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

Protective Effect of Hepatocyte Growth Factor on Interferon- Gamma-Induced Cytotoxicity in Mouse Hepatocytes

MASAHIKO MORITA, YOSHIFUMI WATANABE, AND TOSHIHIRO AKAIKE

We examined the interactive effect of several cytokines (interleukin-1 beta [IL-lp], tumor necrosis factor alpha [TNF-a], interferon gamma [IFN-y], IL-6, IFN-dp, and hepatocyte growth factor [HGF]) presumably involved in hepatitis, on primary cultured murine hepatocytes. Among these cytokines, only IFN-y induced LDH release from hepatocytes in both time- and dose-dependent fashions. The cytotoxic effect was inhibited by antiserum-containing anti-mouse IFN-y monoclonal antibodies (R4-6A2). Moreover, intriguingly, IFN-y 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-y on hepatocytes was caused by inductive apoptosis. The LDH release and DNA fragmentation induced by IFN-y were inhibited by HGF in a dose-dependent manner, whereas they seemed to be accelerated by TNF-a. 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-y but recovered by hepatocyte growth factor. Taken together, IFN-y is presumed to be a critical cytokine in hepatic damage, and the network composed of IFN-y, TNF-a, and HGF may play an important role in the regulation of liver injury. (HEPATOLOGY 1995;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-2y1)-2,5- diphenyl tetrazolium bromide: EGTA, ethyleneglycol bis(2-aminothyl etherltetraacetic acid; IXS, 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 ofBiomo-

lecular Enb?neering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori- ku, Yokohama 226, Japan.

Copyright 0 1995 by the American Association for the Study of Liver Diseases.

0270-9 i 3 9 m 2 io6-a80i6$3.00/o

cally involved in the hepatic damage. It has been reported that tumor necrosis factor alpha (TNF-a), interleukin (1L)-1 and -6, and interferon-gamma (IFN- y ) are important mediators in inflammation. Increased production of these inflammatory cytokines is often de- tected in the cases of patients with hepatitis"' or in a liver infected with bacteria,3 and they are considered to play important roles in the onset of he pa ti ti^.^ For example, there are some cases in which TNF-a or IFN- y is proven to be a critical factor in the induction of hepatitis in m i ~ e . ~ , ~ In addition, IFN- y receptors were expressed on hepatocytes in a diseased liver but not in a normal liver.7 It is also suggested that IFN-7 makes hepatocyte antigen presenting cells in the liver and initiates an inflammatory reaction because IFN- y induced MHC class I1 expression on human hepatocytes.*

Hepatocyte growth factor (HGF ) was originally found to be a potent mitogenic factor for hepato~ytes .~ After this discovery, the factor has been reported to be a multifunctional cytokine having mitogenic, motogenic, morphogenic, and tumoricidal a c t i ~ i t i e s . ~ Moreover, it has a protective effect on hepatocytes from experimental hepatic 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 embryogene- sis." The characteristic features of apoptosis are con- densation and fragmentation of nuclear chromatin, ac- companied by compaction of cellular organelles, dilatation of the endoplasmic reticulum, and a marked reduction in cell volume.'' It is also presumed to be involved in the mechanisms of cell death by antitumor drugs,13 cytotoxic T cells or natural killer cells,I4 and antitumor cytokines such as TNF-a.l5 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 t ransd~ct ion ,~""~ is expressed on hepatocytes and may play an important role in inducing fulminant hepatitis via apoptosis." Therefore, apoptosis is expected to be an important mechanism in the pathogenesis of hepatitis.

1585

1586 MORITA, WATANBE, AND AKAIKE HEPAToLOCYJune 1995

In this study, we examined the direct effects of cytokines supposedly involved in hepatitis on hepatocytes in vitro and found that IFN-y is a cytotoxic cytokine for primary cultured mouse hepatocytes and induces apoptosis in these cells. Moreover, the cytotoxic, apoptosis-inducible function of IFN- y was inhibited by HGF. The interaction between these cytokines is envis- aged to play important roles in the pathogenesis of hepatitis.

