Interferon gamma plays a critical role in T cell-dependent liver injury in mice initiated by concanavalin A

GASTROENTEROLOGY 1996;111:462–471

Interferon Gamma Plays a Critical Role in T Cell–Dependent Liver Injury in Mice Initiated by Concanavalin A ¨ STERS, FLORIAN GANTNER, GERALD KU ¨ NSTLE, and GISA TIEGS SABINE KU Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz, Germany

Background & Aims: T cell–dependent liver injury involving endogenous tumor necrosis factor (TNF) a can be induced by either concanavalin A in naive mice or by activating anti-CD3 antibody or staphylococcal enterotoxin B in D-galactosamine–sensitized mice. In this study, the role of interferon gamma (IFN-g ) in these Tcell models was addressed. Methods: Mice were pretreated with a neutralizing anti-mouse IFN-g antiserum before injection of T cell–activating agents. Plasma cytokine and transaminase levels were determined. Apoptotic cell death was assessed by hepatic DNA fragmentation. Results: Anti–IFN-g antiserum significantly protected mice from concanavalin A–induced liver injury. Circulating IFN-g was completely suppressed, and TNF was reduced by 50%. Recombinant TNF-a administered to mice treated with concanavalin A and anti– IFN-g antiserum failed to initiate liver injury. Similar results were obtained with recombinant IFN-g in concanavalin A–challenged mice under the condition of TNF neutralization. Neither hepatic DNA fragmentation nor release of transaminases was inhibited by anti–IFN-g antiserum when liver injury was induced by staphylococcal enterotoxin B or anti-CD3 antibody in D-galactosamine–sensitized mice. Conclusions: Both TNF as well as IFN-g are critical mediators of liver injury in concanavalin A–treated mice, whereas hepatic DNA fragmentation and liver failure in the D-galactosamine models depend only on TNF.

I

nterferon gamma (IFN-g) is a cytokine secreted by activated T cells and natural killer cells that regulates host defense, inflammation, and autoimmunity.1 The biological properties of IFN-g are induction of major histocompatibility complex class I and class II expression, stimulation of tumor necrosis factor (TNF) production, costimulation of nitric oxide synthase expression, and induction of high affinity receptors for the constant moiety of immunoglobulins on monocytes and/or macrophages, thus promoting clearance of microbial pathogens.1 Experimentally, administration of IFN-g increased mortality of endotoxic shock2 and rendered lipopolysaccharide (LPS)-resistant mice responsive towards LPS,3,4 whereas neutralizing anti–IFN-g antibodies2 or IFN-g / 5e10$$0016

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receptor deficiency5 prevented mice from LPS lethality. These effects were mediated by enhanced2 or reduced5,6 TNF production, respectively. Natural killer cells were suggested as the cellular source of LPS-induced IFN-g, which in turn primes macrophages to produce enhanced amounts of TNF.1 In a T cell–dependent model, i.e., staphylococcal enterotoxin B (SEB)-induced shock in Nnitro-L-arginine methyl ester–treated mice, TNF as well as IFN-g were shown to mediate lethality.7 In liver injury, increased production of IFN-g was observed in patients with autoimmune8,9 or viral hepatitis10,11 and liver allograft rejection.12 IFN-g receptors were shown to be expressed on hepatocytes in pathological human liver tissue.13 Because IFN-g induces major histocompatibility complex class II expression on human hepatocytes, it has been suggested that class II–carrying hepatocytes found in patients with chronic liver disease may serve as antigen-presenting cells.14 In addition, transgenic mice expressing IFN-g in the liver develop liver injury resembling chronic active hepatitis.15 In vitro, IFN-g induced apoptotic cell death in mouse hepatocytes that was accelerated by TNF.16 From this observation, it was concluded that IFN-g as such is cytotoxic to hepatocytes. Three experimental models of T cell–dependent apoptotic and necrotic liver injury in mice were described recently. D-Galactosamine (GalN)-sensitized mice challenged with either activating anti-CD3 monoclonal antibody (MAb) or with the superantigen SEB developed severe apoptotic and secondary necrotic liver injury as assessed by histological examination, cytosolic DNA fragmentation, and determination of plasma transaminases.17,18 Injection of the T-cell mitogenic plant lectin concanavalin A (con A) to nonsensitized mice resulted also in hepatic apoptosis that preceded necrosis.19 – 21 Abbreviations used in this paper: con A, concanavalin A; ELISA, enzyme-linked immunosorbent assay; GalN, D-galactosamine; IFN-g, interferon gamma; LPS, lipopolysaccharide; MAb, monoclonal antibody; SEB, staphylococcal enterotoxin B; TNF, tumor necrosis factor. 䉷 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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Anti-CD3 MAb as well as SEB or con A induced the release of systemic TNF, IFN-g, and various other cytokines. Passive immunization against TNF protected mice from liver injury.17,20,22 Moreover, direct administration of recombinant murine TNF to mice resulted in hepatocellular apoptosis and necrosis under the condition of either actinomycin D – or GalN-induced arrest of hepatic transcription.21,23 Because con A induced TNF-mediated liver injury without inhibiting the rates of hepatic transcription or protein synthesis21 and because IFN-g was frequently shown to act synergistically with TNF,7,24 we wondered whether con A–stimulated endogenously produced IFN-g would sensitize the liver towards the endogenously produced hepatotoxic mediator TNF. In comparison to this, we investigated the role of IFN-g in the two models of T cell–dependent hepatic failure in GalNsensitized mice, i.e., in GalN/anti-CD3 MAb- and GalN/ SEB-induced liver injury.

