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The Journal of Immunology, 2001, 167: 514-523.
Copyright © 2001 by The American Association of Immunologists

Dissection of the Intracellular Pathways in Hepatocytes Suggests a Role for Jun Kinase and IFN Regulatory Factor-1 in Con A-Induced Liver Failure1

Konrad Streetz, Bastian Fregien, Jörg Plümpe, Kerstin Körber{dagger}, Stefan Kubicka, G. Sass{dagger}, Stephan C. Bischoff, Michael P. Manns, Gisa Tiegs{dagger} and Christian Trautwein2

* Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Germany; and {dagger} Institute of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen, Erlangen, Germany.


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Con A administration results in dose-dependent immune-mediated liver injury. Cytokines are important to determine the outcome of liver failure in this model, and especially TNF-{alpha} and IFN-{gamma} directly contribute to hepatocyte damage. The intracellular pathways of these two cytokines, which eventually result in tissue destruction, are not well defined. Here we used anti-IFN-{gamma} Abs and adenoviral vectors that express molecules inhibiting distinct TNF-{alpha}-dependent pathways in hepatocytes to better understand the relevance of specific intracellular signaling cascades for Con A-induced liver failure. We show that activation of TNF-{alpha}- and IFN-{gamma}-dependent intracellular pathways occurs prior to the influx of immune-activated cells into the liver and that anti-TNF-{alpha} and anti-IFN-{gamma} neutralizing Abs cannot block infiltration of these cells. Blocking experiments with Abs and adenoviral vectors showed that NF-{kappa}B activation and the Fas-associated death domain protein/caspase 8 cascade in hepatocytes during Con A-induced liver failure have no impact on tissue injury. Additionally, STAT1 activation alone after Con A injection in liver cells does not result in liver damage. In contrast, IFN-{gamma}-dependent expression of IFN regulatory factor-1 and TNF-{alpha}-dependent activation of c-Jun N-terminal kinase in liver cells correlates with liver cell damage after Con A injection. Therefore, our experiments indicate that IFN regulatory factor-1 and the c-Jun N-terminal kinase pathway are involved in determining hepatocyte damage during Con A-induced liver failure and thus may provide new targets for therapeutic intervention.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Con A injection in mice results in dose-dependent immune-mediated liver injury. After in vivo exposure of Con A, different distinct cell types become activated, which eventually leads to the destruction of liver cells. The high affinity of Con A to the hepatocyte sinus seems specifically involved in attracting cells of the immune system to the liver (1, 2). Several reports contributed to our current understanding of this immune-mediated process, and there is evidence that these mechanisms are also relevant for the initial events during the pathophysiology of liver diseases found in humans like autoimmune or viral hepatitis (3, 4, 5, 6, 7, 8).

Recent studies indicated that liver NK T cells, i.e., NK1.1 CD4+CD8-TCR{alpha}{beta}+ and NK1.1. CD4-CD8-TCR{alpha}{beta}+ are the essential T cells early involved during Con A-induced liver injury (7, 8), and it seems that this T cell population is sufficient for the development of Con A-induced hepatitis. However, Con A injection results in the depletion of liver NK T cells during the first 4 h after administration. At this early period the Fas/Fas ligand and the perforin-granzyme B system contribute to liver NK T cell elimination after Con A injection (7, 8).

Besides the elimination of liver NK T cells (during this initial process) different cytokines, namely, IL-2, TNF-{alpha}, IFN-{gamma} IL-6, GM-CSF, and IL-1 become elevated in the serum of these animals (1, 2, 9, 10). The role of some of these cytokines during Con A-induced liver failure has been characterized in more detail. TNF-{alpha} and IFN-{gamma} have direct implications for the induction of liver cell injury, as anti-TNF-{alpha} and anti-IFN-{gamma} Abs protect from Con A-induced liver injury (9, 11). In contrast, IL-6 family members, e.g., IL-6 and IL-11, have a protective role as administration before Con A injection prevents mice from liver cell injury (2, 12).

After liver NK T cells have become eliminated and maximal TNF-{alpha} serum levels were present there is an influx of CD4-positive T lymphocytes and activation of polymorphonuclear cells. This event is not crucial in contributing to Con A-induced liver failure (13).

In contrast to the events that contribute to the activation of cells of the immune systems resulting in liver damage, the mechanisms eventually leading to hepatocyte apoptosis/necrosis are not well defined. The Ab-blocking experiments suggest that TNF-{alpha} and IFN-{gamma} directly contribute to liver cell damage in this model. TNF-{alpha} via the intracellular adapter molecules TNFR-associated death domain protein, Fas-associated death domain protein (FADD),4 TNFR-associated factor-2, and receptor interacting protein (for review, see Ref. 14) activates three main intracellular pathways resulting in caspase 8, NF-{kappa}B, or c-Jun N-terminal kinase (JNK) activation. IFN-{gamma} through different mechanisms may contribute to liver cell damage. Mice overexpressing IFN-{gamma} in the liver suffer from chronic hepatitis (15), and activation of STAT1 has been linked to the induction of apoptosis (16, 17).

