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*ACETAMINOPHEN
The Journal of Immunology, 2007, 178: 3777-3785.
Copyright © 2007 by The American Association of Immunologists, Inc.

Overexpression of Suppressor of Cytokine Signaling-3 in T Cells Exacerbates Acetaminophen-Induced Hepatotoxicity1

Kosuke Numata*,{dagger}, Masato Kubo{ddagger}, Hiroyuki Watanabe*,{dagger}, Katsumasa Takagi{dagger}, Hiroshi Mizuta{dagger}, Seiji Okada§, Steven L. Kunkel, Takaaki Ito* and Akihiro Matsukawa2,*,||

* Departments of Pathology and Experimental Medicine and {dagger} Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; {ddagger} Laboratory for Signal Network, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan; § Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto, Japan; Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and || Department of Pathology and Experimental Medicine, Graduate School of Medical, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cytokines have been implicated in the progression of acetaminophen (APAP)-induced acute liver injury. Suppressors of cytokine signaling (SOCS) proteins are negative regulators of cytokine signaling by inhibiting the JAK-STAT pathway, but their role in APAP hepatotoxicity is unknown. In this present study, we attempted to explore the role of SOCS3 in T cells in APAP-induced liver injury. Mice with a cell-specific overexpression of SOCS3 in T cells (SOCS3Tg, in which Tg is transgenic) exhibited exaggerated hepatic injury after APAP challenge, as evidenced by increased serum alanine aminotransferase levels, augmented hepatic necrosis, and decreased survival relative to the wild-type mice. Adaptive transfer of SOCS3Tg-CD4+ T cells into T and B cell-deficient RAG-2–/– mice resulted in an exacerbated liver injury relative to the control. In SOCS3Tg mice, hepatocyte apoptosis was enhanced with decreased expression of antiapoptotic protein bcl-2, whereas hepatocyte proliferation was reduced with altered cell cycle-regulatory proteins. Levels of IFN-{gamma} and TNF-{alpha} in the circulation were augmented in SOCS3Tg mice relative to the control. Studies using neutralizing Abs indicated that elevated IFN-{gamma} and TNF-{alpha} were responsible for the exacerbated hepatotoxicity in SOCS3Tg mice. Activation of STAT1 that is harmful in liver injury was augmented in SOCS3Tg hepatocytes. Alternatively, hepatoprotective STAT3 activation was decreased in SOCS3Tg hepatocytes, an event that was associated with augmented SOCS3 expression in the hepatocytes. Altogether, these results suggest that forced expression of SOCS3 in T cells is deleterious in APAP hepatotoxicity by increasing STAT1 activation while decreasing STAT3 activation in hepatocytes, possibly through elevated IFN-{gamma} and TNF-{alpha}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Acetaminophen (APAP)3 is a widely used analgesic and antipyretic agent. Although considered safe at therapeutic doses, accidental or intentional APAP overdose frequently causes acute liver failure characterized by centrilobular hepatic necrosis with high morbidity and mortality (1). The hepatotoxicity of APAP is initiated by a toxic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), that is generated by cytochrome P450 CYP2E1. NAPQI is usually detoxified by glutathione in the liver. However, APAP overdose depletes hepatic glutathione insomuch that NAPQI covalently binds to cellular proteins, leading to mitochondrial dysfunction and DNA damage, resulting in cell damage or cell death (2, 3). After acute toxic hepatic injury, several cytokines produced by injured hepatocytes and nonparenchymal inflammatory cells including lymphocytes contribute to the APAP-induced hepatotoxicity (4, 5).

Cytokines exert their biological functions through the JAK/STAT pathway, which is implicated in a variety of immune and inflammatory diseases (6). We have thus far provided clear evidence that Stat proteins are important in innate inflammatory responses (7, 8, 9). STAT proteins also play critical roles in the liver innate system during antiviral defense, acute phase response, hepatic injury, and regeneration (10). In the liver, STAT1 is activated in response to IFN-{gamma} in Con A-induced hepatitis and LPS/D-galactosamine-induced hepatitis model and plays a harmful role in these models of liver injury (11, 12). STAT3 mainly activated by IL-6 contributes to liver regeneration not only in physiological conditions (13) but also in pathological conditions including Con A-induced hepatitis by down-regulating IFN-{gamma} signaling and suppression of hepatocyte apoptosis (11, 14). Thus, activation of STAT1 and STAT3 is important in these T cell-mediated liver injury model. Because APAP-induced hepatotoxicity is also dependent on IFN-{gamma} (15), STAT1 and STAT3 could contribute to the progression of APAP-induced hepatotoxicity. The JAK/STAT pathway is regulated by several mechanisms including suppressors of cytokine signaling (SOCS) proteins, a family of SH2-containing cytoplasmic proteins (16). Among these, SOCS3 is induced by a variety of cytokines including IFN-{gamma}. IFN-{gamma} activates a STAT-binding element in the SOCS3 promoter though STAT1 activation (17). SOCS3 in turn inhibits STAT3 activation by inhibiting JNK, which binds to tyrosine residues of cytokine receptors (18). These findings lead to an assumption that SOCS3 may play a role in the liver injury though inhibiting the JAK-STAT pathway. In the present studies, we have attempted to explore the role of SOCS3 with special interest to T cells in the evolution of APAP-induced liver injury. The generation of a cell-specific overexpression of SOCS3 in T cells (19) allowed us to explore the role of SOCS3 in T cells in a murine model of APAP-induced hepatotoxicity. We demonstrate here that forced expression of SOCS3 in T cells is deleterious in APAP-induced liver injury. Our findings suggest that the exacerbated liver injury in these mice was based on enhanced hepatocyte apoptosis and impaired hepatocyte regeneration, an event that was associated with augmented STAT1 activation and in contrast decreased STAT3 activation in the hepatocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The myc-tagged SOCS3 was expressed under the control of the lck proximal promoter Eµ enhancer and backcrossed with C57BL/6J mice over 10 generations (19). In the mice, SOCS3 was overexpressed in T cells but not in B cells (data not shown). RAG-2 knockout mice (RAG-2–/–, lacking T and B cells, C57BL/6J strain) were a gift from Dr. T. Taniguchi (University of Tokyo, Tokyo, Japan). C57BL/6J mice were used as the wild-type (WT) mice. These mice were transferred to the Center for Animal Resources and Development, Kumamoto University (Kumamoto, Japan) and then bred at the facility. Male mice (6–8 wk) were used in this study under specific pathogen-free conditions. The animal use committee at Kumamoto University approved all experiments conducted in this study.