Materials and Methods Mice Animals received humane care according to the guidelines of the National Institutes of Health as well as to the legal requirements in Germany, were maintained for at least 10 days under controlled conditions (22⬚C, 55% humidity, and 12-hour day/night rhythm), and were fed a standard laboratory chow (Altromin 1313; Altromin, Lage, Germany). Male BALB/c mice (age, 6–8 weeks; weight range, 25–30 g) were obtained from the animal facility of the University of Konstanz (Konstanz, Germany). Male BALB/c/OLaHsd nu/nu mice were purchased from Harlan (CPB, Austerlitz, The Netherlands).

Application Regimen and Sampling of Material All animals were fasted overnight before the experiments and challenged at 7 AM to 8 AM. A single dose of 25 mg/kg con A (Sigma Chemical Co., Deisenhofen, Germany) was injected intravenously (IV) in a volume of 300 mL pyrogenfree saline. SEB (Sigma Chemical Co.) was administered intraperitoneally (IP) in 300 mL pyrogen-free saline containing

0.1% human serum albumin at a single dose of 2 mg/kg. The activating anti-CD3 MAb specific for the murine CD3e chain was obtained as described by Gantner et al.17 from the mouse and hamster hybridoma clone 145 2C1125 and was injected in a single dose of 10 mg/kg IV in 300 mL pyrogen-free saline containing 0.1% human serum albumin. GalN (Roth, Karlsruhe, Germany) was administered IP 10 minutes before antiCD3 MAb or SEB application, respectively, in 200 mL pyrogen-free saline in a dose of 700 mg/kg. Aminoguanidine (Sigma Chemical Co.) was injected IP in 300 mL pyrogen-free saline in a dose of 15 mg/kg. Sheep anti-murine TNF polyclonal antiserum (50 mL/ mouse; in-house preparation; specific neutralizing activity was measured with a bioassay using the murine fibrosarcoma cell line WEHI 164 clone 13 according to Espevic and NissenMeyer26: a 1:2,000,000 dilution neutralized 8 ng/mL recombinant murine TNF-a), rabbit anti-IFN-g polyclonal antiserum (200 mL/mouse; in-house preparation; specific binding activity was measured using enzyme-linked immunosorbent assay [ELISA] technique: a 1:70,000 dilution bound 25 ng/mL recombinant murine IFN-g), or corresponding control sera were injected IV 15 minutes before challenge. The sera injected alone did not induce cytokine or plasma enzyme release (data not shown), and control sera did not have any influence on the effect of the stimuli (compare Table 1 and Gantner et al.20). Recombinant murine TNF-a or IFN-g (kindly provided by Dr. G. R. Adolf, Bender & Co., Vienna, Austria) were injected IV in a dose of 2.5 or 5 mg/kg, respectively, 30 minutes after con A administration in 300 mL pyrogen-free saline containing 0.1% human serum albumin. Recombinant murine IFN-g (50 mg/kg) was injected 15 minutes after GalN application. For determination of circulating TNF, blood samples were taken from the tail vein 90 minutes after challenge. Eight hours after challenge, blood was withdrawn by cardiac puncture into heparinized syringes under lethal Nembutal anesthesia (Sanofi-Ceva; Wirtschaftsgenossenschaft deutscher Tiera¨rzte eG, Hannover, Germany; 150 mg/kg IV) for determination of circulating IFN-g as well as for assessment of liver injury by measurement of plasma transaminase and sorbitol dehydrogenase levels according to the method of Bergmeyer.27 Plasma samples were stored at 080⬚C until determination of enzyme activity or measurement of cytokines by ELISA. For determina-

Table 1. Protection by Anti–IFN-g Antiserum Against con A–Induced Liver Injury in Mice Treatment

n

Alanine aminotransferase (U/L)

Aspartate aminotransferase (U/L)

con A con A / preimmune serum con A / anti–IFN-g

9 6 14

4500 { 1000 2700 { 680 125 { 17a

2010 { 450 1300 { 325 180 { 41a

Sorbitol dehydrogenase (U/L) 1500 { 350 1100 { 230 130 { 26a

NOTE. Two hundred microliters per mouse of either preimmune serum or antiserum was IV injected 15 minutes before IV administration of 25 mg/kg con A. Plasma enzyme activities of alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase were determined 8 hours after the con A challenge (values of saline control: alanine aminotransferase, 40 { 20 U/L; aspartate aminotransferase, 80 { 30 U/ L; sorbitol dehydrogenase, 30 { 10 U/L). Data are expressed as mean { SEM. a P õ 0.001 vs. con A–treated or P õ 0.01 vs. con A / preimmune serum–treated control.

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tion of DNA fragmentation, livers were perfused for 10 seconds with cold buffer (50 mmol/L phosphate, 120 mmol/L NaCl, and 10 mmol/L ethylenediaminetetraacetic acid; pH 7.4) before excision and treated with three strokes of an Elvehjemtype homogenizer. The 20% homogenate (in perfusion buffer) was centrifuged at 13,000g for 20 minutes. The supernatant was either further diluted 250-fold and used directly in an ELISA designed to detect DNA fragmentation or DNA was precipitated from 400 mL of supernatant by the addition of 50 mL NaCl (5 mol/L) plus 1 mL ethanol (020⬚C) and stored at 020⬚C for further analysis on an agarose gel.

Cytokine Determination by ELISA All incubations of the sandwich ELISAs were performed in flat-bottom high-binding polysterene microtiter plates (Greiner, Nu¨rtingen, Germany) using specific rat antimouse MAb pairs (biotinylated detecting MAb) purchased from Pharmingen (Hamburg, Germany). For determination of TNF, a protein G/-purified polyclonal sheep anti-mouse TNFa capture antibody (protein content, 20 mg/mL; in-house preparation) was used, replacing the Pharmingen capture MAb. Steptavidin-peroxidase (Jackson Immuno Research, West Grove, PA) and the peroxidase chromogen teramethylbenzidine (Boehringer Mannheim, Mannheim, Germany) were used to detect the immunocomplex.