MATERIALS AND METHODS Reagents and Animals

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and propidium iodide were purchased from Sigma (St. Louis, MO). Murine recombinant IFN-y (mrIFN- y ) and TNF-a were gifts from Genentech Inc. (South San Francisco, CA). Human recombinant HGF (hrHGF) was a gift from Snow Brand Milk Products Co. Ltd. (Tokyo, Japan). MrIL-10 and IL-6 were purchased from R & D Systems (Min- neapolis, MN). The hybridoma (R4-6A2,") producing anti- murine IFN-y immunoglobulin (Ig) G2 antibodies was obtained from American Type Culture Collection (Maryland, USA). The cells were transplanted into the peritoneum of female Balbk nude mice (7 weeks of age) (Jc1:AF'-nu, Clea Japan, Inc., Kanagawa, Japan), and the antiserum was em- ployed 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 Japan Inc. (Kanagawa, Japan). All animal experiments were performed in accordance with local institutional guide- lines 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.20,21 Briefly, the liver was first perfused in situ through the thoracic infe- rior vena cava with Ca2+-free Hank's solution supplemented with 5 mmollL ethyleneglycol bis(2-aminoethyl ether)tetra- acetic acid (EGTA) and 5 mmoUL glucose a t 37C 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 a t 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 ), lo-' mol/L insulin and antibiotics in RPMI1640 with 10% FCS and were plated at a density of 2.7 x lo5 cells per well, 1.3 x lo5 cells per well and 3 x lo4 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 mi- croscopic 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 a t 37C 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 lymphokines, EGF and insulin were

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

Cytotoxic Assay of Hepatocytes Lactose Dehydrogenase Release Assay. The activity of lac-

tose dehydrogenase (LDH), a stable cytosolic enzyme that is released on cell lysis and one of the commonly used hallmarks of cellular cytotoxicity,22 in the supernatants of treated hepatocytes was measured using a CytoTox 96 Nonradioactive Cytotoxicity Assay Kit (Promega, Madison, WI),23 following the manufacturer's instructions. The percentage of lysis was calculated using the formula:

5% LDH release = 100 x (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 (BrdUlz4 with a BrdU labeling and detection kit I11 (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 a t 37C. The cells were then washed and fixed with HC1-ethanol a t -20C 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 a t 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.25 Briefly, MTT was added to treated hepatocytes plated in a 96-well, flat-bottomed plate a t a final concentration of 500 mg/mL, and the cells were incubated for 4 hours a t 37C. One hundred pL 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 a12" with minor modification. Briefly, cells were incubated with the lysis buffer (10 pg/mL; proteinase K [Sigma, St. Louis, MO], 10 mmol Tris, 150 mmoL/L NaC1,l mmoVL edetic acid [EDTAI, 1% SDS) for 15 hours a t 37C. Chromosomal DNA was obtained by phenoUchloroform (1: 1) extraction and ethanol-precipitation. The samples in TE solution (10 mM Tris-HC1 [pH 8.01, 1 mmoVL EDTA) with 1 pg/mL RNase were incubated for 1 hour a t 37C. The same amount of DNA from each sample was subjected to electrophoresis through 1.0% agarose gel containing 0.1 pg/mL ethidium bromide. Flow Cytometry Analysis. Flow cytometry analysis of nu-

clei from hepatocytes was performed as described by Nicoletti et aLZ7 Hepatocytes were suspended in 0.1 moUL citrate

HEFATOLOGY Vol. 21, No. 6, 1995 MORITA, WATANABE, AND AKAIKE 1587

?A L I::::I

FIG. 1. Comparative effects of various cytokines on LDH release of hepatocytes. Murine hepatocytes (3 X lo4 cells per well) were treated with IFN-y, TNF-a, IL-lp, HGF, IFN-a/P, or IL-6 at various concentrations for 48 hours, and then the LDH activities of the supernatants were measured. The LDH release was expressed as % release as described in the Materials and Methods section. W, IFN-y (U/mL); 0, TNF-a (ng/mL); + , IL-lp (ng/mL); 0, HGF (ng/mL); 0, IFN-(a/ p ) (U/mL); and A, 11,-6 (ng/mL).

buffer (pH 7.2) containing 0.1% Triton X-100 and incubated a t 37C for 30 minutes. The tubes were vortexed and centri- fuged, and the resultant pellets were washed twice with ethanol and stained with 10 ,ug/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 (Ja- pan Spectroscopic Co. Ltd., Tokyo, Japan) a t wavelengths of 488 nm for excitation and 530 nm for emission.