APAP-induced liver injury

Fresh suspensions of APAP (Sigma-Aldrich) were made immediately before use by dissolving the compound in PBS warmed to 40°C (20). In all experiments, mice were allowed free access to water and food or to only water for 16 h before an i.p. injection of APAP. APAP (400 mg/kg) was used as otherwise noted. At appropriate time intervals after APAP injection, mice were anesthetized, bled, and sacrificed. The livers were excised, snap frozen in liquid nitrogen, and stored at –50°C for subsequent analyses. Part of the tissues was fixed in 4% paraformaldehyde and embedded in paraffin, and the tissue sections were stained with H&E. For immunofluorescence staining, tissues were frozen in Tissue-Tek OCT compound (Sakura Finetek). For neutralization of endogenous IFN-{gamma} or TNF-{alpha}, neutralizing rabbit anti-murine IFN-{gamma} or anti-murine TNF-{alpha} IgG was administered i.p. 2 h before (1 mg/mouse) and at a time of APAP treatment (500 µg/mouse). Control rabbit IgG was used as control. In the different set of experiments, APAP was given to each mouse by i.p. injection at a dose of 500 mg/kg, after which the mouse survival was monitored for 5 days. In other experiments, CD4+ T cells from nontreated WT or SOCS3Tg (Tg, transgenic) mice (3 x 106 cells/mouse) were administered into the tail vein of RAG-2–/– mice. The mice received CD4+ T cells were fasted for 16 h, after which they were injected i.p. with APAP. CD4+ T cells were purified from spleen cells using a MACS separation column (Miltenyi Biotec) and CD4 Microbeads (Miltenyi Biotec). Most of the purified cells were alive (>97%) and routinely >95% CD4+ T cells. NKT cells were <2.5%, as assessed by flow cytometry (not shown).

Serum alanine aminotransferase (ALT) measurement

Acute hepatocellular injury results in elevated levels of circulating ALT. Serum levels of ALT were measured using standardized techniques.

DNA fragmentation assays

DNA fragmentation is a hallmark of apoptosis. To detect DNA fragmentation in paraffin-embedded liver sections, an MEBSTAIN Apoptosis kit direct (Medical and Biological Laboratories) and an ApopTag peroxidase in situ apoptosis detection kit (Chemicon International) were used, according to manufacturers’ instructions. For DNA ladder assay, genomic DNA was isolated from livers (10 mg) using a genomic DNA Purification Kit (Promega), and samples were separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light. Cell death detection ELISA (Roche Diagnostics) was used for the quantitative assessment. Livers were homogenized in lysis buffer provided by the kit, and 20 µg of protein were subjected to the ELISA. Protein concentrations in the extracts were measured by protein-dye binding assay (Bio-Rad Laboratories).

Cytochrome c release and caspase activity

Cytochrome c content in cytoplasm fractions of liver extracts (4 µg of protein) was analyzed with a cytochrome c ELISA kit (Medical and Biological Laboratories). Cytoplasmic fractions were extracted with a mitochondria/cytosol fractionation kit (MBL). Caspase-3, -8, and -9 activities in cleared supernatants of liver homogenates (100 µg protein) were measured with a colorimetric assay kit (Medical and Biological Laboratories), according to the manufacturer’s instructions.

Western blotting

Cleared supernatants of liver homogenates were boiled with sample buffer that contained 1% SDS, 10 mmol/L Tris-HCl (pH 7.5), fractionated on SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. After blocking with TBST containing 5% skim milk for 1 h at room temperature, the membrane was incubated with Abs to STAT1, STAT3, tyrosine-phosphorylated STAT3 (Cell Signaling), tyrosine-phosphorylated STAT1, p21cip1, cyclin D1 (Santa Cruz Biotechnology), Bcl-2 (Medical and Biological Laboratories), or beta-actin (Sigma-Aldrich) for 1 h at room temperature. After washing with TBST, the membrane was incubated with anti-HRP-linked Ab for 1 h at room temperature and visualized with an ECL system (Cell Signaling), photographed, and digitized, and the band densities were measured with NIH Image.

Immunofluorescence staining

Frozen liver tissues were cut with a cryostat at 4 µm and mounted on slides. The sections were air dried and fixed with acetone. After blocking with 10% normal goat serum in TBST for 20 min at room temperature, the slides were incubated with rabbit polyclonal Abs to STAT1, STAT3, tyrosine-phosphorylated STAT3, tyrosine-phosphorylated STAT1, SOCS3 (Santa Cruz Biotechnology), or control IgG (5 µg/ml) for 1 h at room temperature. After washing with TBST, the sections were incubated with biotinylated anti-rabbit IgG (Chemicon) for 30 min at room temperature, followed by treatment with FITC-conjugated avidin (Sigma-Aldrich). Nuclear staining was done with 4',6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich).

In vivo BrdU labeling

Mice were injected i.p. with BrdU (100 mg/kg; Sigma-Aldrich) 2 h before sacrifice. Immunohistochemical staining for BrdU was performed in liver sections, using a DAKO EnVision System (DakoCytomation). Anti-BrdU mouse mAb (Sigma-Aldrich) and biotin-labeled goat anti-mouse IgG polyclonal Ab (DakoCytomation) were used as the primary and secondary Abs, respectively. As a chromogen, diaminobenzidine (DakoCytomation) was used. BrdU-labeled hepatocytes were counted under a light microscope. The cell counting was conducted in all areas of the sections and expressed as the positive cells per square millimeter.