DNA Fragmentation DNA fragmentation was measured by quantitation of cytosolic oligonucleosome-bound DNA using a cell death detection ELISA kit (Boehringer Mannheim) as described previously.23 Briefly, the 13,000g supernatant from liver homogenates was used as the antigen source in the sandwich ELISA with an antihistone capture MAb and an anti-DNA–detecting MAb coupled to peroxidase. The OD values of samples from con A-, GalN/antiCD3 MAb–, or GalN/SEB-challenged mice were compared with those samples from saline- or GalNtreated control mice, respectively. Alternatively, semiquantitative determination of DNA fragmentation28 was performed by analyzing the pattern of low-molecular-weight DNA, which was stained with ethidium bromide after extraction by phenol/ chloroform, precipitation in ethanol, and subsequent electrophoresis on 1% agarose gels. The n 1 123–base pair molecular weight marker used for gel electrophoresis was obtained from Gibco Laboratories (Eggenstein, Germany).

Nitrite Determination in Serum Nitrite in serum was measured essentially according to the method of Misko et al.,29 using the Griess assay replacing the fluorimetric assay as described previously.30 Briefly, plasma obtained by cardiac puncture 8 hours after con A administration into heparinized syringes was filtered through an Ultrafree-MC microcentrifuge filter unit (Millipore, Bedford, MA) for 1 hour at 14,000 rpm to remove hemoglobin that might interfere with the colorimetric assay. Serum nitrate was reduced to nitrite by incubation with nitrate reductase from

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aspergillus species (Sigma Chemical Co., St. Louis, MO). Fourteen milliunits of nitrate reductase (in 20 mmol/L Tris-HCl, pH 7.6) was added to 10 mL of filtrate, and the reaction was started by the addition of reduced nicotinamide adenine dinucleotide phosphate in Tris-HCl buffer to a final concentration of 40 mmol/L and a final volume of 50 mL. After 5 minutes of incubation at room temperature, the reaction was terminated by dilution with 50 mL of distilled water. The whole reaction volume was transferred to 96-well microtiter plates, 10 mL sulfanilamide (1% in 1.2 mol/L HCl) and 10 mL N-(1-naphthyl) ethylenediamine (0.1% in H2O) were added, and absorbance was read after 3 minutes of incubation time at 560/ 690 nm on an ELISA reader.

Statistical Analysis Data are expressed as mean { SEM and were analyzed by one-way analysis of variance; in case of differences among groups (P õ 0.05), data were subjected to Dunnett multiple comparisons test of the control against all other groups with the program INSTAT 2 (GraphPad Software, Inc., San Diego, CA).

Results Protection by Anti–IFN-g Antiserum Against con A–Induced Liver Failure in Mice After IV injection of 25 mg/kg con A, male BALB/c mice developed severe liver injury as assessed by determination of enhanced activities of circulating liver enzymes, i.e., plasma alanine aminotransferase, plasma aspartate aminotransferase, and plasma sorbitol dehydrogenase.19,20 As observed previously,20 systemically administered con A also induced the release of TNF and IFN-g into the circulation with maximal plasma concentrations at 1.5 hours and 8 hours, respectively (Figure 1). Both anti-mouse TNF antiserum20 as well as antimouse IFN-g antiserum significantly protected against con A, whereas 200 mL preimmune serum failed to prevent liver injury (Table 1 and Figure 2A). Protection by the anti–IFN-g antiserum was associated by a complete suppression of plasma IFN-g and by a 50% reduction of peak levels of circulating TNF (Figure 2B). Conversely, anti-TNF antiserum completely neutralized peak levels of systemic TNF and lowered maximal circulating IFNg concentrations by 50% (Figure 2B). The appearance of fragmented oligonucleosome-bound DNA in 13,000g supernatants of 20% murine liver homogenates was shown to correlate with histological signs of apoptosis, such as formation of hepatocyte hyperchromatic nuclear membranes and apoptotic bodies.17,20,21,30 Detection of oligonucleosome-bound DNA fragments by ELISA turned out to be highly sensitive for the assessWBS-Gastro

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ment of organ injury. As shown in Figures 2C and 3, con A induced hepatic DNA fragmentation, which was significantly reduced by either anti-TNF antiserum,21 anti–IFN-g antiserum, or a combination thereof. However, a residual amount of DNA fragmentation was still observed in all antiserum-treated groups, suggesting that additional signals besides IFN-g and TNF may contribute to con A–induced hepatic apoptosis. We recently described that TNF induces internucleosomal DNA cleavage in mouse liver as well as histological features indicative of hepatocyte apoptosis.21,23 We also showed that it is TNF that mediates con A–induced liver injury and hepatic DNA fragmentation (compare Figures 2 and 320,22). Therefore, we asked the question of whether the protective effect of anti–IFN-g antiserum was merely caused by the inhibition of con A–induced TNF production or whether IFN-g may have also contributed directly to hepatic DNA fragmentation and liver cell death. To answer this question, we neutralized IFNg and injected mice with 2.5 mg/kg recombinant murine

Figure 1. Time course of (A ) TNF and (B ) IFN-g release into plasma of mice treated with 25 mg/kg con A IV. Data are expressed as mean { SEM (n Å 3 for each time point). *P õ 0.05 and **P õ 0.01 vs. time at 0 hours.

