, AND TOSHIHIRO AKAIKE

We examined the interactive effect of several eytokines (interleukin-1 beta [IL-lfi], tumor necrosis factor alpha [TNF-a], interferon gamma [IFN-T], IL-6, IFN-a/~, and hepatocyte growth factor [HGF]) presumably involved in hepatitis, on primary cultured murine hepatoeytes. Among these cytokines, only IFN- T induced LDH release from hepatoeytes in both time- and dose-dependent fashions. The eytotoxic effect was inhibited by antiserum-containing anti-mouse IFN-~, monoclonal antibodies (R4-6A2). Moreover, intriguingly, IFN-~/induced DNA fragmentation in the hepatocytes in a time- and dose-dependent fashion according to the gel electrophoresis of genomic DNA and flow cytometry analysis. These results suggest that the cytotoxic effect of IFN-T on hepatocytes was caused by inductive apoptosis. The LDH release and DNA fragmentation induced by IFN-T were inhibited by HGF in a dose-dependent manner, whereas they seemed to be accelerated by TNF-~. Flow cytometry analysis of the nuclei of treated hepatocytes confirmed the interactions in DNA degradation. The DNA synthesis of cultured hepatocytes was also reduced by IFN-?/but recovered by hepatocyte growth factor. Taken together, IFN-~/is presumed to be a critical cytokine in hepatic damage, and the network composed of IFN-~/, TNF-~, and HGF may play an important role in the regulation of liver injury. (HEPATOLOGY1995;21:1585-1593.)

Hepatitis is an inflammatory liver disease induced by various causes (such as viral infection, bacterial infection, alcohol, and drug injury). Although there are many unsolved problems relative to the mechanisms, it is clear that hepatocytes are the major target cells damaged in hepatitis. However, it is not clear what kind of molecules and what kind of regulation are criti-

Abbreviations: TNF-a, tumor necrosis factor alpha; IL, interleukin; IFN, interferon; HGF, hepatocyte growth factor; mr, murine recombinant; hr, human recombinant; Ig, immunoglobulin; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5diphenyl tetrazolium bromide; EGTA, ethyleneglycol bis(2-aminothyl ether)tetraacetic acid; FCS, fetal calf serum; EGF, epidermal growth factor; LDH, lactose dehydrogenase; BrdU, 5'-bromo-2'-deoxy-uridine; EDTA, edetic acid. From the Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohoma, Japan. Received March 9, 1994; accepted December 19, 1994. Address reprint requests to: Dr Yoshifumi Watanabe, Department of BiomolecuIar Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226, Japan. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2106-001653.00/0

cally involved in the hepatic damage. It has been reported that tumor necrosis factor alpha (TNF-a), interleukin (IL)-I and -6, and interferon-gamma (IFN-~) are important mediators in inflammation. Increased production of these inflammatory cytokines is often detected in the cases of patients with hepatitis 1'2 or in a liver infected with bacteria, z and they are considered to play important roles in the onset of hepatitis. 4 For example, there are some cases in which TNF-a or IFNis proven to be a critical factor in the induction of hepatitis in m i c e Y In addition, IFN-~/receptors were expressed on hepatocytes in a diseased liver but not in a normal liver. 7 It is also suggested that IFN-~ makes hepatocyte antigen presenting cells in the liver and initiates an inflammatory reaction because IFN-~ induced MHC class II expression on h u m a n hepatocytes, s Hepatocyte growth factor (HGF) was originally found to be a potent mitogenic factor for hepatocytes. 9 After this discovery, the factor has been reported to be a multifunctional cytokine having mitogenic, motogenic, morphogenic, and tumoricidal activities. 9 Moreover, it has a protective effect on hepatocytes from experimental hepatic injury. 1°'11 However, the questions of which cytokine is the substantial cytotoxic or protective factor for hepatocytes and how the network of these cytokines functions in the regulation of hepatic injury have not been resolved. Apoptosis is a typical form of programmed cell death to eliminate unwanted cells in the development of the immune system, organ formation, and embryogenesis. ~2 The characteristic features of apoptosis are condensation and fragmentation of nuclear chromatin, accompanied by compaction of cellular organelles, dilatation of the endoplasmic reticulum, and a marked reduction in cell volume. ~2 It is also presumed to be involved in the mechanisms of cell death by antitumor drugs, ~3 cytotoxic T cells or natural killer cells, ~4 and antitumor cytokines such as TNF-a. 1~ Although in the liver, apoptosis is considered to be involved in the normal regulation of liver size, it has been reported recently that Fas (also designated APO-1) antigen, which is a membrane-associated antigen that induces apoptosis through signal transduction, ~6'17is expressed on hepatocytes and m a y play an important role in inducing fulminant hepatitis via apoptosis, is Therefore, apoptosis is expected to be an important mechanism in the pathogenesis of hepatitis.