RT-PCR

The tissues were homogenized in Trizol reagent (Invitrogen Life Technologies), and total RNA was isolated according to the manufacturer’s instructions. First-strand cDNA was constructed from 2 µg of total RNA with oligo(dT)12–18 as primers, and the first-strand cDNAs were then amplified by each PCR in the presence of Taq polymerase (Invitrogen Life Technologies) and specific primers. RT-PCR was performed using the ThermoScript RT-PCR System (Invitrogen Life Technologies), according to the manufacturer’s instruction. The primers were designed to amplify murine SOCS3, c-fos, c-myc, Fas, Fas ligand (FasL), GAPDH, and beta-actin referred to the cDNA sequence from the National Center for Biotechnology Information database. The primers were as follows. SOCS3: sense: 5'-CGCCTCAAGACCTTCAGCTC-3'; antisense, 5'-CTGATCCAGGAACTCCCGAA-3'. c-fos: sense, 5'-TCAACGCCGACTACGAGGCG-3'; antisense, 5'-GTTCCCTTCGGATTCTCCGT-3'. c-myc: sense, 5'-CTATGTTGCGGTCGCTACGT-3'; antisense, 5'-GGAATCGGACGAGGTACAGGA-3'. Fas: sense, 5'-GAGAATTGCTGAAGACATGACAATCC-3'; antisense, 5'-GTAGTTTTCACTCCAGACATTGTCC-3'. FasL: sense, 5'-GAGAAGGAAACCCTTTCCTG-3'; antisense, 5'-ATATTCCTGGTGCCCATGAT-3'. GAPDH: sense, 5'-TGTTCCAGTATGACTCCACTCACG-3'; antisense, 5'-TCTGGGTGGCAGTGATGGCATGGA-3'. beta-actin: sense, 5'-ATCGTGGGCCGCCCTAGGCACCA-3'; antisense, 5'-TTGGCCTTAGGGTTCAGGGGGG-3'. The PCR was conducted at 35 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min. Ten microliters of PCR products were subjected to electrophoresis on a 2% agarose gel in presence of ethidium bromide, photographed, and digitized, and the band densities were measured with NIH Image. Results are expressed as a ratio of each PCR product/GAPDH or beta-actin band density; and this represents semiquantitative analysis.

Flow cytometry analysis

Spleens and livers were excised from WT and SOCS3Tg mice before and after APAP treatment. The splenocytes were dispersed into single-cell suspensions. After lysing RBC, the cells (1 x 106 cells/ml) were suspended in PBS supplemented with 2% FCS and 0.1% sodium azide. Cells were stained with mAb specific for mouse CD4, CD25, CD44, CD62L, and CD69 (BD Pharmingen). Liver leukocytes were isolated from the cell suspension by using Percoll centrifugation as described elsewhere (21, 22). Cells (1 x 106 cells/ml) were suspended in PBS supplemented with 2% FCS and 0.1% sodium azide, and were stained with FITC-conjugated anti-CD3 (145-2C11; BD Pharmingen) and PE-conjugated anti-NK1.1 (PK136; BD Pharmingen) for detection of NK and NKT cells. For neutrophils and macrophages, cells were incubated with mAb specific for mouse neutrophils (7/4; Cytotech) or macrophages (F4/80; Serotec) and stained with PE-conjugated goat F(ab')2 anti-rat IgG (Beckman Coulter). Stained cells were analyzed using a FACSCalibur (BD Biosciences).

Cell culture

Spleens were excised from WT and SOCS3Tg mice at 12 h after APAP treatment, and the splenocytes were dispersed into single-cell suspensions. After lysing RBC, the cells (5 x 106 cells/ml) were suspended in RPMI 1640 supplemented with 5% FCS, glutamine, and antibiotics and cultured for 48 h without stimulation. The culture supernatants were used for measurement of cytokines.

Measurement of cytokines

Murine cytokines were measured using a standard method of sandwich ELISA, as described (7, 23). The captured Abs, detection Abs, and the recombinant cytokines were purchased from R&D Systems. The ELISAs used in this study did not cross-react with other murine cytokines available. For cytokine measurement in the liver, livers were homogenized in PBS containing 0.1% Triton X-100 and complete protease inhibitor (Roche Diagnostics), centrifuged, and the cleared supernatants were obtained.

Statistics

Statistical significance was evaluated by ANOVA. In case of a survival curve, the data were analyzed by the log rank test. p < 0.05 was regarded as statistically significant. All data were expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Exacerbated liver injury in SOCS3Tg mice

APAP overdose provokes acute liver injury as represented by elevated serum levels of ALT. To examine the potential role of SOCS3 in T cells in APAP hepatotoxicity, we examined serum the ALT level after APAP challenge in WT and SOCS3Tg mice. There was no significant difference in serum ALT level between WT and SOCS3Tg mice before APAP challenge (42.3 ± 8.1 vs 44.3 ± 9.5 IU/dl, respectively; six mice each). The data in Fig. 1A demonstrated that the ALT level at 6 h after APAP challenge was substantially augmented in SOCS3Tg mice relative to the WT mice in a dose-dependent manner. Augmented levels of ALT in SOCS3Tg mice were seen until 48 h after 400 mg/kg APAP challenge, with a considerable disparity at 6 h, resulting in a 4.3-fold increase relative to the WT mice (Fig. 1B). Histological examination revealed that centrilobular liver necrosis in SOCS3Tg mice was more severe than that in WT mice (Fig. 1, C and D). In addition, survival rate in SOCS3Tg mice after 500 mg/kg APAP challenge was significantly lower than that in WT mice. Twenty-one of 30 SOCS3Tg mice were dead, whereas 25 of 40 WT mice were alive on day 5 (Fig. 1E). These findings indicate that forced expression of SOCS3 in T cells was deleterious in APAP-induced liver injury.