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TNF-a IV 30 minutes after the con A challenge. In this experimental setting, the systemic TNF concentration 90 minutes after con A injection was similar to the one observed after injection of con A alone (Figure 4B). However, recombinant murine TNF-a failed to cause liver injury under these conditions, as indicated by plasma

Figure 2. Modulation of (A ) plasma enzyme activities, (B ) plasma cytokine levels, and (C ) hepatic DNA fragmentation by anti–IFN-g antiserum or anti-TNF antiserum in con A–treated mice. Either 200 mL anti–IFN-g antiserum or 50 mL anti-TNF antiserum or a combination thereof was IV injected per mouse 15 minutes before IV administration of 25 mg/kg con A. Plasma TNF concentrations (B, ) were measured 90 minutes after injection with con A. Plasma activities of alanine aminotransferase (A, ), aspartate aminotransferase (A, ), and sorbitol dehydrogenase (A, 䊐), circulating IFN-g concentrations (B, 䊐), or DNA fragmentation in 13,000g supernatants of 20% liver homogenate were determined 8 hours after the con A challenge. Oligonucleosomebound DNA was measured by ELISA. Data are expressed as mean { SEM (n Å 3). *P õ 0.05 and **P õ 0.01 vs. con A–treated control. n.d., Not detectable.

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transaminase activities comparable to those observed in the con A plus anti–IFN-g antiserum–treated group (Figure 4A). In line with this statement, DNA fragmentation was not significantly increased by recombinant murine TNF-a when IFN-g activity was blocked (Figure 4C). These experiments show that recombinant murine TNFa cannot induce liver injury caused by con A treatment in the absence of IFN-g. We obtained similar results when we injected 5 mg/kg recombinant murine IFN-g IV together with con A and neutralizing anti-TNF antiserum (data not shown). Under these conditions, i.e., in the absence of TNF, IFN-g also failed to induce liver injury in con A–treated animals. These findings show that either cytokine, i.e., TNF and IFN-g, is necessary but not sufficient to induce liver injury in con A–treated mice.

TNF and IFN-g was caused by apoptosis of activated infiltrating T lymphocytes,22 DNA fragmentation was examined in the livers of con A–resistant BALB/c nu/ nu mice.19 In contrast to wild-type BALB/c mice, BALB/ c nu/nu mice failed to release TNF and other cytokines after con A injection (TNF peak levels, 850 { 160 pg/ mL vs. undetectable amounts). 20 Thus, the amount of DNA fragmentation in livers of nu/nu mice was significantly reduced compared with control mice (1010 { 160

Hepatic DNA Fragmentation in con A– Resistant Nude Mice To rule out that the residual hepatic DNA fragmentation after con A challenge and neutralization of

Figure 3. DNA fragmentation in livers of BALB/c mice 8 hours after challenge with 25 mg/kg con A. DNA was prepared from 13,000g supernatants of liver homogenates and stained with ethidium bromide after analysis on a 1% agarose gel. M, marker of 123–base pair DNA ladder; lane 1, saline control; lane 2, con A only; lane 3, con A pretreated with 200 mL anti–IFN-g antiserum per mouse; lane 4, con A pretreated with 50 mL anti-TNF antiserum per mouse.

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Figure 4. Lack of induction of liver injury by recombinant murine TNFa in con A–challenged mice pretreated with 200 mL of anti–IFN-g antiserum. Recombinant murine TNF-a (2.5 mg/kg) was injected IV 30 minutes after administration of 25 mg/kg con A. Plasma TNF concentrations (B, ) were measured 90 minutes after injection with con A. Plasma activities of alanine aminotransferase (A, ), aspartate aminotransferase (A, ), and sorbitol dehydrogenase (A, 䊐), circulating IFN-g concentrations (B, 䊐), or oligonucleosome-bound DNA in 13,000g supernatants of 20% liver homogenate were determined 8 hours after the con A challenge. Data are expressed as mean { SEM (n Å 3). *P õ 0.05 vs. con A–treated control. n.d., Not detectable.

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millioptical density vs. 1470 { 90 millioptical density; P ° 0.05). These results also show that a residual fragmentation of DNA was detectable and comparable to the amounts observed in normal mice after injection of antiTNF antiserum or anti–IFN-g antiserum (Figure 2C). Hence, it seems unlikely that the residual DNA fragmentation observed after injection of con A together with the cytokine-neutralizing antibodies was caused by infiltrating apoptotic T cells or by T cell–mediated cytokineindependent hepatocyte apoptosis.

CD3 MAb–or GalN/SEB-treated mice protected by neutralizing anti-TNF antiserum.17 We were interested in studying the effect of IFN-g neutralization in the two GalN models, which should protect at least by inhibition of TNF production. As shown in Figure 5, pretreatment of GalN/anti-CD3

Lack of Protection by the Inducible NOS Inhibitor Aminoguanidine Against con A– Induced Liver Injury TNF and IFN-g were described to induce synergistically the formation of NO,31 which has been found to induce programmed cell death.32 Because TNF and IFN-g are likely to synergistically induce liver injury in con A–treated mice, we wondered whether NO was involved in mediating hepatic damage. We injected mice IP with 15 mg/kg aminoguanidine, a relatively selective inhibitor of the cytokine inducible NO synthase, 30 minutes, 3 hours, and 6 hours after the con A challenge. This dose and application regimen of aminoguanidine was shown recently to improve survival in murine endotoxemia.33 Unlike in this latter animal model, aminoguanidine failed to prevent con A–inducible liver injury (aminoguanidine / 25 mg/kg con A IV; 3470 { 630 U/L alanine aminotransferase vs. con A; control, 7070 { 3600 U/L alanine aminotransferase; n Å 3; NS), although the drug significantly suppressed plasma nitrite concentrations (aminoguanidine / con A, 1.2 { 1.2 mmol/L vs. con A; control, 25.0 { 4.9 mmol/L; n Å 3; P õ 0.001). These data provide circumstantial evidence that endogenously produced NO is unlikely to mediate con A–induced liver injury. Lack of Protection by Anti–IFN-g Antiserum Against T Cell–Dependent Liver Injury in GalN-Sensitized Mice Activating antibodies directed against the CD3 signal transducing subunit of the T-cell receptor or the bacterial superantigen SEB were shown to induce severe liver injury only in GalN-sensitized mice, i.e., by concomitant inhibition of hepatic transcription.17 Liver injury was characterized by TNF-mediated hepatic internucleosomal DNA cleavage as well as by formation of hyperchromatic structures of hepatocyte nuclei and apoptotic bodies. In contrast to the con A model, DNA fragmentation was completely abrogated in GalN/anti/ 5e10$$0016