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I n t h i s study, we e x a m i n e d t h e direct effects of cytok i n e s s u p p o s e d l y i n v o l v e d in h e p a t i t i s on h e p a t o c y t e s i n vitro a n d f o u n d t h a t I F N - ~ is a cytotoxic c y t o k i n e for p r i m a r y c u l t u r e d m o u s e h e p a t o c y t e s a n d i n d u c e s a p o p t o s i s in t h e s e cells. M o r e o v e r , t h e cytotoxic, a p o p t o s i s - i n d u c i b l e f u n c t i o n of I F N - ~ w a s i n h i b i t e d b y H G F . T h e i n t e r a c t i o n b e t w e e n t h e s e c y t o k i n e s is envisa g e d to p l a y i m p o r t a n t roles in t h e p a t h o g e n e s i s of hepatitis. MATERIALS AND METHODS

Reagents and Animals MTT (3-(4,5-dimethylthiazol-2-y])-2,5-diphenyl tetrazolium bromide) and propidium iodide were purchased from Sigma (St. Louis, MO). Murine recombinant IFN-~/(mrIFN7) and TNF-a were gifts from Genentech Inc. (South San Francisco, CA). H u m a n recombinant HGF (hrHGF) was a gift from Snow Brand Milk Products Co. Ltd. (Tokyo, Japan). MrIL-1/~ and IL-6 were purchased from R & D Systems (Minneapolis, MN). The hybridoma (R4-6A2,19) producing antimurine IFN-~ immunoglobulin (Ig) G2 antibodies was obtained from American Type Culture Collection (Maryland, USA). The cells were transplanted into the peritoneum of female Balb/c nude mice (7 weeks of age) (Jcl:AF-nu, Clea Japan, Inc., Kanagawa, Japan), and the antiserum was employed in the experiments. Collagen type I for coating culture dishes was a gift from Kawasumi Laboratories Inc. (Tokyo, Japan). Female Balb/c (6 to 12 weeks of age) mice used in the experiments of this study were purchased from Charles River J a p a n Inc. (Kanagawa, Japan). All animal experiments were performed in accordance with local institutional guidelines for the care and use of laboratory animals.

Cell Preparation Parenchymal hepatocytes were isolated from an adult mouse by the modified in situ perfusion method. 2°'21 Briefly, the liver was first perfused in situ through the thoracic inferior vena cava with Ca2+-free Hank's solution supplemented with 5 mmol/L ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid (EGTA) and 5 mmol/L glucose at 37°C until the blood in the liver was completely removed. Then the solution was exchanged with 0.0125% collagenase solution. After a few minutes of perfusion, the liver was excised, dispersed in cold Hank's solution, and the resulting cell suspension was filtered through 300-gauge mesh. Parenchymal hepatocytes were separated from nonparenchymal cells by differential centrifugation at 50 g for 90 seconds. After being washed once, the dead parenchymal hepatocytes were removed by density gradient centrifugation on Percoll (Pharmacia). The live parenchymal hepatocytes were suspended in Williams' E medium containing 10% FCS, 20 ng/mL epidermal growth factor (EGF), 10 -9 mol/L insulin and antibiotics in RPMI1640 with 10% FCS and were plated at a density of 2.7 × 10 ~ cells per well, 1.3 × 105 cells per well and 3 × 104 cells per well in flat-bottomed 6-well, 24-well and 96-well (respectively) plates (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) precoated with collagen. The purity of the hepatocytes was confirmed by microscopic observation counting and flow cytometry analysis. Only when the purity was more than 98% were the isolated hepatocytes subjected to the following experiments. The hepatocytes were incubated at 37°C for 10 hours so that they would adhere to the collagen-coated plates and were washed before being subjected to the experiments. When the hepatocytes were treated with ]ymphokines, EGF and insulin were

HEFATOLOGYJune 1995 removed from the medium to eliminate the effect of potent interaction between lymphokines and these growth factors on hepatocytes.

Cytotoxie Assay of Hepatoeytes Lactose Dehydrogenase Release Assay. The activity of lactose dehydrogenase (LDH), a stable cytosolic enzyme that is released on cell lysis and one of the commonly used hallmarks of cellular cytotoxicity, 2e in the supernatants of treated hepatocytes was measured using a CytoTox 96 Nonradioactive Cytotoxicity Assay Kit (Promega, Madison, W I ) y following the manufacturer's instructions. The percentage of lysis was calculated using the formula: % LDH release = 100 × (Experimental release - Spontaneous release) (Maximum release - Spontaneous release). Maximum release was obtained by complete solubilization of hepatocytes with 0.1% Triton X-100. Evaluation of DNA Synthesis Induced by Cytokines. The DNA synthesis of treated hepatocytes was also evaluated by the incorporation of 5-bromo-2'-deoxy-uridine (BrdU) 24 with a BrdU labeling and detection kit III (Boehringer Mannheim Biochemica, Mannheim, Germany), following the manufacturer's instructions. Briefly, BrdU was added to treated hepatocytes plated in a 96-well, flat-bottomed microtiter plate, and the cells were incubated for 4 hours at 37°C. The cells were then washed and fixed with HCl-ethanol at -20°C for 40 minutes. The cells were treated with nuclease, and they reacted with peroxidase conjugated monoclonal anti-BrdU antibodies. The substrate (ABTS) for peroxidase was added after washing, and the mixture was incubated at room temperature for 5 minutes with an enhancer. The absorbance of each well at 415 nm was measured using a micro plate reader, MTP-120 (Corona Electronic Co., Ltd., Ibaragi, Japan).