In the Con A model TNF-{alpha} and IFN-{gamma} are essential to trigger liver cell damage (9, 11). Thus knowledge of the intracellular targets of these cytokine-dependent pathways will help to understand the pathogenesis of this model. To address this question we used different tools, namely, blocking Abs and adenoviral vectors that specifically inhibit the activation of intracellular pathways to evaluate the relevance of these signals in hepatocytes during Con A-induced liver cell injury. We show that the FADD, the NF-{kappa}B, and the STAT1 pathway in hepatocytes are not involved in Con A-induced liver damage. However, our results indicate that there is a correlation between activation of JNK and IFN regulatory factor-1 (IRF-1) with hepatocyte damage during Con A-induced liver injury.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Animals, Con A injection, and preparation of liver nuclear extracts

Pathogen-free male BALB/c mice were obtained from the Animal Research Institute of the Medizinische Hochschule Hannover (Hannover, Germany). All the experiments were started between 8 and 10 a.m. and were performed in agreement with the German legal requirements. Animals were anesthetized by an i.p. injection of a combination of rompun and ketamine as indicated earlier (10). For each time point at least four animals were treated in parallel.

Con A (20 mg/kg) was injected i.v. Anti-INF-{gamma} Abs were generated in the laboratory of G. Tiegs (Erlangen, Germany) in rabbits (11). Anti-INF-{gamma} was administered 0.5 h before Con A injection i.v. when indicated. Recombinant mouse TNF-{alpha} (2.5 µg/kg) was injected i.v. 15 min after Con A injection as described (11). In control-treated animals only the carrier solution (NaCl) was administered. At the indicated time points a small subxyphoid incision was made, blood was taken, and the liver was removed. The livers from animals treated in parallel were pooled. A part of the liver was frozen for Northern blot analysis, immunofluorescence, and DNA fragmentation assays. The remaining liver was used to prepare liver nuclear extracts.

For preparation of nuclear extracts the pooled livers were rinsed in ice-cold PBS, and liver nuclear proteins were prepared as described previously (10). All the steps were performed at 4°C. Nuclear proteins were aliquoted and immediately frozen in liquid nitrogen.

Cytokine and aminotransferase determinations

For cytokine and aminotransferase determinations blood was withdrawn into heparinized syringes by puncture of the right atrium. After a short spin at 200 x g plasma was recovered and stored at -80°C until use for determination of cytokine levels and aminotransferases.

TNF-{alpha} and IFN-{gamma} were determined by commercially available ELISA as described before (11, 18) according to manufacturer’s instructions. Alanine aminotransferase activities in plasma were determined by an automated enzyme assay.

SDS-PAGE and Western blot analysis

Nuclear extracts were separated on a 10% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane (Schleicher & Schuel, Keene, NH) in 1% SDS, 20% methanol, 400 mM glycine, 50 mM Tris-HCl (pH 8.3), at 4°C for 2 h at 200 mA as described previously (18). Western blot analysis was performed. Membranes were probed with anti-STAT1 Abs, which were a generous gift from T. Decker (Vienna Biocenter, Vienna, Austria) and anti-NF-{kappa}B p65 Abs (Santa Cruz Biotechnology, Santa Cruz, CA). The Ag-Ab complexes were visualized using the ECL detection system as recommended by the manufacturer (Amersham, Braunschweig, Germany). Western blot analysis was performed for each protein of interest at least three times.

Northern blot analysis

Northern blot analysis was performed according to standard procedures. Total RNA was isolated by the guanidiumisothiocyanate method (19). RNA (20 µg) was run in a 1% agarose formaldehyde gel, followed by transfer to Hybond-N membranes (Amersham). The IRF-1 and GAPDH cDNA probes were labeled with [{alpha}-32P]dATP using a random priming kit (Boehringer Mannheim, Mannheim, Germany). Hybridization procedure was performed as described previously (19). For quantification, blots were exposed for autoradiography and to an Image plate (Fuji, Nakanuma, Japan). The counts of the IRF-1 signal were distributed through the counts of the GAPDH signal and set to 1 in untreated animals.

Detection of mRNA by RT-PCR

To analyze gene expression by RT-PCR, mRNA was transcribed into cDNA using SuperScript II RNase H- Reverse Transcriptase (Life Technologies, Grand Island, NY). Oligonucleotides and Taq polymerase for subsequent PCRs were also obtained from Life Technologies. The following oligonucleotide pairs were used: 5' IFN-{alpha}: GAT GAG CTA CTG GTC AAT (39–56) 3' IFN-{alpha}: GCT GCA TCA GAC AGG T (376–361; GenBank NM010503), 5' {beta}-actin: TGG AAT CCT GTG GCA TCC ATG AAA (729–752); 3' {beta}-actin: TAA AAC GCA GCT CAG TAA CAG TCC G (1076–1053 in GenBank X03765). Semiquantitative evaluation was performed using the Gel Doc 2000 system (Bio-Rad, München, Germany).

In vitro JNK assays

JNK activity was assessed by an in vitro kinase assay as previously described (18) using recombinant GST-c-Jun protein 1–79(1–79). The proteins were fractionated using 12.5% SDS-PAGE and visualized/quantitated using phosphorimager analysis. Coomassie staining was used to demonstrate equal protein loading.

Gel retardation assays

For gel retardation assays, liver nuclear extracts were used as indicated. Binding reaction was performed for 20 min on ice (19). Binding buffer consisted of 25 mM HEPES (pH 7.6), 5 mM MgCl2, 34 mM KCl, 2 mM DTT, 0.2 mM PMSF, 1 µg/µl poly(dl:dC), and 2 µg/µl BSA. A p32-labeled oligonucleotide representing the NF-{kappa}B consensus site (5'-TAGTTG AGG GGA CTT TCC CAG GCA-3') was used as a probe. Free DNA and DNA-protein complexes were resolved on a 6% polyacrylamide gel. Supershift experiments were performed with Abs directed against the p50 and p65 NF-{kappa}B protein (Santa Cruz, Biotechnology).