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Figure 5. Lack of protection by anti–IFN-g antiserum against T cell– dependent liver injury in GalN-sensitized mice. Anti-CD3 MAb (10 mg/ mouse IV) or SEB (2 mg/kg IP) was administered 10 minutes after GalN (700 mg/kg IP). Two hundred microliters of anti–IFN-g antiserum per mouse was IV injected 5 minutes before GalN application. Plasma TNF concentrations (B, ) were measured 90 minutes after injection with con A. Plasma activities of alanine aminotransferase (A, ), aspartate aminotransferase (A, ), and sorbitol dehydrogenase (A, 䊐), circulating IFN-g concentrations (B, 䊐), or oligonucleosome-bound DNA in 13,000g supernatant of 20% liver homogenate were determined 8 hours after GalN injection. Data are expressed as mean { SEM (n Å 6). n.d., Not detectable.

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MAb– or GalN/SEB-challenged mice with anti–IFN-g antiserum neutralized circulating IFN-g but failed to attenuate TNF release and to significantly protect mice from liver injury as assessed by plasma transaminases or hepatic DNA fragmentation. Thus, it seems likely that GalN specifically sensitizes the liver towards TNF, whereas con A–induced liver injury depends at least on one additional mediator, i.e., IFN-g. Recombinant Murine IFN-g Fails to Induce Liver Injury in GalN-Sensitized Mice IV administration of 4–10 mg/kg recombinant murine TNF-a to GalN-sensitized mice was shown to induce early hepatocyte apoptosis and subsequent development of liver injury21,34 comparable to the severity of hepatic failure observed after GalN/anti-CD3 MAb or GalN/SEB challenge (Figure 5). These TNF doses corresponded approximately to a magnitude of 60–150-fold of systemic TNF peak concentrations measured after administration of SEB to GalN-sensitized mice (Figure 5). In contrast to administration of recombinant murine TNF-a, IV injection of 50 mg/kg recombinant murine IFN-g to GalN-sensitized mice, i.e., a magnitude of 75fold of SEB-induced maximal circulating IFN-g concentrations, resulted neither in TNF production (TNF peak levels were less than the detection limit of the ELISA) nor in hepatic DNA fragmentation (values of GalN/IFNg–treated mice and GalN control mice were near the detection limit of the ELISA) nor in development of liver injury (alanine aminotransferase, 20 { 3 U/L of GalN/ IFN-g–treated mice vs. 15 { 6 U/L of GalN control). These results suggest again that GalN sensitized the liver towards TNF but not towards IFN-g after T-cell activation in mice.

Discussion In this study, we identified IFN-g as playing a key role in the con A–inducible liver injury model that is initiated by activation of CD4/ lymphocytes.19 An early cytokine response was observed (Figure 120) before hepatic apoptosis was detectable by specific internucleosomal DNA cleavage, and transaminase release indicated severe organ destruction.20,21 At least two proinflammatory cytokines, TNF and IFN-g, mediate liver injury in this model. Our results in Figure 1 show an early increase of circulating TNF and IFN-g levels. The time course of IFN-g release differs from previously published data in showing a substantial increase of plasma IFN-g concentrations at later time points.20 This may be explained by a higher sensitivity of the ELISA used in this study. However, as published previously,20 the time course of / 5e10$$0016

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IFN-g release showed a steady further increase throughout the time of the experiment. Several mechanisms are to be considered by which IFN-g may destroy cells or may evoke organ destruction. These mechanisms are activation of macrophage functions,35 including synergistic stimulation of NO production together with TNF or LPS,31,32 direct induction of lymphocyte cytotoxicity,36 and direct activation of genes involved in signaling of cell death.37,38 In an experimental mouse model in which a specific cytotoxic T lymphocyte response towards hepatitis B surface antigen (HBsAg) on hepatocytes of HBsAg transgenic mice induced fulminant hepatitis, the most destructive pathogenic process was shown to be mediated by IFN-g.39 As a consequence of antigen recognition, cytotoxic T lymphocyte was induced to release IFN-g, which activated macrophages and finally destroyed the liver. In contrast to cytotoxic T lymphocyte class I restricted liver injury of HBsAg transgenic mice, con A– induced liver injury is mediated by CD4/ cells19 infiltrating the portal area of the hepatic tissue.22 However, as in the case of the transgenic model, macrophages are also required to mediate con A–induced hepatic failure.19 Moreover, con A substantially augmented cytokine production of lymphocytes in the presence of primary murine liver cell cultures. This lymphocyte/liver cell crosstalk was mostly pronounced in cocultures of murine lymph node cells and hepatic macrophages (Gantner et al.20 and Gantner et al., manuscript submitted for publication). Thus, either CD8/ or CD4/ cells, the latter were also suggested to regulate hepatitis B virus disease activity by a Th1-like response,40 produce IFN-g, which mediates liver injury possibly by macrophage activation and induction of a cytokine-response syndrome. TNF was shown to induce apoptosis and DNA fragmentation in mouse liver, and the early DNA fragmentation observed in the con A model was proposed to depend at least in part on TNF.21 The initial release of TNF and IFN-g into the circulation occurred nearly simultaneously after con A administration (Figure 1), suggesting interactions between both proinflammatory cytokines. First of all, both cytokines regulated each other in respect to their production (Figure 2). Because anti–IFN-g antiserum partially inhibited TNF release in vivo, inhibition of DNA fragmentation and prevention of liver injury may have only been caused by reduced TNF concentrations and not by inhibition of IFN-g–mediated hepatocyte death (Figure 2). This interpretation is supported by the finding that recombinant murine IFN-g failed to induce DNA fragmentation and hepatic failure in GalNsensitized mice. However, a direct contribution of IFNWBS-Gastro