RESULTS

IFN- y i s a Cytotoxic Cytokine for Mouse Hepatocytes

First, we investigated the effect of several cytokines (IFN-y, TNF-a, IL-10, IL-6, HGF, and IFN-a/P) that are presumed to be involved in hepatitis or hepatic injury on the LDH release from hepatocytes as a hall- mark of cellular cytotoxicity (Fig. 1). One of the commonly used direct toxins for hepatocytes, CC14,28 as a positive control induced almost 100% LDH release of hepatocytes, whereas only IFN-y among the examined cytokines showed a cytotoxic effect on hepatocytes in a dose-dependent fashion (Fig. 1). The LDH release induced by IFN- y reached a plateau a t a concentration of more than 10 U/mL. Other inflammatory cytokines such as TNF-a, IL-10, 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 transami- nase activities of the supernatants. Furthermore, the results were not influenced by the removal of serum from the medium in the assay.

Because IFN-y has not to our knowledge been reported to be a direct cytotoxic factor for hepatocytes, an inhibition test by monoclonal anti-murine IFN-y

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-y-induced LDH release, anti-IFN- y serum inhibited the cytotoxicity of IFN-y in a dilution-dependent way. These data demon- strate that the substantial cytotoxic factor for hepatocytes is presumed to be IFN-y. Moreover, the time course analysis of LDH release by IFN-y showed that the release was time dependent (Fig. 3). Significant LDH release was observed a t 24 hours after IFN-y treatment, and it gradually increased with time as shown in the figure. Finally, it reached the maximum release, 100% a t 72 hours.

HGF Suppresses IFN- y Induced Hepatic Cytotoxicity It has been reported that TNF-a is one of the critical

cytokines involved in hepatic i n j ~ r y . ~ . ~ On the other hand, HGF has been reported to have a protective effect on hepatocytes from experimental hepatic Therefore, we investigated the effects of these cytokines combined with IFN-y on the LDH release of hepatocytes. 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,

60

T

control no-Ab rat IgG a-mouse IFN-)I

antiserum

IFN-)I ( lOOU/ml)

FIG. 2. Inhibition by anti-IFN-y antiserum of IFN-y-induced LDH release from cultured hepatocytes. Mouse hepatocytes were cultured with 100 U/mL of IFN-y in the absence or presence of di- luted anti-IFN-y antiserum or rat IgG (10 pg/mL) as a control for 48 hours. The LDH activities of the supernatants were measured and compared as percentage release.

MORITA, WATANABE, AND AKAIKE:

2o I 0 1 I I I I I I I

0 12 24 36 48 60 72 Time (h)

FIG. 3. Time-course analysis of the LDH release from IFN-y- treated hepatocytes. Murine hepatocytes were cultured with 100 U/ mL of IFN-y for various times, and the LDH activities of the supernatants were measured and expressed as percentage release. (0 __ 0) control, ( A ~ A ) IFN-y.

when these cytokines were used in the presence of IFN- y , 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). DNA Fragmentation Induced by IFN- y 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-y through only morphological observation with phase contrast microscopy, we examined the DNA fragmentation of IFN-y-treated 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-y, 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-a nor IFN-a/@ showed any effect on DNA fragmentation as expected from the results of the LDH release assay (Fig. 1). In addition, neither HGF nor IL-lp 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.

HEPATOLOGYJune 1995

1). The IFN-y-induced 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.

Flow Cytometry Analysis Confirmed that HGF Suppressed the DNA Fragmentation Induced by IFN- y

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

80 I

T T T 60 h

6? v Q)

3 3

5 2 40 X

20

0

TNFa ( n g / d ) +

IFN-y( lOoU/ml)

control o 10 102 103 104 105 HGF ( ng/ml)

+ IFN-)I ( lOoU/ml)

FIG. 4. Opposing effects of TNF-a or HGF on the LDH release by IFN-y-treated hepatocytes. Murine hepatocytes were cultured with 100 U/mL of IFN-y for 48 hours in the presence of either TNF- ru or HGF a t various concentrations, and the LDH activities were measured and expressed as percentage release.