MTT Assay The viability of treated hepatocytes was also evaluated by MTT assay. 2~ Briefly, MTT was added to treated hepatocytes plated in a 96-well, flat-bottomed plate at a final concentration of 500 mg/mL, and the cells were incubated for 4 hours at 37°C. One hundred #L of acidic isopropyl alcohol was then added to each well, and the solution was vigorously mixed to solubilize the reacted dye. The absorbance of each well at 550 nm was measured using a micro plate reader, MTP-120 (Corona Electronic Co., Ltd., Ibaragi, Japan).

Analysis of Chromosomal DNA DNA Isolation and Agarose Gel Electrophoresis. DNA was isolated according to the method described by Sambrook et al 2~ with minor modification. Briefly, cells were incubated with the ]ysis buffer (10 #g/mL; proteinase K [Sigma, St. Louis, MO], 10 mmol Tris, 150 mmo]/L NaC1, I mmol/L edetic acid [EDTA], 1% SDS) for 15 hours at 37°C. Chromosomal DNA was obtained by phenol/chloroform (1:1) extraction and ethanol-precipitation. The samples in TE solution (10 mM Tris-HC1 [pH 8.0], 1 mmol/L EDTA) with 1 #g/mL RNase were incubated for i hour at 37°C. The same amount of DNA from each sample was subjected to electrophoresis through 1.0% agarose gel containing 0.1 #g/mL ethidium bromide. Flow Cytometry Analysis. Flow cytometry analysis of nuclei from hepatocytes was performed as described by Nicoletti et al. 27 Hepatocytes were suspended in 0.1 mol/L citrate

HEPATOLOGY Vol. 21, No. 6, 1995

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antibodies was carried out to eliminate the possibility that any contaminated unknown factors in the sample were the substantial toxins for hepatocytes. According to the results shown in Fig. 2, although control rat IgG did not have any effect on the IFN-3,-induced LDH release, anti-IFN-y serum inhibited the cytotoxicity of IFN-3, in a dilution-dependent way. These data demonstrate that the substantial cytotoxic factor for hepatocytes is presumed to be IFN-T. Moreover, the time course analysis of LDH release by IFN-T showed that the release was time dependent (Fig. 3). Significant LDH release was observed at 24 hours after IFN-7 treatment, and it gradually increased with time as shown in the figure. Finally, it reached the maximum release, 100% at 72 hours.

130

9o 1 ©

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1587

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10-1

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FIG. 1. Comparative effects of various cytokines on LDH release of hepatocytes. Murine hepatocytes (3 × 104 cells per well) were treated with IFN-7, TNF-a, IL-lfl, HGF, IFN-a/fl, or IL-6 at various concentrations for 48 hours, and t h e n the LDH activities of the supern a t a n t s were measured. The LDH release was expressed as % release as described in the Materials and Methods section. II, IFN-3, (U/mL); @, TNF-a (ng/mL); 0 , IL-1/3 (ng/mL); O, HGF (ng/mL); [], IFN-(a/ /3) (U/mL); and A, IL-6 (ng/mL).

HGF Suppresses IFN-T Induced Hepatic Cytotoxieity It has been reported that TNF-a is one of the critical cytokines involved in hepatic injury. 5'6 On the other hand, H G F has been reported to have a protective effect on hepatocytes from experimental hepatic injury, l°'n Therefore, we investigated the effects of these cytokines combined with IFN-~/on the LDH release ofhepatocytes. According to the results of Fig. 1, it is already confirmed that neither TNF-a or HGF alone has any significant effect on hepatic LDH release. However,

buffer (pH 7.2) containing 0.1% Triton X-100 and incubated at 37°C for 30 minutes. The tubes were vortexed and centrifuged, and the resultant pellets were washed twice with ethanol and stained with 10 #g/mL propidium iodide in the citrate buffer at room temperature for 20 minutes. The nuclei were subjected to flow cytometry analysis using Cyto Ace-150 (Japan Spectroscopic Co. Ltd., Tokyo, Japan) at wavelengths of 488 nm for excitation and 530 nm for emission.