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g to con A–induced hepatotoxicity seems likely because injection of recombinant murine TNF-a to anti–IFN-g antiserum and con A–treated mice failed to potentiate transaminase release (Figure 4). Liver injury was also not observed after administration of recombinant murine IFN-g to con A and anti-TNF antiserum–treated mice. These results corroborate the notion that both cytokines acted synergistically to mediate liver injury. Synergism between TNF and IFN-g has been described for LPS lethality; in combination, TNF and IFN-g exerted a lethal effect, and IFN-g mediated the lethality of high TNF doses.24 Moreover, both cytokines have been reported to act synergistically in their cytotoxic antitumor effects and in the killing of intracellular pathogens31,41 possibly mediated by NO. Our results showing the lack of protection by the inducible NO synthase inhibitor aminoguanidine against con A–induced liver injury argue against NO-induced hepatic failure as a major pathogenic mechanism. Likewise, in a T lymphocyte–dependent experimental mouse model using SEB to stimulate the T cells, IFNg synergized with TNF to generate NO.7 In this model, however, endogenous NO conferred protection, probably by vascular effects, as it has also been described for LPS or TNF hepatotoxicity.30,42 One further potential cellular mechanism of IFN-g interaction with TNF activity is the regulation of the TNF receptor number. IFN-g has been reported to increase the TNF receptor number on cell lines in vitro.43,44 Such a mechanism may also underlie TNF-induced hepatic DNA fragmentation in the con A model, i.e., IFNg may have up-regulated TNF receptors on hepatocytes, thereby potentiating TNF-induced DNA fragmentation and hepatotoxicity. However, the lack of protection by anti–IFN-g antiserum against T cell–mediated liver injury in the GalN models, which highly depend on TNF, suggests that IFN-g–induced up-regulation of the TNF receptor number is not a primary mechanism of hepatotoxicity in vivo. Beyond these considerations of IFN-g–dependent indirect cellular toxicity, IFN-g was described to induce hepatocellular apoptosis and DNA fragmentation and leakage in primary mouse16 or rat45 hepatocyte cultures that were potentiated by TNF-a. Moreover, in cell lines, IFN-g induced the activation of genes that were suggested as candidates for mediators of programmed cell death.37,38 With respect to the activities of IFN-g described so far, one possible explanation for IFN-g–mediated liver injury in the con A model is that TNF and IFN-g cooperated to induce direct hepatocytotoxicity independently of NO. In the GalN models, T-cell activation by either / 5e10$$0016

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anti-CD3 MAb or SEB resulted in the production of large quantities of IFN-g (Figure 517,18). However, doses of the anti–IFN-g antibody that prevented con A–induced liver damage failed to inhibit anti-CD3 MAb– or SEB-induced TNF production and to protect GalNsensitized mice from liver failure. In contrast to these results, anti–IFN-g antibodies prevented pathological changes induced by the injection of high doses of either anti-CD3 MAb46 or SEB47 to nonsensitized mice, however, also without affecting TNF release. These observations indicate again that IFN-g plays a critical role in the pathogenesis of cytokine-release syndromes. With respect to mortality of SEB in GalN-sensitized mice, anti–IFN-g antibodies failed to improve survival47 or had only moderate effects.18 Lack of protection by anti– IFN-g antibodies was also observed after administration of LPS to GalN-sensitized mice.6 Together with our finding that anti–TNF-a antibodies completely abrogated hepatic DNA fragmentation, i.e., a highly sensitive parameter of liver damage, in GalN/anti-CD3 MAb– or GalN/SEB-treated mice,17 we conclude that GalN sensitizes the liver exclusively towards TNF. Thus, the GalN models are useful for studying the regulation of TNF release and ensuing mechanisms of TNF-dependent hepatocyte death. Unlike in the GalN models, pathophysiological events initiated by con A injection to mice were also observed in acute hepatitis, where IFN-g produces an antiviral state and may evoke severe hepatic injury.39 The con A model may also reflect inflammatory disease mechanisms where cytokines activate T cells to generate a cytolytic response towards target cells as it has been described for antigen-specific killing induced by Th0 cell clones generated from intrahepatic isolates of patients with chronic hepatitis C.48 Moreover, IFN-g expression in transgenic mice under control of the insulin promoter was shown to evoke islet cell destruction that was mediated by T lymphocytes.36 In a transgenic mouse model, in which IFN-g is regulated by a liver-specific promoter, liver injury was associated by lymphoid cell infiltrations at the portal area.15 Thus, IFN-g and possibly other cytokines that are produced after con A injection to mice may also have stimulated T lymphocytes to generate a cytotoxic response towards hepatocytes. According to the experimental findings and conclusions drawn from this study, con A–induced hepatitis in mice seems to be a suitable model to study basic processes of liver pathology caused by lymphocyte activation and infiltration.

References 1. Farrar MA, Schreiber RD. The molecular cell biology of interferong and its receptor. Annu Rev Immunol 1993;11:571–611.