Immunofluorescence

For immunofluorescence experiments, cryosections (4- to 5-µm thick) were performed and fixed immediately in ice-cold acetone for 5 min, air dried, and either stored at -80°C or used immediately. Immunofluorescence staining was performed as described before (18).

Anti-mouse CD 45.2 FITC-conjugated Ab (1/1000 dilution, clone 104; PharMingen, Hamburg, Germany) and anti-mouse PE-conjugated CD25 Ab (1/250 dilution; PharMingen) was incubated for 3 h at room temperature. Sections were washed three times for 5 min in PBS at room temperature.

As primary Abs an anti-mouse CD4 Ab (1/50 dilution, clone Gk1.5; PharMingen) was incubated at 4°C overnight and an anti-phospho-c-jun polyclonal rabbit Ab (1/300 dilution; New England Biolabs, Beverly, MA) for 2 h at room temperature. Sections were washed three times for 5 min in PBS. As secondary Abs a Texas Red-conjugated anti-rat goat Ab or a Cy-3-conjugated affinity-purified F(ab')2 goat anti-rabbit IgG (H + L) (dilution 1/1000; Dianova, Hamburg, Germany) was added, respectively, for 1 h at room temperature. Subsequently, sections were washed three times for 5 min in PBS at room temperature.

Sections were analyzed with a fluorescence microscope (Olympus, Hamburg, Germany).

Adenovirus preparation

To generate high titer viral stocks, 2 x 108 293 packaging cells at 90% confluence were infected at a multiplicity of infection of 5–10 PFU per cell. The infected cells were cultured for 3–5 days until a strong cytopathic effect could be observed and ~50% of these cells were detached. The cells were then collected by centrifugation, and viral particles were released by four cycles of freezing in liquid nitrogen and rapid thawing at 37°C. For further purification the virus preparation was subjected to a 2-fold CsCl2 banding. CsCl2 banding and determination of infectivity by viral plaquing were performed according to protocols previously described (20). Endotoxin contamination was monitored by the LAL-test kit (Chromogenix, Molndal, Sweden) following the protocol provided by the manufacturer. All virus preparations used for infection experiments were LPS free. Virus preparations were stored at -20°C in 25% glycerol, 10 mM Tris-HCl (pH 7.4), 1 mM MgCl2, and 140 mM NaCl.

The adenoviral vectors (adv) used in this study, adv {beta}-galactosidase ({beta}-gal), I-{kappa}B-AA, and dominant-negative (dn) FADD, have been described before (20, 21).

Cell culture and infection experiments

HepG2 cells were grown in DMEM (Life Technologies) supplemented with 10% FCS. Twenty-four hours before stimulation cells were infected with 100 PFU. HepG2 cells were stimulated with TNF-{alpha} (50 ng/ml) and/or cycloheximide (CHX) (1 mg/ml) for the time points as indicated. Nuclear extracts were prepared by the Dignam C method as described earlier (19). Morphologic changes such as membrane blebbing and cell shrinkage indicating apoptosis were visualized using an Olympus microscope B x60 (Olympus, Hamburg, Germany).

Con A injection and adenoviral infection

Using increasing amounts of the {beta}-gal adv, the efficacy of hepatocyte infection and liver toxicity was tested. A dose of 109 PFU {beta}-gal adv (as titered by plaques assay) infected 70–80% of the hepatocytes. The reliability of the infection was controlled by X-gal-stained liver sections of adv {beta}-gal-infected livers. Transaminases were determined at each point of time after hepatectomy.

Respective adenoviruses (109 PFU) were injected 24 h before Con A injection. Increasing amounts of Con A were administered. Transaminases were determined 8 h after injection to test the sensitivity of adenovirus-pretreated animals vs Con A.

DNA fragmentation

DNA fragmentation was determined by the commercially available ELISA cell death detection kit (Roche, Mannheim, Germany) according to manufacturer instruction. In brief, livers were homogenized 1:5 in Tris EDTA buffer with five strokes of a homogenizer (pestle B). The 20% homogenate was centrifuged at 13,000 x g for 20 min. The supernatant was further diluted 200-fold and directly used to determine DNA fragmentation (20).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
Anti-IFN-{gamma} treatment blocks liver cell damage, but not infiltration of CD4-positive and CD45-positive cells after Con A injection

Earlier results indicated that Con A-induced liver cell damage could be blocked by administration of anti-IFN-{gamma} blocking Abs. To study the IFN-{gamma}-dependent pathways, which are likely to mediate hepatocyte damage, we confirmed these results and substituted the decrease in TNF-{alpha} serum levels, as anti-IFN-{gamma} may reduce TNF-{alpha} serum concentration (11). Therefore, four different treatment groups were included in our analyses (carrier solution alone, Con A alone, Con A with anti-IFN-{gamma}, and Con A with anti-IFN-{gamma} and TNF-{alpha}).