Figure 1
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  		FIGURE 1. APAP-induced liver injury in SOCS3Tg mice. A, WT ({square}) and SOCS3Tg ({blacksquare}) mice were injected i.p. with APAP (200, 300, and 400 mg/kg), and serum levels of ALT were measured at 6 h after the injection. Data are the mean ± SEM of 10–16 estimations from separate mice. B, WT ({circ}) and SOCS3Tg (•) mice were injected i.p. with 400 mg/kg APAP. At indicated time intervals after the injection, serum samples were collected, and ALT levels were measured. Data are the mean ± SEM from 6–16 estimations from separate mice. C, Representative photographs of the liver sections after 400 mg/kg APAP challenge. Original magnification, x200. D, Injured area by 400 mg/kg APAP was measured in WT liver ({square}) and SOCS3Tg liver ({blacksquare}) using H&E sections by NIH Image. Data are the mean ± SEM from 6–16 estimations from separate mice. E, The survival rates in WT ({circ}, 40 mice) and SOCS3Tg (•, 30 mice) were monitored for 5 days after 500 mg/kg APAP challenge. Four independent experiments were pooled. The mortality rates were very similar in individual experiments. {dagger}, p < 0.05; {ddagger}, p < 0.01, vs WT mice.

 
CD4+ T cells play an important role in APAP-induced liver injury

We then asked whether APAP-induced liver injury would be dependent on T cells. To do this, we first examined APAP-induced hepatotoxicity in RAG-2–/– mice, T and B cell-deficient mice. The data in Fig. 2A demonstrated that ALT level in RAG-2–/– mice was significantly lower than that in WT mice, suggesting a crucial role of lymphocytes in this model of liver injury. Next, CD4+ T cells from nontreated WT or SOCS3Tg mice were transferred into RAG-2–/– mice, after which the mice were injected i.p. with APAP. As shown in Fig. 2B, RAG-2–/– mice demonstrated higher levels of ALT when transferred with WT-CD4+ T cells, although the level was not statistically significant (with vs without WT-CD4+ T cells = 3695 ± 1423 IU/dl, 5 mice vs 570 ± 305 IU/dl, 11 mice; p = 0.119), suggesting a role of CD4+ T cells in the liver injury. Interestingly, ALT level in RAG-2–/– mice harboring SOCS3Tg-CD4+ T cells was significantly higher than those with WT-CD4+ T cells (Fig. 2B). Histological analysis showed an increased area of centrilobular hepatic necrosis in RAG-2–/– mice with SOCS3Tg-CD4+ T cells relative to the control (Fig. 2C). The enhanced ALT level after APAP injection was also seen when WT mice were transferred with WT-CD4+ T cells or SOCS3Tg-CD4+ T cells (3511 ± 943 IU/dl vs 8337 ± 2101 IU/dl, respectively; p < 0.05, eight mice each). These findings provide clear evidence that CD4+ T cells are ascribed to the exacerbated liver injury seen in SOCS3Tg mice.


Figure 2
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  		FIGURE 2. CD4+ T cells play an important role in APAP-induced liver injury. A, WT and RAG-2–/– mice (five mice each) were injected i.p. with 400 mg/kg APAP. Serum levels of ALT were measured at 6 h after the injection. B and C, CD4+ T cells from nontreated WT or SOCS3Tg mice (3 x 106 cells/mouse) were administered into the tail vein of RAG-2–/– mice. RAG-2–/– mice received WT-CD4+ T cells ({square}, 11 mice) or SOCS3Tg-CD4+ T cells ({blacksquare}, 11 mice) were then injected i.p. with 400 mg/kg APAP. Mice were killed at 6 h after APAP injection. B, Serum ALT level was measured. C, left, Representative photographs of liver sections. Original magnification, x200. Right, injured area in the sections was measured using H&E sections by NIH Image. Data are the mean ± SEM from 10–15 estimations from separate mice. {dagger}, p < 0.05, vs RAG-2–/– mice with WT-CD4+ T cells.

 
Augmented hepatocyte apoptosis in SOCS3Tg mice

To analyze the basis in APAP-induced liver injury, WT and SOCS3Tg mice were injected i.p. with APAP, and the hepatocyte apoptosis was examined. TUNEL staining of liver sections at 6 h post-APAP revealed that apoptotic hepatocytes were markedly increased in SOCS3Tg mice, relative to WT mice (Fig. 3A). DNA ladder formation was noted in SOCS3Tg liver, albeit there was no apparent observation in WT liver (Fig. 3B). To quantitate DNA fragmentation, histone-associated DNA fragments in mono- and oligonucleosomes were measured in the cytoplasmic fractions of liver lysates, which demonstrated that values of the fragmented DNA in SOC3Tg liver were significantly higher than those in WT liver (Fig. 3C).


Figure 3
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  		FIGURE 3. Hepatocyte apoptosis in APAP-induced liver injury. WT and SOCS3Tg mice were injected i.p. with 400 mg/kg APAP. At 6 h after the injection, mice were killed. A, left, Representative fluoromicroscopic photographs of liver sections with TUNEL staining. Original magnification, x200. Right, The numbers of TUNEL-positive hepatocytes/mm2 were counted (10 mice each). B, Representative photograph of DNA ladder pattern from three independent experiments. M, Marker; C, nontreated control liver from WT mice. C, Quantitative evaluation of apoptosis was done using cell death ELISA (10 mice each). All data represent the mean ± SEM. {ddagger}, p < 0.01 vs WT mice; {dagger}, p < 0.05 vs WT mice.