HEPATOI,O(:Y Val. 21, NO. 6, 1995

IFN-y-stimulated time ( h )

MORITA, WATANABE, AND AKAIKE 1589

0 12 24 36 48 60 72 IFN-y (U/ml)

FIG. 5. Time-course analysis of DNA fragmentation induced by IFN-y in murine hepatocytes. Murine hepatocytes (4 x 10' cells) were incubated with 100 UimL IFN-y for various times, then the DNA was extracted and subjected to electrophoresis through 19 agarosc gel.

apoptotic nuclei was performed. The reduced DNA con- tent of apoptotic nuclei resulted in an unequivocal hy- podiploid DNA peak in the red fluorescence channels when the nuclei were stained with propidium iodide.27 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-7. These results of flow cytometry analysis confirmed th,ose 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

c lo-' 100 10' 102 103 A

TNFa (ng/ml)

c 1 0 2 lo-' loo 10' 102

FIG. 6. 11"-y but not TNF-cu-induced DNA fragmentation of murine hepatocytes in a dose-dependent fashion. Murine hepatocytes were cultured with (A) IFN-y, (B) TNF-a, or (C) IFN-aIP for 48 hours a t various concentrations. After the incubation, the DNA was extracted and subjected to 1% agarose electrophoresis.

1590 MORITA, WATANABE, AND AKAIKE HEPAToLoGYJune 1995

IFN-)I +

HGF (ng/ml)

c 0 100 10' lo2

FIG. 7. HGF suppressed the DNA fragmentation induced by IFN- y in hepatocytes. Murine hepatocytes (4 x lo5 cells) were incubated with 100 U/mL of IFN-y for 48 hours in the presence of HGF a t 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.24 After 24 hours of incubation with tested cytokines, treatment of hepatocytes with IFN-y almost thoroughly suppressed the DNA synthesis as de~cribed,~' 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 time- course 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-y in hepatocytes, even in the presence of serum, although the mitogenic effect is masked under these conditions.

The Presence of Serum Did Not Affect IFN- y-Induced Cytotoxicity but Enhanced the Protective Effect of HGF

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

rum in the medium, because serum contains multiple factors, including growth factors and cytokines. There- fore, the effects of IFN-y 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-y a t 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 1OC) although the serum alone a t the same concentration did not sup- press the cytotoxicity by IFN-y.

DISCUSSION In this study, we investigated the interactive effects

of cytokines presumably involved in the pathogenesis of hepatic injury to primary cultured mouse hepatocytes. IFN-7, TNF-a, IL-1, and IL-6 are known as inflammatory cytokines and have several effects on hepatocytes, i.e., induction of NOp," changes in glucose metab~lism,~' and acute phase gene e x p r e ~ s i o n . ~ ~ ' ~ ~ Be- cause it has been reported that T lymphocytes and neu- trophils invaded the liver in he pa ti ti^,^^,^^ these immune cells and liver specific tissue macrophages, Kupffer cells,4 are presumed to be the sources of in-

control II IFN-y

IFN-)I +

I

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/mLj, IFN-y (100 U/mLj, IFN- y (100 U/mL) +TNF-a (100 ng/mL) or IFN-y (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.

HEPATOI,O(;Y Val. 211, NO. 6 , 1995 MORITA, WATAIYAES. AND AKAIKE 1591

0.4

3 0.3 2 i; ov p 0.2 3 3 D g 0.1

0

-I 0.6

non-treated IFN-y HGF LFN-y + HGF

FIG. 9. Effect of HGF on IFN-y inducible suppression of DNA synthesis in hepatocytes. Murine hepatocytes (1 x lo4 cells per well) were cultured with [FN-y (100 U/mL), HGF (100 ng/mL) or IFN-y (100 UirnLi +HGF il00 ng/mL) for 24 hours. The DNA synthesis was then measured by BrdU incorporation, and the viability of the hepatocytes with the same treatment was measured by MTT assay as described in the Materials and Methods section.