Transaminase levels were measured to determine the degree of liver cell damage. Injection of Con A resulted in a significant increase in transminases compared to NaCl-treated animals. Maximal levels were found 8 h after injection. At later time points transaminases decreased, but were still significantly elevated (Fig. 1Go, A and B).



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  		FIGURE 1. Anti-IFN-{gamma} blocks Con A-induced liver failure. Mice were treated with carrier solution (control), Con A alone, Con A + anti-IFN-{gamma}, or Con A + anti-IFN-{gamma}/TNF-{alpha}. Serum transaminases (A, alanine aminotransferase; B, aspartate aminotransferase), were determined before and at the indicated time points after injection. In parallel IFN-{gamma} (C) and TNF-{alpha} (D) serum levels were analyzed.

 
Anti-IFN-{gamma} before Con A injection significantly reduced the increase in transaminases compared to the Con A-treated group (10–20% of Con A group). However, the transaminases were higher compared to control-treated animals. Additionally, TNF-{alpha} administration did not significantly change transaminase levels compared to the Con A/anti-IFN-{gamma}-treated mice (Fig. 1Go, A and B).

In further experiments, TNF-{alpha} and IFN-{gamma} serum levels were measured to study the impact of anti-IFN-{gamma} treatment on the expression of these two cytokines (Fig. 1Go, C and D). After Con A injection there was a strong increase in TNF-{alpha} and IFN-{gamma} serum levels. Maximal levels for both cytokines were found 2 h after administration. At later time points, cytokine levels decreased and were at baseline level for TNF-{alpha} after 24 h and still elevated for IFN-{gamma} at this time point.

Anti-IFN-{gamma} treatment blocked elevation of IFN-{gamma} in the Con A/anti-IFN-{gamma} and Con A/anti-IFN-{gamma}/TNF-{alpha}-treated animals. TNF-{alpha} serum levels were first significantly reduced 2 h after injection compared to the Con A only group. At this time point, TNF-{alpha} expression was reduced to 35% in the anti-IFN-{gamma} and to 48% in the anti-IFN-{gamma} plus TNF-{alpha} group compared to Con A alone (Fig. 1Go, C and D).

Next, we analyzed whether anti-IFN-{gamma} administration has a direct impact on the recruitment of immune-activated cells to the liver (Fig. 2Go). Immunohistochemistry revealed that there was an increase in CD4-positive, CD25-positive, and CD45-positive cells 8 h after Con A injection, whereas at 2 h the occurrence of immune-activated cells was not changed compared to untreated animals (Fig. 2Go). CD25 serves as a marker of activated T cells.



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  		FIGURE 2. Liver influx of immune-activated cells is independent of IFN-{gamma} and TNF-{alpha}. The influx of immune-activated cells, CD4-positive (A), CD45-positive (B), and CD25-positive (C), was monitored before, 2, and 8 h after treatment by immunohistochemistry. Figures are shown from untreated animals and mice challenged with Con A alone, Con A + anti-IFN-{gamma}, and Con A + anti-IFN-{gamma}/anti-TNF-{alpha}. Different time points are depicted.

 
Anti-IFN{gamma} blocking experiments with or without TNF-{alpha} injection did not change the influx and time course of immune-activated cells (data not shown). The influx of immune-activated cells was also not changed when, besides an anti-IFN-{gamma} blocking Ab, anti-TNF-{alpha} was coinjected indicating that both cytokines are not involved in regulating the liver influx of CD4- and CD45-positive cells after Con A injection.

Anti-IFN{gamma} treatment blocks IRF-1, but not STAT1 activation after Con A injection

To understand how effectively anti-IFN-{gamma} Abs might block IFN-{gamma}-dependent pathways in the liver, different intracellular pathways, which are activated through IFN-{gamma}, were tested. Via the Janus kinases IFN-{gamma} promotes STAT1 translocation into the nucleus (22). Its expression was tested by Western blot analysis in liver nuclear extracts (Fig. 3Go).



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  		FIGURE 3. Nuclear translocation of STAT1 can not be blocked by anti-IFN-{gamma}. Nuclear extracts were prepared from animals treated with the carrier solution (Control) (A), Con A alone (B), Con A + anti-IFN-{gamma} (C), and Con A + anti-IFN-{gamma}/TNF-{alpha} (D) before (N) and at the indicated time points after administration of the respective combinations. Nuclear extracts (10 µg per time point) were used for Western blot analysis. Membranes were incubated with an anti-STAT1 Ab. The position of the STAT1 signal is indicated through an arrowhead. E, RT-PCR for IFN-{alpha} and {beta}-actin was performed from liver RNA 4 h after treatment. The relative IFN-{alpha}/{beta}-actin ratio was calculated, and the average of three independent experiments is shown.

 
In control-treated animals no significant nuclear expression of STAT1 was found (Fig. 3GoA). Con A injection resulted in a strong induction of nuclear STAT1 expression immediately after Con A injection (Fig. 3GoB). Highest nuclear expression of STAT1 was found after 2 h, and a decrease in expression was found at later time points. There was still significant STAT1 expression 24 h after challenge.

Anti-IFN{gamma} did not block the increase in nuclear STAT1 expression after Con A injection (Fig. 3GoC). Nuclear STAT1 expression increased to maximal levels after 1.5 h, and a second peak was evident after 8 h. At later time points STAT1 expression decreased again. Therefore, anti-IFN-{gamma} blocking experiments had some effect on the time course of nuclear STAT1 expression after Con A treatment, but had no impact on the overall expression level.