 
A toxic metabolite of APAP, NAPQI, causes mitochondrial dysfunction (3), leading to cytochrome c release into cytoplasm from the intermembrane space of mitochondria, which in turn initiates activation of caspase-3 next to caspase-9 (24). We then examined cytochrome c release and caspase activities in the APAP liver. The data in Fig. 4A demonstrated that cytochrome c content in the cytoplasm was significantly elevated in SOCS3Tg liver relative to the control. Activities of caspase-3 and -9 in SOCS3Tg liver were significantly higher than those in the control. Caspase-8 activity was also augmented in SOCS3Tg liver (Fig. 4B). The expression of bcl-2, an anti-apoptotic protein that inhibits cytochrome c release from mitochondria and thereby prevents downstream caspase activation (24), was next examined. As shown in Fig. 4C, bcl-2 was notably induced at 3 h after APAP challenge in WT liver, whereas the level was markedly decreased in SOCS3Tg liver. Because caspase-8 activity was induced by death receptor pathway (25), we then examined Fas/FasL expression in the livers. As shown in Fig. 4D, Fas mRNA expression after 6 h of APAP treatment was significantly augmented in SOCS3Tg liver relative to the control, despite no significant difference was found in FasL expression between WT and SOCS3Tg liver (Fig. 4D). These results demonstrate that mitochondrial apoptosis in hepatocytes was enhanced in SOCS3Tg mice, an event that was associated with reduced antiapoptotic protein bcl-2. The Fas-dependent apoptosis pathway was also enhanced in SOCS3Tg mice.


Figure 4
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  		FIGURE 4. Apoptosis pathway was enhanced in SOCS3Tg mice. WT ({square}) and SOCS3Tg ({blacksquare}) mice were injected i.p. with 400 mg/kg APAP. A, The release of cytochrome c in cytoplasm fraction of the livers was quantitatively measured at 0 (4 mice each) and 6 h (10 mice each) after APAP challenge. B, Activities of caspase-3, -8, and -9 were measured at 6 h after APAP challenge (10 mice each). C, Left, Livers were harvested at 3 h after APAP challenge, and the extracts were immunoblotted with anti-bcl-2 IgG and anti-beta-actin IgG. Shown are representative data. Right, The band densities of bcl-2 and beta-actin were digitalized by NIH Image (eight mice each). Results are expressed as percent ratio of beta-actin. D, Livers were harvested at indicated times after APAP challenge, and mRNA expressions of Fas, FasL, and beta-actin in the livers were analyzed by RT-PCR. Top, Representative data; bottom, The band densities were digitalized by NIH Image. {square}, WT mice; {blacksquare}, SOCS3Tg mice, 6–10 mice at each point. Results are expressed as percent ratio of beta-actin. All data represent the mean ± SEM. {dagger}, p < 0.05; {ddagger}, p < 0.01; §, p < 0.001, vs WT liver.

 
Reduced hepatocyte proliferation in SOCS3Tg mice

We next examined the hepatocyte proliferation after APAP treatment, which is an important process in the liver regeneration. The data in Fig. 5A demonstrated that BrdU incorporation in hepatocytes at 48 h after APAP challenge that represents hepatocytes in S phase was significantly lower in SOCS3Tg liver than that in WT liver. Expression of cyclin D1 in the liver, a key regulator that achieves the G1 to S transition in the cell cycle (26), was next examined. At 3 and 6 h post-APAP, no apparent cyclin D1 was detected in WT and SOCS3Tg liver (data not shown). In WT liver, the expression was detected at 24 h and decreased after 72 h. In SOCS3Tg liver, obvious expression was first noted at 48 h and then increased thereafter (Fig. 5B), suggesting that cyclin D1 expression was delayed in SOCS3Tg liver. The hepatic expression of p21cip1, which binds to cyclin-dependent kinases and suppresses their activities (27), was conversely increased in SOCS3Tg liver relative to the control (Fig. 5B). The expression of a group of early growth response genes such as c-fos and c-myc (28) was also examined, which demonstrated that hepatic expressions of c-fos and c-myc after APAP challenge were significantly decreased in SOCS3Tg liver relative to the WT liver. Thus, hepatocyte proliferation was attenuated in SOCS3Tg liver.


Figure 5
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  		FIGURE 5. APAP-induced hepatocyte proliferation was attenuated in SOCS3Tg mice. WT and SOCS3Tg mice were injected i.p. with 400 mg/kg APAP. A, BrdU incorporation of the liver was assessed at 48 h after APAP challenge. Left, Representative photographs. Original magnification, x200. Right, The numbers of BrdU-positive hepatocytes per square millimeter were counted under a microscope (eight mice each). B, At the indicated times after APAP challenge, livers were harvested, and the extracts were immunoblotted with anti-cyclin D1 IgG, anti-p21cip1 IgG, or anti-beta-actin IgG. Top, Representative data; bottom, The band densities were digitalized by NIH Image. {square}, WT mice; {blacksquare}, SOCS3Tg mice, six mice each. Results are expressed as percent ratio of beta-actin. C, Livers were harvested at indicated times after APAP challenge, and mRNA expressions of c-fos and c-myc in the livers were analyzed by RT-PCR. Top, Representative data; bottom, The band densities were digitalized by NIH Image. {square}, WT mice; {blacksquare}, SOCS3Tg mice, 6–10 mice at each point). Results are expressed as percent ratio of GAPDH. All data represent the mean ± SEM. {dagger}, p < 0.05; {ddagger}, p < 0.01, vs WT liver.

 
Enhanced cytokine production in SOCS3Tg mice

APAP overdose causes elevated cytokines in the circulation (29). We next investigated the cytokine response in mice after APAP treatment. As shown in Fig. 6A, the circulating level of IFN-{gamma}, a Th1 cytokine that activate STAT1, was significantly elevated in SOCS3Tg mice relative to WT mice. In liver extracts, there was a trend toward increase in the level of IFN-{gamma} in SOCS3Tg mice (WT vs SOCS3Tg = 6.3 ± 0.3 vs 7.2 ± 0.3 ng/mg protein; p = 0.07, 12 mice each). Serum levels of IL-12, a powerful inducer of IFN-{gamma}, and a potent proinflammatory cytokine TNF-{alpha}, were also measured, which demonstrated that levels of these cytokines were augmented in SOCS3Tg mice relative to WT mice (Fig. 6A), albeit no difference was found in the liver extracts (not shown). Splenocytes from APAP-treated SOCS3Tg mice spontaneously released higher levels of IL-12 and TNF-{alpha} than did cells from the WT mice, although no appreciable level of IFN-{gamma} was detected (Fig. 6B).