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RESULTS

IFN-T is a Cytotoxic Cytokine for Mouse Hepatocytes First, we investigated the effect of several cytokines (IFN-7, TNF-a, IL-1/3, IL-6, HGF, and IFN-a/fl) that are presumed to be involved in hepatitis or hepatic injury on the LDH release from hepatocytes as a hallm a r k of cellular cytotoxicity (Fig. 1). One of the commonly used direct toxins for hepatocytes, C C l 4 , 2 s as a positive control induced almost 100% LDH release of hepatocytes, whereas only IFN-T among the examined cytokines showed a cytotoxic effect on hepatocytes in a dose-dependent fashion (Fig. 1). The LDH release induced by IFN-3, reached a plateau at a concentration of more than 10 U/mL. Other inflammatory cytokines such as TNF-a, IL-1/~, and IL-6, which are frequently suggested to be critical in hepatic injury, did not induce any significant LDH release. We obtained the same results by evaluating other hepatic injury evidence, i.e., glucose oxidase test and glutamic pyruvic transaminase activities of the supernatants. Furthermore, the results were not influenced by the removal of serum from the medium in the assay. Because IFN-3, has not to our knowledge been reported to be a direct cytotoxic factor for hepatocytes, an inhibition test by monoclonal anti-murine IFN-7

40

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IFN-7 ( lOOU/ml ) FIG. 2. Inhibition by anti-IFN-3, a n t i s e r u m of I F N - T - i n d u c e d LDH release from cultured hepatocytes. Mouse hepatocytes were cultured with 100 U/mL of IFN-T in the absence or presence of diluted anti-IFN-T a n t i s e r u m or r a t IgG (10 #g/mL) as a control for 48 hours. The LDH activities of the s u p e r n a t a n t s were measured and compared as percentage release.

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1). The I F N - y - i n d u c e d DNA fragmentation was also inhibited by HGF in a dose-dependent fashion, as in the case of the LDH release (Fig. 7). In this case, 10 ng/mL of HGF conferred complete suppression of the DNA fragmentation by IFN-y.

120

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Flow Cytometry Analysis Confirmed that HGF Suppressed the DNA Fragmentation Induced by I F N - y

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To compare the degree of the DNA degradation induced by cytokines, flow cytometry analysis of the

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Time (h) FIG. 3. Time-course analysis of the LDH release from I F N - y treated hepatocytes. Murine hepatocytes were cultured with 100 U/ mL of IFN-y for various times, and the LDH activities of the supernarants were measured and expressed as percentage release. (© ©) control, (A &) IFN-y.

40

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20 when these cytokines were used in the presence of IFNy, TNF-a slightly enhanced the cytotoxicity induced by IFN-y whereas HGF antagonized IFN-y during the LDH release in a dose-dependent fashion, even though the concentration of HGF required to prevent the cytotoxicity of IFN-y on hepatocytes was relatively high compared with the physiological concentrations (Fig. 4).

control

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DNA Fragmentation Induced by IFN-T in Hepatocytes was Inhibited by HGF

Fragmentation of chromosomal DNA into 180 to 200 base pair pieces results in a ladder pattern in agarose electrophoresis, which is commonly accepted as a typical biochemical qualification of apoptosis. 14 Because it was difficult to determine the form of the hepatocyte death induced by IFN-T through only morphological observation with phase contrast microscopy, we examined the DNA fragmentation of I F N - y - t r e a t e d hepatocytes. Time-course analysis showed that IFN-y induced DNA fragmentation in hepatocytes with incubation time (Fig. 5). DNA fragmentation was observed after 48 hours of incubation with IFN-% and the results of time course analysis of both DNA fragmentation (Fig. 5) and LDH release (Fig. 3) agree. Furthermore, as shown in Fig. 6, only IFN-y induced DNA fragmentation in hepatocytes in a dose-dependent fashion, although neither TNF-c~ nor IFN-a/fl showed any effect on DNA fragmentation as expected from the results of the LDH release assay (Fig. 1). In addition, neither HGF nor IL-lfl by itself induced DNA fragmentation in hepatocytes (data not shown). The result in which 10 U/mL of IFN-y caused DNA fragmentation in hepatocytes also closely matches that of LDH release (Fig.

0

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HGF (ng/ml) + IFN-y ( lOOU/ml ) FIG. 4. Opposing effects of TNF-a or HGF on the LDH release by I F N - y - t r e a t e d hepatocytes. Murine hepatocytes were cultured with 100 U/mL of IFN-y for 48 hours in the presence of either TNFor HGF at various concentrations, and the LDH activities were measured and expressed as percentage release.

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IFN-y-stimulated time ( h ) IFN- 7 (U/mi)

0 12 24 36 48 60 72

C 10 -1 10 o 101 10 2 10 3

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FIG. 5. Time-course analysis of DNA fragmentation induced by IFN-y in murine hepatocytes. Murine hepatocytes (4 x 105 cells) were incubated with 100 U/mL IFN-y for various times, t h e n the DNA was extracted and subjected to electrophoresis t h r o u g h 1% agarose gel.