Coadministration of Con A/anti-IFN-{gamma} and TNF-{alpha} reduced maximal STAT1 expression compared to the other two conditions when Con A was administered (Fig. 3GoD).

STAT1 was still increased in anti-IFN-{gamma}-treated animals. As IFN-{alpha} also results in STAT1 activation, its expression was tested by RT-PCR in liver tissue (Fig. 3GoE). These experiments showed that IFN-{alpha} mRNA is increased after Con A challenge. IFN-{alpha} expression was not completely blocked after anti-IFN-{gamma} injection, and these results might explain why STAT1 is still induced in the liver of these animals (Fig. 3GoE).

In further experiments we investigated changes in IRF-1 mRNA expression. Earlier results indicated that TNF-{alpha} and IFN-{gamma} might induce higher IRF-1 gene transcription (23, 24). In control-treated animals there was no induction in IRF-1 mRNA levels (Fig. 4GoA). In contrast, Con A alone strongly induced the expression of IRF-1 with maximal levels 1.5 and 2 h after treatment. At later time points IRF-1 decreased again (Fig. 4GoB). Anti-IFN-{gamma} with or without TNF-{alpha} treatment significantly reduced IRF-1 mRNA levels (Fig. 4Go, C and D). These results indicate that the protection from Con A-induced liver damage through anti-IFN-{gamma} correlated with the lack of higher IRF-1 mRNA expression.



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  		FIGURE 4. IRF-1 mRNA expression correlates with IFN-{gamma} serum levels and liver cell damage. Northern blot analysis was performed with 15 µg liver RNA prepared from animals before (N) and after injection of the carrier solution (Control) (A), Con A alone (B), Con A + anti-IFN-{gamma} (C), and Con A + anti-IFN-{gamma}/TNF-{alpha} (D). The filters were hybridized with 32P-labeled probes for IRF-1 and GAPDH. The position of the IRF-1 and GAPDH signal is indicated by an arrowhead.

 
Anti-IFN-{gamma} does not inhibit NF-{kappa}B activation after Con A injection

The anti-IFN-{gamma} blocking experiments, besides reducing IFN-{gamma} serum levels, had a direct effect on TNF-{alpha} expression. Thus these experiments provide a direct link to further evaluate the relevance of the TNF-{alpha}-dependent pathways in hepatocytes during Con A-induced liver failure. We thus investigated TNF-{alpha}-activated intracellular signaling cascades. NF-{kappa}B activation was studied by Western blot analysis and gel shift experiments using liver nuclear extracts (Fig. 5Go).



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  		FIGURE 5. NF-{kappa}B activation after Con A injection is independent of liver cell damage. NF-{kappa}B p65 expression was analyzed by Western blot analysis in liver nuclear extracts. Time points are shown before (N) and after injection of the carrier solution (Control) (A), Con A alone (B), Con A + anti-IFN-{gamma} (C), and Con A + anti-IFN-{gamma}/TNF-{alpha} (D). An arrowhead shows the position of NF-{kappa}B p65.

 
Western blot analysis showed that Con A injection strongly induced NF-{kappa}B translocation into the nucleus starting 0.5 h after administration, and maximal expression was found after 1 h. A gradual decrease was found at later time points. However, 24 h after injection, nuclear NF-{kappa}B expression was still higher compared to the pretreatment level (Fig. 5GoB).

In the Con A plus anti-IFN{gamma} group the time kinetic of NF-{kappa}B expression showed no major difference in the nuclear translocation of NF-{kappa}B compared to animals treated with Con A alone (Fig. 5GoC). Additionally, after the administration of anti-IFN-{gamma} and TNF-{alpha} only a minor variation in NF-{kappa}B activation was found (Fig. 5GoD). Nuclear NF-{kappa}B expression was more strongly increased after 0.5 h and remained high for up to 1.5 h. After this time point, NF-{kappa}B expression gradually decreased comparable to the other two conditions when Con A was administered (Fig. 5GoD).

In further experiments these results were evaluated by gel shift experiments. The similar time kinetic in NF-{kappa}B DNA-binding in the four treatment groups was found by gel shift experiments as evidenced by Western blot analysis (data not shown).

TNF-{alpha}-dependent pathways leading to FADD and NF-{kappa}B activation are not involved in triggering Con A-induced liver failure

The NF-{kappa}B experiments showed no strong variations between the three different conditions when Con A was injected alone or in combination with anti-IFN-{gamma}. Therefore, these experiments indicate that after the infiltration of CD4- and CD45-positive cells activation of NF-{kappa}B alone is not sufficient for inducing liver cell damage. However, the experiments did not exclude that NF-{kappa}B is required to trigger Con A-induced liver failure. To address this point we wanted to selectively block NF-{kappa}B activation in hepatocytes. We thus used an adv overexpressing the I-{kappa}B super-repressor (I-{kappa}B-AA) in hepatocytes. This mutant I-{kappa}B construct cannot be phosphorylated efficiently and thus ubiquitination and degradation of I-{kappa}B does not occur after TNF-{alpha} stimulation. Additionally, we used an adv expressing a dn form of FADD (adv dn FADD), which inhibits activation of the downstream caspase cascade (20).