flammatory cytokines including IFN-y 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 candi- date because TNF-a production is increased in the serum of patients with alcohol-induced hepatitis,' with chronic liver or in mice treated with galactos- aminelendotoxin in which a fulminant-type hepatic necrosis is observed.35 In addition, TNF-a instead of lipo- polysaccharide induced hepatic necrosis in these mice.5 Moreover, it is intriguing that either TNF-a or IFN-y has been reported to induce hepatitis in transgenic mice with a hepatitis B virus envelope.6 It has also been reported that IFN-y receptors are expressed on hepatocytes in a liver with hepatic disease but not in a normal liver7 and that IFN-y suppressed liver regeneration after partial h e p a t e ~ t o m y . ~ ~ These reports suggest that TNF-a and IFN-y play significant roles in the regulation of hepatocyte proliferation in uiuo. IFN- y and TNF-a function synergistically in some cases? but antagonistically in others.38 However, to our knowledge, these cyto'kines 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-y a t various concentrations in thse presence (0 ~ 0) or absence of 10% serum (.----.I for 48 hours, then LDH activities in the supernatants were measured. (B) Hepatocytes were treated for 48 hours with various combinations of IFN-y (100 U/mL), HGF (lo3 ng/mL) and serum (lo%), and LDH activities in the supernatants were measured. (C) Hepatocytes were cultured for 48 hours with the combinations of IFN-y (100 UlmL), HGF (100 ng/mL), and serum (1081, then the DNAs were extracted and subjected to electrophoresis.

C

loo 75 : 50

25

0 I 0 0.1 1.0 10 100

IFN-)I (U/ml)

loo I 75 t 50 -

25 -

0 IFN-y - + + + + + + - HGF - FCS - - -

IFN-y

FCS + - - - + + HGF - - - + - +

1592 MORITA, WATANABE, AND AKAIKE

a direct cytotoxic effect on hepatocytes in uitro. We showed that IFN-y clearly has a cytotoxic effect on primary cultured mouse hepatocytes, although TNF-a did not have any direct cytotoxicity on hepatocytes in uitro (Fig. 1). The effect was time- and dose-dependent and was eliminated by the anti-serum containing monoclonal anti-IFN-y antibodies (Figs. 2 and 3). These results confirmed that the substantial lethal factor to hepatocytes in those experiments was IFN-y. However, TNF-a seemed to slightly increase the cytotoxic effect of IFN-y 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 uiuo experiments, it is noteworthy that TNF-a by itself was not cytotoxic to hepatocytes in uitro. However, Satoh and Yamazaki3' reported that TNF-a stimulates DNA synthesis of primary cultured mouse hepatocytes. On the other hand, IFN-y suppressed the DNA synthesis, and the stimulatory effect of TNF-cu is inhibited by IFN- y. Moreover, Akerman et aI4' also reported that TNF- a is a critical cytokine for liver regeneration after hepa- te~tomy.~" These results in which TNF-a by itself is not the substantial cytotoxic factor but rather a proliferat- ing 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-y, although IFN-y 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 (Shin- zawa 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 antagoniz- ing IFN-y in three aspects, namely, LDH release (Fig. 41, DNA fragmentation (Figs. 7 and 81, and DNA synthesis (Fig. 9). The required concentration of HGF to antagonize IFN-y on LDH release seemed to be relatively high compared with physiological concentrations; however, it is presumed that in microenviron- ments 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. lo), it is possible that HGF suppresses the cytotoxicity of IFN- y at lower concentrations in viuo than in uitro. 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 uiuo 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-y (Fig. lOA), but it enhanced the protective effect of HGF (Fig.

HEPATOLOGYJune 1995

10B and 1OC). It is likely that serum contains growth- hormone-like factors cooperating with HGF in the protection against IFN- y -induced cytotoxicity. The mechanism(s) by which HGF is cytoprotective against IFN-y -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-p,43 a ~ t i v i n ~ ~ and IFN-y (in this study). A recent study reported by Oga- sawara et a1 showed that the administration of anti- Fas antibodies induced fulminant hepatitis-like disease in mice." Fas (also designated APO-1) antigen was found to be a membrane-associated antigen inducing apoptosis in mainly immune cell^.^^.^^ 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 Fas antigen is not thought to be involved in the IFN-y -induced apoptosis in hepatocytes. The biochemical mechanisms of the IFN- y -induced apoptosis remain to be solved.

Although we suggested in this study that IFN-y was an initiating factor of hepatitis by inducing apoptosis, whether IFN-y functions as a regulator of the regeneration or hyperplasia in uiuo is still an unsolved problem. The engagement and the significance of the apoptosis induced by IFN-y in hepatocytes in uiuo are under in- vestigation.

While the manuscript for this article was being re- viewed, Toyonaga et al reported that transgenic mice expressing IFN-y 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 con- cept that IFN-y is a cytotoxic cytokine to hepatocytes.

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