Figure 6
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  		FIGURE 6. Augmented cytokine production in SOCS3Tg mice. WT and SOCS3Tg mice were injected i.p. with 400 mg/kg APAP. A, At 6 and 24 h after APAP challenge, mice were killed and serum levels of IFN-{gamma}, IL-12, and TNF-{alpha} were measured. {square}, WT mice; {blacksquare}, SOCS3Tg mice, 12–16 mice. B, Mice were killed at 12 h after APAP challenge. Spleen cells from APAP-treated WT and SOCS3Tg mice (five mice each) were cultured for 48 h without any stimulation. Levels of IFN-{gamma}, IL-12, and TNF-{alpha} in the culture supernatants were measured. {dagger}, p < 0.05; {ddagger}, p < 0.01; ¶, p < 0.0001, vs WT mice; n.d., not detected.

 
The different response of splenocytes may result from altered activation of CD4+ T cells. We then examined the expression of CD69, an early activation marker, along with other surface markers at 6 h post-APAP. However, APAP did not cause a significantly increase in CD4+CD69+ double-positive cells in WT and SOCS3Tg mice, and the level was similar between the mouse groups (1.6% vs 1.6%). There were no significant differences in the memory T cells (CD4+CD44+, 1.0% vs 1.2%) and naive T cells (CD4+CD62L+, 9.1% vs 9.1%) between the groups. There was a trend toward decrease in CD4+CD25+ double-positive T cells (regulatory T cells) in SOCS3Tg mice (2.7% vs 1.6%), but the percentage was low, and it was unchanged after APAP challenge. Altered cytokine response may affect the numbers of leukocytes in the liver. The data in Fig. 7 demonstrated that the number of NK (CD3–NK1.1+ cells) and NKT cells (CD3+NK1.1+ cells) was not different between WT and SOCS3Tg liver. There were no statistical differences in the numbers of neutrophils (7/4+ cells) and macrophages (F4/80+ cells) between the groups (Fig. 7). Thus, SOCS3Tg mice showed increased IFN-{gamma}, IL-12, and TNF-{alpha} after APAP challenge, in which the splenic T cell repertoire and hepatic leukocyte populations were unchanged.


Figure 7
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  		FIGURE 7. Liver leukocytes after APAP challenge. WT and SOCS3Tg mice were injected i.p. with 400 mg/kg APAP. At 12 h after APAP challenge, mice were killed, and liver leukocytes were isolated as described in Materials and Methods. The leukocytes were stained with FITC- or PE-conjugated mAbs and analyzed for NK cells (CD3–NK1.1+), NKT cells (CD3+NK1.1+), neutrophils (7/4+), and macrophages (M{phi}; F4/80+) by flow cytometry. A, Percentages for individual cell populations. B, The number per liver for individual cell type was calculated by multiplying the percentage of individual cell type by the total number of isolated liver leukocytes per liver. {square}, WT mice; {blacksquare}, SOCS3Tg mice; four mice each). The numbers of leukocytes in untreated WT and SOCS3Tg liver were too low to show in the figures. PMN, Polymorphonuclear neutrophils.

 
Altered STAT1 and STAT3 activation and SOCS3 expression in hepatocytes of SOCS3Tg mice

To understand the contribution of altered cytokine response to an exacerbated liver injury in SOCS3Tg mice, SOCS3Tg mice were pretreated with polyclonal Abs against IFN-{gamma} and TNF-{alpha}, and the mice were i.p. injected with APAP. Six hours later, the mice were killed, and the serum levels of ALT were measured. As shown in Fig. 8A, neutralization of IFN-{gamma} by anti-IFN-{gamma} IgG substantially decreased the ALT level relative to the control. The ALT level was also decreased by anti-TNF-{alpha} IgG by 55% although it was not statistically significant (Fig. 8B, p = 0.22). Thus, IFN-{gamma} and TNF-{alpha} appear to be responsible for the enhanced APAP hepatotoxicity.


Figure 8
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  		FIGURE 8. IFN-{gamma} and TNF-{alpha} play a role in the exacerbated liver injury. SOCS3Tg mice were pretreated with neutralizing rabbit anti-mouse IFN-{gamma} IgG or anti-TNF-{alpha} IgG (1 mg/mouse i.p.) 2 h before APAP challenge. At time 0, the mice were injected i.p. with 400 mg/kg APAP together with anti-IFN-{gamma} IgG or anti-TNF-{alpha} IgG (500 µg/mouse i.p.). Rabbit IgG was used as control. Six hour later, the mice were killed, and serum levels of ALT were measured. A, SOCS3Tg mice were treated with control IgG (six mice) or anti-IFN-{gamma} IgG (six mice). B, SOCS3Tg mice were treated with control IgG (12 mice) or anti-TNF-{alpha} IgG (14 mice). {ddagger}, p < 0.01, vs WT mice.

 
IFN-{gamma} and TNF-{alpha} activate STAT1 (30, 31), and apoptosis induced by IFN-{gamma} and TNF-{alpha} depends on STAT1 (12, 32, 33). STAT3s play counterregulatory roles in apoptosis against STAT1, given that STAT1 inhibits anti-apoptotic protein bcl-2 whereas STAT3 activates the protein (34). STAT3 suppresses Fas transcription (35) and plays an important role in the liver regeneration (13, 36). To investigate the molecular basis behind the augmented apoptosis and reduced hepatocyte proliferation in SOCS3Tg liver, activation of STAT1 and STAT3 in the livers was examined. As shown in Fig. 9A, STAT1 activation in the liver, represented by phosphorylated STAT1 expression, was increased in SOCS3Tg liver relative to the control. Hepatocytes were the cells responsible for the enhanced STAT1 activation, given that phosphorylated STAT1-positive cells were more evident in nuclei of SOCS3Tg hepatocytes (Fig. 9B). No apparent positive cells were found in nonparenchymal cells. In contrast, STAT3 activation was significantly decreased in SOCS3Tg liver relative to the control (Fig. 9C). Histological examination revealed that a degree of phosphorylated STAT3-positive nuclei of hepatocytes was weaker in SOCS3Tg liver (Fig. 9D).