Before the advs were injected in mice, we performed experiments in cell culture. Hepatoma cells (HepG2) were infected with adv I-{kappa}B-AA, dn FADD, or a control adv expressing {beta}-gal. Cells were treated with PBS, TNF-{alpha}, or TNF-{alpha}/CHX. Eighteen hours after treatment the morphology of the cells was studied (Fig. 6GoA). These results showed that the adv I-{kappa}B-AA sensitized cells toward TNF-{alpha}-induced apoptosis, whereas adv dn FADD protected cells from apoptosis. Additionally, gel shift experiments with a consensus oligonucleotide for NF-{kappa}B and nuclear extracts derived from cells infected with the different advs and plus or minus stimulation with TNF-{alpha} were performed. These studies showed that only the adv I-{kappa}B-AA was able to block TNF-{alpha}-dependent NF-{kappa}B activation (Fig. 6GoB).



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  		FIGURE 6. Con A-dependent liver failure is independent of pathways triggering FADD- and NF-{kappa}B-dependent signals. A, and B, HepG2 cells were infected with adenoviruses expressing {beta}-gal, I-{kappa}B-AA, dn FADD, or with carrier solution (uninfected). A, Twenty-four hours after infection cells were treated with PBS, TNF-{alpha} (50 ng/ml), or a combination of TNF-{alpha} and CHX (1 µg/ml) for 18 h. Morphologic changes such as membrane blebbing and cell shrinkage indicate apoptotic cells. B, HepG2 cells were infected with the adenoviruses as indicated for 24 h. Cells were treated for 30 min with 50 ng/ml TNF-{alpha} (+) or the carrier control (-). Nuclear extracts were prepared and gel shift experiments were performed with a 32P-labeled oligonucleotide representing a NF-{kappa}B consensus sequence. An arrowhead shows the position of bound DNA (C–E), Mice were infected with 109 PFU adv {beta}-gal 24 h before Con A challenge. C, Increasing amounts of Con A were injected as indicated in four mice in parallel, and serum transaminases were determined 8 h after Con A injection. D, Mice were infected with 109 PFU adv expressing {beta}-gal, I-{kappa}B-AA, or dn FADD as a transgene. Twenty-four hours after adv infection the animals were treated with 40 mg/kg body weight Con A for 8 h. Transaminases were analyzed. E, Using the ELISA cell death detection kit DNA fragmentation of the different conditions was measured as shown in B.

 
For the in vivo experiments, we first performed dose-kinetic experiments with a {beta}-gal-expressing adenovirus. A dose of 109 PFU was determined where 70–80% of the hepatocytes were infected and which did not cause signs of hepatitis (20). This dose was used in further experiments with increasing amounts of Con A to evaluate whether after adenoviral gene transfer the same doses of Con A can be used as injected in naive animals. These experiments revealed that in contrast to naive animals 40 mg/kg Con A were required to induce liver cell damage in animals pretreated with adenoviral constructs (Fig. 6GoC).

After challenging adv {beta}-gal-infected mice with Con A transaminases increased compared to animals treated with the carrier solution. In further experiments the adv I-{kappa}B-AA was injected. After 8 h transaminases were moderately higher in the I-{kappa}B-AA-treated animals compared to the controls (adv {beta}-gal group) (Fig. 6GoD).

The FADD/caspase 8 pathway is used by different TNF family members to induce apoptosis (25) and has been shown before to trigger apoptosis of hepatocytes via TNF. In earlier studies we showed that an adenovirus expressing a dn FADD molecule is able to block galactosamine/TNF-{alpha}-induced liver failure in vivo (20). Here, we studied the relevance of this pathway for Con A-induced liver failure. As shown in Fig. 6GoB, the adv dn FADD had no impact on the course of Con A-induced liver failure compared to the adv {beta}-gal control group. These results indicated that the signaling cascade involved in the induction of TNF-{alpha}-dependent apoptosis in hepatocytes is not essential for determining the outcome of liver cell damage after Con A injection.

In parallel, DNA fragmentation was determined in the liver of these animals. As shown for the increase in transaminase levels the I-{kappa}B-AA and dn FADD did not significantly inhibit DNA fragmentation compared to the adv {beta}-gal-treated control after Con A administration (Fig. 6GoE).

The Con A-dependent activation of JNK correlates with liver cell damage

Besides activating NF-{kappa}B and the caspase cascade TNF-{alpha} also activates JNK (14). JNK was monitored by an in vitro JNK assay, and incorporated p32-radioactivity was measured for quantification (18).

In control-treated animals no significant change in JNK activity was found at any time point (Fig. 7GoA). After Con A treatment alone there was a very strong increase in JNK activity starting at 0.5 h after administration. JNK further increased, and maximal levels were detected 4 h after injection (58-fold compared to pretreatment level). At later time points JNK activity rapidly decreased and nearly returned to pretreatment levels 12 h after Con A injection (Fig. 7Go, B and E).



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  		FIGURE 7. JNK activity correlates with Con A-induced liver failure. JNK activity was analyzed in liver nuclear extracts prepared from animals before (N) and after injection of the carrier solution (Control) (A), Con A alone (B), Con A + anti-IFN-{gamma} (C), and Con A + anti-IFN-{gamma}/TNF-{alpha} (D). E, Relative increase in JNK activity. JNK activity of untreated animals was set to 1 and the relative changes in JNK activity during the different treatment conditions are shown as fold activation. F, Phosphorylation of c-jun detected in the liver with an anti-phospho-c-Jun Ab by immunohistochemistry. The treatment conditions and the time points are indicated.