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2. Heinzel FP. The role of IFN-gamma in the pathology of experimental endotoxemia. J Immunol 1990;145:2920–2924. 3. Beutler B, Tkacenko V, Milsark I, Krochin N, Cerami A. Effect of gamma interferon on cachectin expression by mononuclear phagocytes. Reversal of the lpsd (endotoxin resistance) phenotype. J Exp Med 1986;164:1791–1796. 4. Katschinski T, Galanos C, Coumbos A, Freudenberg MA. Gamma interferon mediates Propionibacterium acnes –induced hypersensitivity to lipopolysaccharide in mice. Infect Immun 1992;60: 1994–2001. 5. Car BD, Eng VM, Schnyder B, Ozmen L, Huang S, Gallay P, Heumann D, Aguet M, Ryffel B. Interferon g receptor deficient mice are resistant to endotoxic shock. J Exp Med 1994;179:1437– 1444. 6. Heremans H, Van Damme J, Dillen C, Dijkmans R, Billiau A. Interferon gamma, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice. J Exp Med 1990; 171:1853–1869. 7. Florquin S, Amraoui Z, Dubois C, Decuyper J, Goldman M. The protective role of endogenously synthesized nitric oxide in staphylococcal enterotoxin B–induced shock in mice. J Exp Med 1994; 180:1153–1158. 8. Napoli J, Bishop GA, McCaughan GW. Increased intrahepatic messenger RNA expression of interleukins 2, 6, and 8 in human cirrhosis. Gastroenterology 1994;107:789–798. 9. Hussain MJ, Mustafa A, Mowat AP, Mieli-Vergani G, Vergani D. Immunohistochemical detection of tumor necrosis factor– a (TNFa) and interferon-g (IFN-g) in the liver of children with autoimmune chronic active hepatitis (aCAH) and primary sclerosing cholangitis (PSC) (abstr). J Hepatol 1992;16:S59. 10. Ferrari C, Mondelli MU, Penna A, Fiaccadori F, Chisari FV. Functional characterization of cloned intrahepatic, hepatitis B virus nucleoprotein-specific helper T cell lines. J Immunol 1987;139: 539–544. 11. Ferrari C, Chisari FV, Ribera E, Penna A, Mondelli MU. Functional modulation of hepatitis B core antigen-specific T lymphocytes by an autoreactive T cell clone. J Immunol 1988;141:1155–1160. 12. Tilg H, Vogel W, Aulitzky WE, Herold M, Ko¨nigsrainer A, Margreiter R, Huber C. Evaluation of cytokines and cytokine-induced secondary messages in sera of patients after liver transplantation. Transplantation 1990;49:1074–1080. 13. Volpes R, van den Oord JJ, Vos RD, Depla E, Ley MD, Desmet VJ. Expression of interferon-g receptor in normal and pathological human liver tissue. J Hepatol 1991;12:195–202. 14. Franco A, Barnaba V, Natali P, Balsano C, Musca A, Balsano F. Expression of class I and II major histocompatibility antigens on human hepatocytes. Hepatology 1988;8:449–454. 15. Toyonaga T, Hino O, Sugai S, Wakasugi S, Abe K, Shichiri M, Yamamura K-I. Chronic active hepatitis in transgenic mice expressing interferon-g in the liver. Proc Natl Acad Sci USA 1994; 91:614–618. 16. Morita M, Watanabe Y, Akaike T. Protective effect of hepatocyte growth factor on interferon-gamma–induced cytotoxicity in mouse hepatocytes. Hepatology 1995;21:1585–1593. 17. Gantner F, Leist M, Jilg S, Germann PG, Freudenberg MA, Tiegs G. Tumor necrosis factor–induced hepatic DNA fragmentation as an early marker of T cell–dependent liver injury in mice. Gastroenterology 1995;109:166–176. 18. Nagaki M, Muto Y, Ohnishi H, Yasuda S, Sano K, Naito T, Maeda T, Yamada T, Moriwaki H. Hepatic injury and lethal shock in galactosamine-sensitized mice induced by the superantigen staphylococcal enterotoxin B. Gastroenterology 1994;106:450– 458. 19. Tiegs G, Hentschel J, Wendel A. A T-cell dependent experimental liver injury in mice inducible by concanavalin A. J Clin Invest 1992; 90:196–203.