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B apoptotic nuclei was performed. The reduced DNA content of apoptotic nuclei resulted in an unequivocal hypodiploid DNA peak in the red fluorescence channels when the nuclei were stained with propidium i o d i d e s The results were the same as those of electrophoretic analysis; IFN-y but not TNF-a alone induced DNA degradation in the nuclei of hepatocytes (Fig. 8C and 8D), and HGF clearly inhibited the degradation (Fig. 8F). However, as shown in Fig. 8E, the addition of TNF-a seemed to increase the degree of the DNA degradation induced by IFN-% These results of flow cytometry analysis confirmed those of DNA fragmentation through gel electrophoresis in Figs. 6 and 7. These results taken together show that both LDH release and DNA fragmentation induced by IFN-y are suppressed by HGF but slightly accelerated by TNF-a, although neither of the cytokines has any effect on the DNA of hepatocytes independently. Effect of HGF on IFN- y-Inducible Suppression of DNA Synthesis in Hepatocytes

Finally, the DNA synthesis of hepatocytes treated with cytokines was measured by the incorporation of BrdU, which is supposed to be compatible with the

FIG. 6. IFN-y but not T N F - a - i n d u c e d DNA fragmentation of m u r i n e hepatocytes in a dose-dependent fashion. Murine hepatocytes were cultured with (A) IFN-% (B) TNF-a, or (C) IFN-a//3 for 48 hours at various concentrations. After the incubation, the DNA was extracted and subjected to 1% agarose electrophoresis.

IFN-(a/{3) (ng/ml)

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HEPATOLOGYJune 1995

r u m in the medium, because serum contains multiple factors, including growth factors and cytokines. Therefore, the effects of IFN-T or HGF were compared in the presence or absence of serum for both LDH release and DNA fragmentation. As shown in Fig. 10A, the presence of serum at 10% in the medium did not affect the cytotoxicity of IFN-7 at any concentration examined for LDH release; however, the protective effect of HGF was enhanced by serum for both LDH release and DNA fragmentation (Fig. 10B and 10C) although the serum alone at the same concentration did not suppress the cytotoxicity by IFN-%

DISCUSSION

FIG. 7. HGF suppressed the DNA fragmentation induced by IFNin hepatocytes. Murine hepatocytes (4 × 10~ cells) were incubated with 100 U/mL of IFN-T for 48 hours in the presence of HGF at various concentrations (0, 1, 10, 100 ng/mL). After the incubation, the DNA was extracted and subjected to electrophoresis.

incorporation of 3H-thymidine in most cases. ~4After 24 hours of incubation with tested cytokines, t r e a t m e n t of hepatocytes with IFN-~/almost thoroughly suppressed the DNA synthesis as described, ~9 and the addition of HGF completely restored the DNA synthesis to the control level (Fig. 9). To normalize these results for cell number, the viability of the same treated cells was measured by MTT assay because it was possible that some treatments affect cell viability. However, as shown in Fig. 9, the viability of treated hepatocytes was invariable among these treatments within 24 hours, which is consistent with the results of timecourse analysis (Figs. 3 and 5). This effect is specific for HGF because other hepatic mitogens, EGF, or basic fibroblast growth factor did not show the same effect as HGF (data not shown). In this case, DNA synthesis stimulated by HGF appears to be relatively low compared with the control, because the experiment was carried out in the presence of 10% serum and hepatocytes were plated at a low cell density, under which conditions the DNA synthesis of the control hepatocytes is relatively active. Therefore, it is also noteworthy that HGF recovered the suppressed DNA synthesis by IFN-T in hepatocytes, even in the presence of serum, although the mitogenic effect is masked under these conditions. The Presence of Serum Did N o t Affect IFN-v-Induced Cytotoxicity but E n h a n c e d the P r o t e c t i v e Effect of HGF

The question should be asked whether the effect of IFN-T or HGF is derived from the interaction with se-

In this study, we investigated the interactive effects of cytokines presumably involved in the pathogenesis of hepatic injury to primary cultured mouse hepatocytes. IFN-% TNF-a, IL-1, and IL-6 are known as inflammatory cytokines and have several effects on hepatocytes, i.e., induction of NO-, 29 changes in glucose m e t a b o l i s m y and acute phase gene expression. 31'~2Because it has been reported that T lymphocytes and neutrophils invaded the liver in hepatitis, 33'34 these imm u n e cells and liver specific tissue macrophages, Kupffer cells, 4 are presumed to be the sources of in-

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log fluoresense intensity FIG. 8. Flow cytometry analysis of nuclei from hepatocytes treated with various cytokines. Murine hepatocytes were cultured with HGF (100 ng/mL), TNF-a (100 ng/mL), IFN-T (100 U/mL), IFN(100 U/mL) +TNF-a (100 ng/mL) or IFN-T (100 U/mL) +HGF (100 ng/mL) for 48 hours, and the nuclei were extracted, stained with propidium iodide, and then analyzed with the flow cytometry.