 
Anti-IFN-{gamma} blocking experiments resulted in a strong reduction of JNK activation. JNK first increased only 1 h after Con A injection, and maximal activation was found after 1.5 up to 4 h. At the 8-h time point JNK activity was dramatically reduced to nearly pretreatment levels (Fig. 7Go, C and E). Thus, maximal JNK levels and the length of JNK activation was strongly reduced in the anti-IFN-{gamma}-treated animals, which correlated with the reduction in transaminases (Fig. 7Go, C and E).

In the anti-IFN-{gamma} and TNF-{alpha} group JNK activity did not further increase compared to the Con A- and IFN-{gamma}-treated animals. In this group also at the time point 0.5 h after Con A injection, when TNF-{alpha} levels were higher compared to the maximal levels found in the Con A alone group, JNK activity did not rise to levels as found in the Con A group (Fig. 7Go, D and E). Thus the JNK results indicate that the duration and the maximal levels of JNK activation correlated with liver damage after Con A injection.

To localize the cell type in the liver with higher JNK activation immunofluorescence studies with an anti-phospho-c-Jun Ab were performed (Fig. 7GoF). A lack of phospho-c-Jun expression was found in untreated animals. Higher nuclear phospho-c-Jun expression in hepatocytes was found 2 and 8 h after Con A injection. The increase in phospho-c-Jun expression could be blocked through pretreating the animals with anti-TNF Abs.


   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
Con A injection into mice results in liver failure. Several cytokines, especially TNF-{alpha} and IFN-{gamma}, are involved in this process (4, 9, 11, 26). Our earlier experiments with anti-TNF-{alpha} blocking Abs defined the TNF-{alpha}-dependent intracellular signaling cascades that become activated during Con A-induced liver failure in liver cells (18). In the present study we were interested to further characterize the intracellular pathways that are activated in this model. We used anti-IFN-{gamma} blocking Abs and adv expressing specific molecules, which are able to inhibit the activation of one of the TNF-{alpha}-dependent intracellular pathways to further identify cascades resulting in liver damage after Con A injection.

Anti-IFN-{gamma} treatment blocked Con A-induced liver failure. However, anti-IFN-{gamma} and anti-TNF-{alpha} blocking experiments did not inhibit liver influx of CD4-positive and CD45-positive cells. For several reasons this result is especially important for the understanding of Con A-induced liver failure. First, TNF-{alpha} and IFN-{gamma} seem not to significantly contribute to the influx of immune-activated cells into the liver after Con A administration. Second, the influx of these cells occurs after the pathways leading to liver cell damage have been activated; and third, as these cells infiltrate independent of liver cell damage this step is not a consequence of liver cell destruction.

The next interest was to investigate the TNF-{alpha}- and IFN-{gamma}-dependent intracellular pathways. Several signaling cascades can be activated through the IFN/IFN receptor system (27, 28). Therefore, we concentrated on two pathways that might have implication in mediating liver cell damage in the Con A model. Earlier experiments showed that STAT1 can be involved in triggering apoptosis (16, 17) and for IRF-1 several functions have been described that might have a direct link to trigger immune-mediated tissue damage and cell death (29, 30, 31, 32, 33).

In contrast to STAT1, the increase in IRF-1 directly correlated with a strong induction of transaminases and was blocked when anti-IFN{gamma} was administered. Higher IRF-1 expression was observed clearly before the increase in transaminases and thus these results indicate that IRF-1 could be involved in triggering liver cell damage in this model. The mechanisms how IRF-1 might contribute to liver cell damage cannot be directly deducted from these data. However, earlier experiments showed that IRF-1 induces IL-1 converting enzyme gene transcription, which is directly involved in inducing apoptosis (29, 34) and that IRF-1 directly contributes to tissue damage in conditions like ischemia (30). Stasis of the blood flow is relevant during Con A-induced liver failure (35), and inhibition of IL-1 converting enzyme through caspase inhibitors modifies the outcome of Con A-induced liver failure (36). Therefore, both mechanisms could be relevant in determining the outcome after Con A administration. Recent results showing that IRF-1 knockout mice are resistant to Con A-induced liver failure also point to the relevance of our findings (37). Additionally, our results show that for the regulation of IRF-1 expression in liver cells IFN-{gamma} and not TNF-{alpha} is important in this model.

We studied the impact of the anti-IFN-{gamma} blocking experiments on the activation of the TNF-{alpha}-dependent signaling cascades. Anti-IFN-{gamma} administration did not block NF-{kappa}B activation. To further elucidate the role of NF-{kappa}B, an adenovirus expressing the I-{kappa}B-AA was injected. Adenoviruses very effectively infect hepatocytes, but to a lesser extent other cells of the body (38). However, as the adenoviral infection activates immune-mediated mechanisms (39, 40), higher doses of Con A were required for these experiments.