/ 5e10$$0016

06-29-96 08:43:45

gasas

20. Gantner F, Leist M, Lohse AW, Germann PG, Tiegs G. Concanavalin A–induced T cell–mediated hepatic injury in mice: the role of tumor necrosis factor. Hepatology 1995;21:190–198. 21. Leist M, Gantner F, Bohlinger I, Tiegs G, Germann PG, Wendel A. Characterization of TNF-induced murine hepatic apoptosis as a pathomechanism of septic liver failure. Am J Pathol 1995;146: 1220–1234. 22. Mizuhara H, O’Neill E, Seki N, Ogawa T, Kusunoki C, Otsuka K, Satoh S, Miwa M, Senoh H, Fujiwara H. T cell activation–associated hepatic injury: mediation by tumor necrosis factor and protection by interleukin 6. J Exp Med 1994;179:1529–1537. 23. Leist M, Gantner F, Bohlinger I, Germann PG, Tiegs G, Wendel A. Murine hepatocyte apoptosis induced in vitro and in vivo by TNFa requires transcriptional arrest. J Immunol 1994;153: 1778–1787. 24. Doherty GM, Lange JR, Langstein HN, Alexander HR, Buresh CM, Norton JA. Evidence for IFN-g as a mediator of the lethality of endotoxin and tumor necrosis factor– a. J Immunol 1992;149: 1666–1670. 25. Leo O, Foo D, Sachs DH, Bluestone JA. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc Natl Acad Sci USA 1987;34:1374–1378. 26. Espevik T, Nissen-Meyer J. A highly senstive cell line, WEHI 164 clone 13, for measuring cytotoxic factor tumor necrosis factor from human monocytes. J Immunol Meth 1986;95:99–105. 27. Bergmeyer HU. Methods of enzymatic analysis. 3rd ed. Weinheim, Germany: Verlag Chemie, 1984. 28. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980; 284:555–556. 29. Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorimetric assay for the measurement of nitrite in biological samples. Anal Biochem 1993;214:11–16. 30. Bohlinger I, Leist M, Barsig J, Uhlig S, Tiegs G, Wendel A. Interleukin-1 and nitric oxide protect against tumor necrosis factor a– induced liver injury through distinct pathways. Hepatology 1995; 22:1829–1837. 31. Liew FY, Li Y, Millot S. Tumor necrosis factor–a synergizes with IFNg in mediating killing of Leishmania major through the induction of nitric oxide. J Immunol 1990;145:4306–4310. 32. Messmer UK, Lapetina EG, Bru¨ne B. Nitric-oxide–induced apoptosis in RAW 264.7 macrophages is antagonized by protein kinase C– and protein kinase A–activating compounds. Mol Pharmacol 1995;47:757–765. 33. Wu CC, Chen SJ, Szabo´ C, Thiemermann C, Vane JR. Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock. Br J Pharmacol 1995; 114:1666–1672. 34. 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. 35. Young HA, Hardy KJ. Role of interferon-g in immune cell regulation. J Leukoc Biol 1995;58:373–381. 36. Sarvetnick N, Shizuru J, Liggitt D, Martin L, McIntyre B, Gregory A, Parslow T, Stewart T. Loss of pancreatic islet tolerance induced by b-cell expression of interferon-g. Nature 1990;346: 844–847. 37. Deiss LP, Feinstein E, Berissi H, Cohen O, Kimchi A. Identification of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the g interferon–induced cell death. Genes Dev 1995;9:15–30. 38. Kissil JL, Deiss LP, Bayewitch, Raveh T, Khaspekov G, Kimchi A. Isolation of DAP3, a novel mediator of interferon-g–induced cell death. J Biol Chem 1995;270:27932–27936. 39. Ando K, Moriyama T, Guidotti LG, Wirth S, Schreiber RD, Schlicht HJ, Huang S, Chisari FV. Mechanisms of class I restricted immu-

WBS-Gastro

IFN-g MEDIATES CON A HEPATITIS IN MICE 471

August 1996

40.

41.

42.

43.

44.

45.

46.

nopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med 1993;178:1541–1544. Lo¨hr HF, Weber W, Schlaak J, Goergen B, Meyer zum Bu¨schenfelde K-H, Gerken G. Proliferative response of CD4/ T cells and hepatitis virus clearance in chronic hepatitis with or without hepatitis B e-minus hepatitis B virus mutants. Hepatology 1995;22: 61–68. Esparza I, Mannel D, Ruppel A, Falk W, Krammer P. Interferongamma and lymphotoxin or tumor necrosis factor act synergistically to induce macrophage killing of tumor cells and schistosomula of Schistosoma mansoni. J Exp Med 1987;166:589– 600. Harbrecht BG, Billiar TR, Stadler J, Demetris AJ, Ochoa JB, Curran RD, Simmons RL. Nitric oxide synthesis serves to reduce hepatic damage during acute murine endotoxemia. Crit Care Med 1992; 20:1568–1574. Aggarwal BB, Eessalu TE, Hass PE. Characterization of receptors for tumor necrosis factor and their regulation by gamma-interferon. Nature 1985;318:665–667. Ruggiero V, Tavernier J, Fiers W, Baglioni C. Induction of the synthesis of tumor necrosis factor receptors by interferongamma. J Immunol 1986;136:2445–2450. Shinagawa T, Yoshioka K, Kakumu S, Wakita T, Ishikawa T, Itho Y, Takayanagi M. Apoptosis in cultured rat hepatocytes: the effects of tumor necrosis factor a and interferon g. J Pathol 1991; 165:247–253. Matthys P, Dillen C, Proost P, Heremans H, Van Damme J, Billiau A. Modification of the anti-CD3–induced cytokine release syndrome by anti–interferon-g or anti–interleukin-6 antibody treat-

/ 5e10$$0016

06-29-96 08:43:45

gasas

ment: protective effects and biphasic changes in blood cytokine levels. Eur J Immunol 1993;23:2209–2216. 47. Matthys P, Mitera T, Heremans H, Van Damme J, Billiau A. Anti– gamma interferon and anti–interleukin-6 antibodies affect staphylococcal enterotoxin B–induced weight loss, hypoglycemia, and cytokine release in D-galactosamine–sensitized and unsensitized mice. Infect Immun 1995;63:1158–1164. 48. Minutello MA, Pileri P, Unutmaz D, Censini S, Kuo G, Houghton M, Brunetto MR, Bonino F, Abrignani S. Compartmentalization of T lymphocytes to the site of disease: intrahepatic CD4/ T cells specific for the protein NS4 of hepatitis C virus in patients with chronic hepatitis C. J Exp Med 1993;178:17–25.

Received February 23, 1996. Accepted April 17, 1996. Address requests for reprints to: Gisa Tiegs, Ph.D., Institute of Pharmacology and Toxicology, University of Erlangen-Nu¨rnberg, Universitaetsstr 22, D-91054 Erlangen, Germany. Fax: (49) 9131-206119. Supported by grant Ti 169/3-3 from the Deutsche Forschungsgemeinschaft. Dr. Ku¨sters’ new affiliation is: Institute of Pharmacology and Toxicology, University of Erlangen-Nu¨rnberg, Erlangen, Germany. The authors thank Dr. Albrecht Wendel (Biochemical Pharmacology, Faculty of Biology, University of Konstanz) for his encouragement and helpful discussion; U. Gebert, I. Linge, M. Ullmann, and E. Schmid for perfect technical assistance; and Dr. G. R. Adolf, Bender & Co. (Vienna, Austria) for generously providing murine tumor necrosis factor a and interferon gamma.

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