HEPATOLOGYVol. 21, No. 6,1995

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0.1

1.0

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75 flammatory cytokines including IFN-3, and TNF-a. It has been a controversial problem as to which cytokine (or cytokines) among these cytokines is substantially lethal to hepatocytes. TNF-a has been a potent candidate because TNF-a production is increased in the serum of patients with alcohol-induced hepatitis, 1 with chronic liver disease, 2 or in mice treated with galactosamine/endotoxin in which a fulminant-type hepatic necrosis is observed. 3~ In addition, TNF-a instead of lipopolysaccharide induced hepatic necrosis in these mice. ~ Moreover, it is intriguing t h a t either TNF-a or IFN-7 has been reported to induce hepatitis in transgenic mice with a hepatitis B virus envelope. 6 It has also been reported t h a t IFN-~/ receptors are expressed on hepatocytes in a liver with hepatic disease but not in a normal liver 7 and t h a t IFN-3, suppressed liver regeneration after partial hepatectomy. 36 These reports suggest t h a t TNF-a and IFN-~ play significant roles in the regulation of hepatocyte proliferation in vivo. IFN-~/ and TNF-ce function synergistically in some cases 37 but antagonistically in others. 3s However, to our knowledge, these cytokines have not been reported to have

FIG. 10. The protective effect of HGF was enhanced by the presence of serum. (A) Hepatocytes were treated with IFN-3, at various concentrations in the presence ((3 ©) or absence of 10% serum (111 II) for 48 hours, t h e n LDH activities in the supernatants were measured. (B) Hepatocytes were treated for 48 hours with various combinations of IFN-3, (100 U/mL), HGF (103 ng/mL) and serum (10%), and LDH activities in the supernatants were measured. (C) Hepatocytes were cultured for 48 hours with the combinations of IFN-3, (100 U/mL), HGF (100 ng/mL), and serum (10%), t h e n the DNAs were extracted and subjected to electrophoresis.

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a direct cytotoxic effect on hepatocytes in vitro. We showed that IFN-~ clearly has a cytotoxic effect on primary cultured mouse hepatocytes, although TNF-a did not have any direct cytotoxicity on hepatocytes in vitro (Fig. 1). The effect was time- and dose-dependent and was eliminated by the anti-serum containing monoclonal anti-IFN-~ antibodies (Figs. 2 and 3). These results confirmed that the substantial lethal factor to hepatocytes in those experiments was IFN-% However, TNF-a seemed to slightly increase the cytotoxic effect of IFN-~ in both LDH release and DNA fragmentation, although TNF-a alone did not show any significant cytotoxic effect on hepatocytes (Figs. 4, 6, and 8). Contrary to the results reported for the in vivo experiments, it is noteworthy that TNF-a by itself was not cytotoxic to hepatocytes in vitro. However, Satoh and Yamazaki 39 reported that TNF-a stimulates DNA synthesis of primary cultured mouse hepatocytes. On the other hand, IFN-~/suppressed the DNA synthesis, and the stimulatory effect of TNF-a is inhibited by IFNT. Moreover, Akerman et aP ° also reported that TNFis a critical cytokine for liver regeneration after hepatectomy.4° These results in which TNF-a by itself is not the substantial cytotoxic factor but rather a proliferating factor for murine hepatocytes coincide with our data. On the contrary, Shinagawa et a141 reported that TNF-a induced both apoptosis and LDH release in cultured rat hepatocytes, and the effect was increased by IFN-% although IFN-~ alone has only a weak cytotoxic effect. We assume that the contradiction between this report and our data is caused by spontaneous apoptosis in hepatocytes. Because hepatocytes have the property such that apoptosis occurs in a cell-density dependent manner, 42 the failure to evenly disperse cells at seeding results in DNA fragmentation and LDH release (Shinzawa et al, Unpublished observations, February 1994) that make the effects of cytokines indistinguishable or misunderstood. Only careful manipulations made it possible to clarify the contradiction. We showed in this study that HGF showed a protective effect on cultured mouse hepatocytes by antagonizing IFN-~ in three aspects, namely, LDH release (Fig. 4), DNA fragmentation (Figs. 7 and 8), and DNA synthesis (Fig. 9). The required concentration of HGF to antagonize IFN-~/on LDH release seemed to be relatively high compared with physiological concentrations; however, it is presumed that in microenvironments in which HGF-producing cells contact or interact nearby with hepatocytes, such a concentration would be achievable. Moreover, because the presence of serum enhanced the protective effect of HGF (Fig. 10), it is possible that HGF suppresses the cytotoxicity of IFN~/at lower concentrations in vivo than in vitro. We used the assay system with 10% serum in this study because the system is common for many cell types, and it is presumed to reflect the in vivo situation, including the presence of unknown factors more than an artificial culture without serum. As described, the presence of serum did not affect the cytotoxicity of IFN-~/ (Fig. 10A), but it enhanced the protective effect of HGF (Fig.