Figure 9
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  		FIGURE 9. Altered STAT1 and STAT3 activation and SOCS3 expression in the livers of SOCS3Tg mice. WT and SOCS3Tg mice were injected i.p. with 400 mg/kg APAP. At indicated time points after APAP challenge, livers were harvested and then extracted. A, The extracts were immunoblotted with anti-phosphorylated STAT1 IgG and anti-STAT1 IgG. Left, Representative data. Right, The band densities were digitalized by NIH Image. {square}, WT liver; {blacksquare}, SOCS3Tg liver, eight mice each). Results are expressed as %pSTAT1/STAT1. Data are the mean ± SEM. {dagger}, p < 0.05, vs WT control. B, Liver sections at 3 h after APAP challenge were stained with Abs against pSTAT1. Original magnification, x200. Representative fluoromicroscopic photographs are shown. C, Liver extracts were immunoblotted with anti-phosphorylated STAT3 IgG and anti-STAT3 IgG. Left, Representative data. Right, The band densities were digitalized by NIH Image. {square}, WT liver; {blacksquare}, SOCS3Tg liver, eight mice each. Results are expressed as percent pSTAT3/STAT3. Data are the mean ± SEM. {ddagger}, p < 0.01, vs WT control. D, Liver sections at 3 h after APAP challenge were stained with Abs against pSTAT3. Original magnification, x200. Shown are representative fluoromicroscopic photographs. E, Livers were harvested at indicated times after APAP challenge, and mRNA expressions of SOCS3 and GAPDH in the livers were analyzed by RT-PCR. Left, Representative data. Right, The band densities were digitalized by NIH Image. {square}, WT liver; {blacksquare}, SOCS3Tg liver, six mice each). Results are expressed as percent ratio of GAPDH. Data are the mean ± SEM. {dagger}, p < 0.05; {ddagger}, p < 0.01, vs WT liver. F, At 6 h after APAP challenge, liver sections from WT and SOCS3Tg mice were stained with anti-SOCS3 IgG. Original magnification, x200. Shown are representative photographs.

 
STAT3 activation is regulated by SOCS3 (37). Experiments were further conducted to investigate SOCS3 expression in the APAP-treated liver. The data in Fig. 9E demonstrated that APAP induced a transient expression of SOCS3 in the liver at 3 h in WT mice. Interestingly, the expression was more prominent and prolonged in SOCS3Tg liver during the observation periods (Fig. 9E). Immunohistochemically, protein expression of SOCS3 was detected mainly in the hepatocytes, and the staining intensity was more intense in SOCS3Tg hepatocytes relative to the control (Fig. 9F). Thus, forced expression of SOCS3 in T cells resulted in increased STAT1 activation and SOCS3 expression and in contrast decreased STAT3 activation in the hepatocytes after APAP treatment, possibly through IFN-{gamma} and TNF-{alpha}, which leads to exacerbated liver injury via augmented hepatocyte apoptosis and impaired hepatocyte proliferation.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The toxic response to APAP is primarily initiated by a highly reactive metabolite, NAPQI. However, evidence suggests that inflammatory mediators produced by injured hepatocytes and nonparenchymal inflammatory cells contribute to the APAP-induced hepatotoxicity (4, 5). Recently, Liu et al. (21) have demonstrated that NK and NKT cells produce IFN-{gamma} and play a critical role in APAP-induced liver injury. In this study, we have demonstrated that RAG-2–/– mice were resistant to the injury and that RAG-2–/– mice that received CD4+ T cells exhibited an increased ALT level. SOCS3 was deleterious by enhancing hepatocyte apoptosis and in contrast reducing hepatocyte proliferation when forced expressed in T cells. Although SOCS3 can be overexpressed in NKT cells, isolated CD4+ T cells used for transfer experiments contained <2.5% NKT cells. In addition, there was no difference in the number of NKT cells in WT and SOCS3Tg liver after APAP treatment. Thus, these findings indicate that CD4+ T cells are important in the liver injury, in which SOCS3 has profound effects on APAP-induced hepatotoxicity.

In the mechanistic level, augmented STAT1 activation and decreased STAT3 activation in the hepatocytes appeared to be responsible for the exacerbated liver injury. In a Con A-induced hepatitis model, disruption of STAT1 attenuates the liver injury, suppresses CD4+ T and NKT cell activation, and down-regulates proapoptotic proteins. IL-6-deficient mice represented decreased STAT3 activation and thereby STAT3-controlled antiapoptotic signals were attenuated in the mice (11). Thus, activation of STAT1 in hepatocytes is deleterious, whereas activation of STAT3 in hepatocytes is beneficial in liver injury. In this study, the mechanism behind the enhanced STAT1 activation in hepatocytes appeared to be ascribed to the elevated IFN-{gamma} and TNF-{alpha} in SOCS3Tg mice. IFN-{gamma} activates STAT1 (31), which initiates inflammatory signals in Con A-induced hepatitis as well as APAP-induced fulminant hepatitis via induction of multiple cytokines/chemokines, adhesion molecules, and Fas/FasL (15, 32, 38). TNF-{alpha} also activates STAT1 (30) and STAT1 null cells are resistant to apoptosis by TNF-{alpha} (33). Although STAT3 can be activated by either IFN-{gamma} or TNF-{alpha} under specific conditions (39, 40), STAT3 activation in hepatocytes was conversely decreased in our present model. This can be explained by the SOCS3 induction by IFN-{gamma}/TNF-{alpha}-STAT1 activation. In WT liver, SOCS3 expression was transiently expressed at 3 h post-APAP, whereas it was prolonged by at least 72 h in SOCS3Tg liver. Correspondingly, decreased STAT3 activation was noted after 3 h. SOCS3 is known to negatively regulate the activation of STAT3 (37). A recent study by Ogata et al. (41) demonstrated that a tissue-specific disruption of SOCS3 in hepatocytes resulted in a sustained and strong activation of hepatic STAT3 after Con A injection, leading to resistance to the liver injury through suppression of IFN-{gamma} signaling and hepatocyte apoptosis. In our current study, the serum level of hepatoprotective IL-6 was elevated in SOCS3Tg mice (WT vs SOCS3Tg mice at 6 h post-APAP = 1.0 ± 0.3 ng/ml vs 3.0 ± 0.7 ng/ml; p < 0.05, 11 mice each). However, IL-6 appeared not to fulfill its function in this model by decreased STAT3 activation, thereby leading to the attenuated liver regeneration.