Infection studies with I-{kappa}B-AA, but also the adv dn FADD did not inhibit Con A-induced liver failure. These results indicate that these two pathways in hepatocytes do not significantly contribute to Con A-induced liver cell damage. TNF through TNF-R1 can induce hepatocyte apoptosis via the FADD/caspase 8 cascade (20, 41, 42). However, in normal hepatocytes, as TNF-{alpha} also activates NF-{kappa}B, the induction of apoptosis is prevented through the induction of NF-{kappa}B-dependent anti-apoptotic pathways (21). Our results show that these mechanisms are not of major relevance for the degree of liver injury after Con A administration. Consistent with our observations, earlier reports showed that a caspase 3 inhibitor is unable to block Con A-induced liver failure (3). However, in other models of TNF-{alpha}-mediated liver failure, e.g., LPS/galactosamine and TNF-{alpha}/galactosamine, caspase 3 activation is essential to determine the outcome of these animals (3, 20). FADD through caspase 8, mitochondrial permeability transition, and caspase 9 activates cleavage of caspase 3 (21) resulting in apoptosis. Therefore, together with the results of Künstle et al. (3) our experiments provide evidence that this caspase cascade is not involved in Con A-induced liver damage. Additionally, as TNF-{alpha}-dependent NF-{kappa}B activation stimulates anti-apoptotic pathways blocking the FADD/caspase 8 cascade, this mechanism also provides an explanation why I-{kappa}B-AA did not further increase Con A-induced liver cell damage.

In contrast to the other TNF-{alpha}-dependent pathways, there was a correlation between JNK activation and liver cell damage in this model. Induction of JNK after Con A treatment compared to other TNF{alpha}-dependent pathophysiological conditions in the liver, e.g., liver regeneration is dramatically increased (D. A. Brenner, unpublished observations). Additionally, JNK activation is specific, as other related pathways, as shown for extracellular signal regulated kinase, are not involved (18). Administration of TNF-{alpha} in the Con A and anti-IFN-{gamma} groups could not induce JNK activation to levels as found in the Con A group; even maximal TNF-{alpha} expression was higher in these animals. These results suggest that through the administration of Con A in the liver, an environment is created that sensitizes a strong TNF-{alpha}-dependent response vs JNK. As activation of JNK was found before the infiltration of immune-mediated cells, especially CD4-positive T cells, it can be excluded that "contaminating" liver-infiltrating cells are critical to explain our findings.

The role of JNK for tissue damage, necrosis/apoptosis, is not unique. JNK can exert pro- and anti-apoptotic stimuli (43, 44, 45). Our results showed that not the classical apoptosis cascade of Fas and TNF-R1 using the FADD/caspase 8 caspase is involved in Con A-induced hepatocyte damage. However, from our data it cannot be excluded that the FADD/caspase 8 cascade might play a role in nonparenchymal liver cells and lymphocytes, as adenoviruses infect predominantly hepatocytes. Additionally, liver histology of Con A-treated animals is different compared to other models resulting in TNF-{alpha}-dependent apoptosis (3). The mechanisms how JNK may mediate tissue damage are less defined and there is evidence for tissue-specific differences (42). There are reports showing the involvement of mitochondrial permeability transition and caspase 9/3 activation (46); however, there is also clear evidence that TNF vs TAJ (toxicity and JNK inducer) activates JNK-dependent/caspase-independent cell death (47). This mechanism would also explain results showing that Con A-induced liver cell damage is caspase 3 independent (3).

In hepatocytes there is clear evidence that JNK activation is a crucial event to stimulate DNA synthesis and it also seems to be involved in inducing anti-apoptotic pathways (48, 49). In contrast, there are reports showing that JNK may stimulate necrosis or apoptosis of hepatocytes (50, 51). Together these results indicate that JNK is involved in determining the balance between proliferation and apoptosis/necrosis of hepatocytes. Therefore, our present findings indicate that in the Con A model therapeutic strategies to block JNK activation seem promising to prevent liver cell damage.

In liver cells almost exclusively JNK 1 and 2 isoforms are expressed (52). At least one of these two isoforms is required for normal liver development as JNK1-/- and JNK 2-/- double-knockout mice are not viable (53). JNK1-/- and 2-/- display their phenotype in defective T cell differentiation (54, 55, 56). Therefore, conventional JNK knockout mice are not useful to study T cell-dependent liver injury (e.g., the Con A model). Thus to ultimately determine the role of JNK for Con A-induced liver cell damage hepatocyte-specific JNK conditional knockout mice will be required.

In summary, our experiments are essential in defining the pathways involved in hepatocyte damage in the Con A model. We show that STAT1, NF-{kappa}B, and the FADD/caspase 8 cascade are not important for hepatocyte damage in this model. However, our results indicate that activation of IRF-1 and JNK correlates with liver injury after Con A administration.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft Grants DFG Tr 285 3-4 and SFB 566. Back

2 Address correspondence and reprint requests to Dr. Christian Trautwein, Professor of Medicine, Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. E-mail address: Trautwein.Christian{at}mh-hannover.de Back

3 Abbreviations used in this paper: FADD, Fas-associated death domain protein; IRF-1, IFN regulatory factor-1; JNK, c-Jun N-terminal kinase; adv, adenoviral vector; CHX, cycloheximide; I-{kappa}B-AA, I-{kappa}B superrepressor; dn, dominant negative; {beta}-gal, {beta}-galactosidase. Back

4 Abbreviations used in this paper: FADD, Fas-associated death domain protein; IRF-1, IFN regulatory factor-1; JNK, c-Jun N-terminal kinase; adv, adenoviral vector; CHX, cycloheximide; I-{kappa}B-AA, I-{kappa}B superrepressor; dn, dominant negative; {beta}-gal, {beta}-galactosidase. Back

Received for publication October 18, 2000. Accepted for publication April 27, 2001.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

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