HEPATOLOGYJune 1995

10B and 10C). It is likely that serum contains growthhormone-like factors cooperating with HGF in the protection against IFN-~/-induced cytotoxicity. The mechanism(s) by which HGF is cytoprotective against IFN-~/-induced cell injury and the interaction between HGF and serum factors are unclear and should be investigated. Apoptosis in hepatocytes is induced by several sources of stimulation such as TGF-/~,43 activin44 and IFN-T (in this study). A recent study reported by Ogasawara et al showed that the administration of antiFas antibodies induced fulminant hepatitis-like disease in mice. is Fas (also designated APO-1) antigen was found to be a membrane-associated antigen inducing apoptosis in mainly immune cells. 16'~7 The report suggested that hepatocytes also express Fas antigen and that the apoptosis is involved in the onset of hepatitis. However, we obtained the same results from the experiment using MRL/lpr mice as using ICR mice (data not shown). Because these mice were defective in Fas antigen, ls'45 Fas antigen is not thought to be involved in the IFN-~-induced apoptosis in hepatocytes. The biochemical mechanisms of the IFN-~-induced apoptosis remain to be solved. Although we suggested in this study that IFN-~/was an initiating factor of hepatitis by inducing apoptosis, whether IFN-~ functions as a regulator of the regeneration or hyperplasia in vivo is still an unsolved problem. The engagement and the significance of the apoptosis induced by IFN-T in hepatocytes in vivo are under investigation. While the manuscript for this article was being reviewed, Toyonaga et al reported that transgenic mice expressing IFN-~/ in liver showed chronic hepatitis (Proc Natl Acad Sci U S A 1994;91:614-618). These results are in agreement with ours and with the concept that IFN-~ is a cytotoxic cytokine to hepatocytes. REFERENCES

1. McClain CJ, Cohen DA. Increased tumor necrosis factor production by monocytes in alcoholic hepatitis. HEPATOLOGY 1989; 9:349-351. 2. Yoshioka K, Kakumu S, Arao M, Tsutsumi Y, Inoue M. Tumor necrosis factor a production by peripheral blood mononuclear cells of patients with chronic liver disease. HEPATOLOGY 1989; 10:769-773. 3. Ehlers S, Mielke MEA, Blankenstein T, Hahn H. Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with listeria monocytogenes. J Immunol 1992; 149: 3016-3022. 4. Peters M, Vierling J, Gershwin ME, Milich D, Chisari FV, Hoofnagle JH. Immunology and the liver. HEPATOLOGY1991; 13:977994. 5. Tiegs G, Wolter M, Wendel A. Tumor necrosis factor is a terminal mediator in galactosamine/endotoxin-induced hepatitis in mice. Biochem Pharmacol 1989;38:627-631. 6. Gilles PN, Guerrette DL, Ulevitch RJ, Schreiber RD, Chisari FV. HBsAg retention sensitizes the hepatocyte to injury by physiological concentrations of interferon-~. HEPATOLOGY1992;16: 655-663. 7. Volpes R, Oord JJvd, Vos RD, Depla E, Ley MD, Desmet VJ. Expression of interferon-~, receptor in normal and pathological human liver tissue. J Hepatol 1991; 12:195-202. 8. Franco A, Barnaba V, Natali P, Balsano C, Musca A, Balsano

HEPATOLOGYVol. 21, No. 6, 1995

9. 10. 11.

12. 13.

14. 15. 16.

17.

18. 19. 20. 21.

22.

23. 24. 25. 26. 27.

28.

F. Expression of class I and II major histocompatibility antigens on human hepatocytes. HEPATOLOGY1988; 8:449-454. Michalopoulos GK. Hepatocyte growth factor. HEPATOLOGY 1992; 15:149-155. Takehara T, Nakamura T. Protective effect ofhepatocyte growth factor on in vitro hepatitis in primary cultured hepatocytes. Biomed Res 1991; 12:335-338. Ishiki Y, Ohnishi H, Mute Y, Matsumoto K, Nakamura T. Direct evidence that hepatocyte growth factor is a hepatotrophic factor for liver regeneration and has a potent antihepatitis effect in rive. HEPATOLOGY1992;16:1227-1235. Ellis RE, Yuan J, Horvitz HR. Mechanisms and functions of cell death. Annu Rev Cell Biol 1991;7:663-698. Ohmori T, Podack ER, Nishio K, Takahashi M, Miyahara Y, Takeda Y, Kubota N, et al. Apoptosis of lung cancer cells caused by some anti-cancer agents (MMC, CPT-11, ADM) is inhibited by bcl-2. Biochem Biophys Res Commun 1993; 192:30-36. Cohen JJ, Duke RC, Fadok VA, Sellins KS. Apoptosis and programmed cell death in immunity. Annu Rev Immunol 1992:267293. Tartaglia LA, Ayres TM, Wong GHW, Goeddel DV. A novel domain within the 55kd TNF receptor signals cell death. Cell 1993; 74:845-853. Yonehara S, Ishii A, Yonehara M. A cell-killingmonoclonal antibody (anti-Fas) to a cell surface antigen co-downregu]ated with the receptor of tumor necrosis factor. J Exp Med 1989; 169:17471756. Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991; 66:233-243. Ogasawara J, Watanabe-Fukunaga R, Adachi M, Matsuzawa A, Kasugai T, Kitamura Y, Itoh N, et al. Lethal effect of the antiFas antibody in mice. Nature 1993;364:806-809. Spitalny GL, Havell EA. Monoclonal antibody to murine gamma interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities. J Exp Med 1984; 159:1560-1565. Michalopoulos G, Pitot HC. Primary culture ofparenchymal liver cells on collagen membranes. Exp Cell Res 1975;94:70-78. Shimaoka S, Nakamura T, Ichihara A. Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells. Exp Cell Res 1987; 172:228-242. Decker T, Lohmann-Matthes ML. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods 1988;115:61-69. Korzeniewski C, Callewaert DM. An enzyme-release assay for natural cytotoxicity. J Immunol Methods 1983;64:313-320. Muir D, Varon S, Manthorpe M. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures. Anal Biochem 1990; 185:377-382. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63. Sambrook J, Fritsch EF, Maniantis T. Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press, 1989:9.16-9.21. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991; 139:271-280. Nakamura T, Fujii T, Ichihara A. Enzyme leakage due to change of membrane permeability of primary cultured rat hepatocytes treated with various hepatotoxins and its prevention by glycyrrhizin. Cell Biol Toxicol 1985; 1:285-295.