A question arises as to why IFN-{gamma} production is enhanced in mice with overexpression of SOCS3 in T cells. SOCS3 is predominantly expressed in Th2 cells (42), and forced expression of SOCS3 in T cells enhanced Th2-mediated allergic immune responses while inhibiting Th1 response (19, 43). Mice received cell-penetrating forms of SOCS3, in which the SOCS3 protein was effectively distributed mainly in T cells, were protective to Con A-induced liver injury by reducing IFN-{gamma}/STAT1 pathway (44). The results are contrary to our current findings. In APAP-induced liver injury, we found exacerbated hepatotoxicity in mice with forced expression of SOCS3 in T cells. These findings highlight the discrepancies between different models of liver injury, which differ in their complexity and in their pathogenesis. Unlike with Con A, somehow APAP may directly enhance the production of IFN-{gamma} from SOCS3Tg T cells. However, this is not likely inasmuch as SOCS3Tg-CD4+ T cells failed to produce IFN-{gamma} upon stimulation with APAP (not shown). In the preset study, SOCS3Tg mice exhibited elevated IL-12 level in the circulation, whereas the hepatic IL-12 level was similar between WT and SOCS3Tg mice. Spleen cells harvested from SOCS3Tg mice after APAP treatment spontaneously produced higher levels of IL-12, suggesting that extrahepatic IL-12, in part splenocytes, may be responsible for the enhanced IL-12 in the circulation. Alternatively, IFN-{gamma} was not detected from APAP-treated splenocytes. Liu et al. have demonstrated that hepatic NK and NKT cells produce IFN-{gamma} in APAP hepatotoxicity, as evidenced by FACS with intracellular staining and NK/NKT cell depletion with anti-NK1.1 Abs (21). IL-12 is a Th1-promoting cytokine that induces IFN-{gamma} (45). These findings suggest that elevated IL-12 in the circulation might stimulate hepatic NK and NKT cells to produce higher levels of IFN-{gamma}. The IFN-{gamma} level in the liver was not very different between WT and SOCS3Tg mice. This can be explained by the fact that the numbers of hepatic NK/NKT cells were similar between WT and SOCS3Tg liver. Augmented IL-12 level in the circulation probably stimulates hepatic NK/NKT cells to release IFN-{gamma} in the circulation. Alternatively, elevated IL-12 in the circulation may stimulate extrahepatic cells to produce IFN-{gamma}. In the current model, the TNF-{alpha} level was also increased in the circulation. This may result from an elevated IFN-{gamma}, as IFN-{gamma} induces the production and release of TNF-{alpha} (46).

Another question arises is the mechanism behind the elevated production of IL-12 in SOCS3Tg mice. Yu et al. proposed the concept that SOCS3 may regulate an important checkpoint that prevents inappropriate activation of Th cells (47). One possibility is that T cells from SOCS3Tg mice may be more activated by APAP treatment. However, early activation marker CD69 was not significantly increased after APAP treatment, and the level was similar between WT and SOCS3Tg-CD4+ T cells, suggesting that APAP in itself does not activate splenic T cells. Th17 differentiation may play a role, given that SOCS3-deficient T cells result in enhanced Th17 generation (48), the development of which is linked to regulatory T cell function (49). Although the number of regulatory T cells was not very different between WT and SOCS3Tg mice, altered function of regulatory T cells might play a role. Identification of the regulatory roles of SOCS3 in T cells remains to be established.

An attractive question is how IFN-{gamma}-deficient x SOCS3Tg mice respond to APAP-induced hepatotoxicity. Could SOCS3 deletion in T cells interfere with hepatocyte apoptosis and regeneration? Does liver injury induced by other chemicals such as Con A, diethyl maleate, or carbon tetrachloride also depend on SOCS3-CD4+ T cells? Further studies are necessary to define the functions of SOCS3 in T cells in hepatotoxicity.

In conclusion, we have shown that forced expression of SOCS3 in T cells is deleterious in APAP-induced liver injury, presumably by augmented STAT1 and reduced STAT3 activation in hepatocytes, leading to an exacerbated hepatic apoptosis and reduced hepatocyte regeneration, respectively. Enhanced productions of IFN-{gamma} and TNF-{alpha} appear to regulate the activation of STAT1 and STAT3 in the liver. APAP overdose with its resulting hepatotoxicity is an important clinical problem. The findings in the present study suggest that an intervention directing SOCS3 in T cells may be effective for the treatment of APAP-induced hepatotoxicity.


   Acknowledgments
 
We thank Mr. Shinji Kudo, Ms. Motoko Kagayama, and Ms. Takako Maeda for their technical assistance.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture and the Ministry of Health and Welfare, Japan. Back

2 Address correspondence and reprint requests to Dr. Akihiro Matsukawa, Department of Pathology and Experimental Medicine, Graduate School of Medical, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1, Shikata, Okayama 700-8558, Japan. E-mail address: amatsu{at}md.okayama-u.ac.jp Back

3 Abbreviations used in this paper: APAP, acetaminophen; ALT, alanine aminotransferase; NAPQI, N-acetyl-p-benzoquinoneimine; SOCS, suppressors of cytokine signaling; Tg, transgenic; WT, wild type; FasL, Fas ligand. Back

Received for publication September 26, 2006. Accepted for publication January 1, 2007.


   References
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 Materials and Methods
 Results
 Discussion
 Disclosures
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