MORITA, WATANABE, AND AKAIKE

1593

29. Nussler AK, Silvio MD, Billiar TR, Hoffman RA, Geller DA, Selby R, Madariaga J, et al. Stimulation of the nitric oxide synthase pathway in human hepatocytes by cytokines and endotoxin. J Exp Med 1992; 176:261-264. 30. Vaartjes WJ, Haas CGMd, Houweling M. Acute effects of interleukin l a and 6 on intermediary metabolism in freshly isolated rat hepatocytes. Biochem Biophys Res Commun 1990; 169:623-628. 31. Perlmutter DH, Dinarello CA, Punsal PI, Colten HR. Cachectin/ tumor necrosis factor regulates hepatic acute-phase gene expression. J Clin Invest 1986;78:1349-1354. 32. Andus T, Geiger T, Hirano T, Kishimoto T, Heinrich PC. Action of recombinant human interleukin 6, interleukin l p and tumor necrosis factor a on the mRNA induction of acute-phase proteins. Eur J Immunol 1988;18:739-746. 33. Tiegs G, Hentschel J, Wendel A. A T cell-dependent experimental liver injury in mice inducible by concanavalinA. J Clin Invest 1992;90:196-203. 34. Doi F, Goya T, Torisu M. Potential role of hepatic macrophages in neutrophil-mediated liver injury in rats with sepsis. HEPATOLoGY 1993;17:1086-i094. 35. Nagakawa J, Hishinuma I, Hirota K, Miyamoto K, Yamanaka T, Tsukidate K, Katayama K, et al. Involvement of tumor necrosis factor-a in the pathogenesis of activated macrophage-mediated hepatitis in mice. Gastroenterology 1990;99:758-765. 36. Sato Y, Tsukada K, Matsumoto Y, Abo T. Interferon-T inhibits liver regeneration by stimulating major histocempatibility complex class II antigen expression by regenerating liver. HEPATOLOGY 1993; 18:340-346. 37. Broxmeyer HE, Williams DE, Lu L, Cooper S, Anderson SL, Beyer GS, Hoffman R, et al. The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: Synergism of tumor necrosis factor and interferon- T. J Immnnol 1986; 136:4487-4495. 38. Watanabe Y, Jacob CO. Regulation of MHC class II antigen expression: Opposing effects of tumor necrosis factor-a on IFN-Tinduced HLA-DR and la expression depends on the maturation and differentiation stage of the ceil J Immunol 1991;146:899905. 39. Satoh M, Yamazaki M. Tumor necrosis factor stimulates DNA synthesis of mouse hepatocytes in primary culture and is suppressed by transforming growth factor ~ and inter]eukin 6. J Cell Phys 1992;150:134-139. 40. Akerman PA, Cote PM, Yang SQ, McClain C, Nelson S, Bagby G, Diehl AM. Long-term ethanol consumption alters the hepatic response to the regenerative effects of tumor necrosis factor-a. HEPATOLOGY1993; 17:1066-1073. 41. Shinagawa T, Yeshioka K, Kakumu S, Wakita T, Ishikawa T, Itoh Y, Takayanagi M. Apoptosis in cultured rat hepatocytes: the effects of tumor necrosis factor a and interferon % J Pathol 1991; 165:247-253. 42. Maeda S, Kimura H, Koga N, Lin KH, Saito T. Cell densitydependent DNA fragmentation and its suppression by heparin in primary culture of adult rat hepatocytes. Biochem Biophys Res Commun 1993;195:270-275. 43. Oberhammer FA, Pavelka M, Sharma S, Tiefenbacher R, Purchio AF, Bursch W, Schulte-Hermann R. Induction of apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor/~1. Proc Natl Acad Sci USA 1992;89:5408-5412. 44. Schwall RH, Robbins K, Jardieu P, Chang L, Lai C, Terrell TG. Activin induces cell death in hepatocytes in rive and in vitro. HEPATOLOGY1993; 18:347-356. 45. Watanabe-FukunagaR, Brannan CI, Copeland NG, Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1992;356:314-317.