Role of IL-6 in an IL-10 and IL-4 Double Knockout ... - ACS Publications

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Chem. Res. Toxicol. 2007, 20, 208-216

Role of IL-6 in an IL-10 and IL-4 Double Knockout Mouse Model Uniquely Susceptible to Acetaminophen-Induced Liver Injury† Mohammed Bourdi,*,‡ Daniel P. Eiras,‡ Michael P. Holt,‡ Marie R. Webster,‡ Timothy P. Reilly,‡,§ Kevin D. Welch,‡,| and Lance R. Pohl‡ Molecular and Cellular Toxicology Section, Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human SerVices, Bethesda, Maryland ReceiVed September 7, 2006

Drug-induced hepatitis remains a challenging problem for drug development and safety because of the lack of animal models. In the current work, we discovered a unique interaction that makes mice deficient in both IL-10 and IL-4 (IL-10/4-/-) highly sensitive to the hepatotoxic effects of acetaminophen (APAP). Male C57Bl/6 wild type (WT) and mice deficient in one or more cytokines were treated with 120 mg/kg APAP. Within 24 h after WT, IL-10-/-, IL-4-/-, or IL-10/4-/- mice were administered APAP, 75% of the IL-10/4-/- mice died of massive hepatic injury while all other genotypes were resistant to liver toxicity at this dose of APAP. The unique susceptibility of IL-10/4-/- mice was associated with reduced levels of liver glutathione and remarkably high serum levels of IL-6 and several proinflammatory factors including TNF-R, IFN-γ, macrophage inflammatory protein-1R (MIP-1R), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and osteopontin (OPN) as well as nitric oxide (NO). IL-6 appeared to have a causal role in controlling the unique susceptibility of IL-10/4-/- mice to APAP-induced liver disease (AILD) because IL-6 neutralizing antibody reversed the high sensitivity of these mice to AILD. Moreover, IL-10/4/6-/- mice were also resistant to the enhanced susceptibility to AILD and expressed relatively low levels of most proinflammatory factor genes that were elevated in the IL-10/4-/- mice. In conclusion, liver homeostasis following AILD appears to be highly dependent on the activities of both IL-10 and IL-4, which together help prevent overexpression of IL-6 and other potential hepatotoxic factors. Introduction Drug-induced liver injury (DILD)1represents a significant source of morbidity and mortality and a substantial challenge to drug therapy and new drug development (1, 2). Despite the low incidence of DILD (1, 2), it is the most frequent reason cited for the withdrawal of drugs from the market, and it accounts for more than 50% of acute liver failure cases in the United States (2). Unfortunately, it remains impossible to accurately predict which new drugs will cause DILD and who will be at risk of developing this disease. This is due in large part to the lack of animal models for most drugs and resultant scarcity of information concerning both mechanisms of liver † Presented in part at the 55th Liver Meeting, Oct 29-Nov 2, 2004, Boston, MA. * To whom correspondence should be addressed. Tel: +1 (301)-4512599/(301)-496-4841; fax: 301-480-4852; e-mail: [email protected]. ‡ National Institutes of Health. § Current address: Immunotoxicology, Drug Safety Evaluation, BristolMyers Squibb, Syracuse, New York. | Current address: USDA Agricultural Research Service, Poisonous Plant Research Laboratory, Logan, Utah. 1 Abbreviations: IL, interleukin; IL-10/4-/-, mice deficient in both IL10 and IL-4; WT, C57Bl/6 wild type mice; APAP, acetaminophen; MIP1R, macrophage inflammatory protein-1R; MCP-1, monocyte chemoattractant protein-1; OPN, osteopontin; NO, nitric oxide; NOS-2, NO synthase 2; STAT3, signal transducer and activator of transcription-3; p-STAT, phosphorylated STAT; AILD, APAP-induced liver disease; DILD, druginduced liver disease; ALT, alanine aminotransferase; GSH, glutathione; AG, aminoguanidine; ELISA, enzyme-linked immunoabsorbant assay; NAPQI, N-acetyl-p-benzoquinone-imine; RT-PCR, reverse transcriptasepolymerase chain reaction; G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

disease and the nature of risk factors that contribute to DILD susceptibility. However, from studies done with those rare drugs that cause liver injury in animals including halothane, ethanol, cocaine, and particularly acetaminophen (APAP), it is clear that DILD can be initiated by reactive metabolites of drugs formed in hepatocytes (2, 3). These metabolites may cause hepatocellular injury directly by covalently altering essential liver proteins or indirectly by promoting the formation of reactive oxygen and nitrogen species, including hydrogen peroxide, hydroxyl radical, lipid peroxides, nitric oxide (NO), and peroxynitrite anion, which may also covalently modify liver proteins (4, 5). Some have hypothesized that liver injury may ultimately be caused by functional changes in enzymes, structural proteins, and transcription factors which can lead to the disruption of calcium and mitochondrial homeostasis, damage to cellular membrane integrity, and the induction of apoptosis (2, 3, 5-8). Alternatively, others have suggested that protein adducts of drugs may be immunogenic in susceptible individuals and may induce the formation of liver-specific antibodies and/or T cells that can cause allergic hepatitis (3, 6, 8, 9). More recent studies of APAP-induced liver disease (AILD) indicate that a wide variety of factors can modulate the progression of AILD. For example, the proinflammatory cytokines macrophage migration inhibitory factor (10), IFN-γ (11), and osteopontin (OPN) (12), as well as lipopolysaccharidebinding protein (13), natural killer cells/natural killer T cells (14), and neutrophils (15, 16) can contribute to the severity of AILD. These effects, however, can be counterbalanced by the up-regulation of hepatoprotective and liver regenerative factors

10.1021/tx060228l CCC: $37.00 © 2007 American Chemical Society Published on Web 01/06/2007

High Susceptibility of IL-10/4-/- Mice to APAP

including IL-6 (17, 18), IL-10, (19) cyclooxygenase-2 (20), the C-C chemokine receptor, CCR2 (21), and Nrf2 (22) (for review, see 8). IL-6 is a multifunctional cytokine that regulates the immune and acute phase responses, hematopoiesis, endocrine function, and inflammation and appears to play a role in a variety of diseases (23). In several animal models of autoimmune disease including insulin-dependent diabetes mellitus, inflammatory bowel disease, multiple sclerosis, and rheumatoid arthritis (24), IL-6 has a pathologic proinflammatory role. In contrast, other animal model studies indicate that IL-6 protects against liver injury caused by CCl4 (25), ethanol (26), concanavalin A (ConA) (27, 28), anti-Fas antibody (29), ischemia/reperfusion (30), and AILD (17, 18). IL-6 hepatoprotection is mediated in part through its antiapoptotic (29) and liver regenerative effects (18, 25, 30) as well as by suppressing natural killer T cell activation in the liver (28), inducing the acute phase response (23), and possibly by increasing the expression of heat shock proteins (17). Many of the hepatoprotective functions of IL-6 appear to be mediated by the IL-6-gp130-STAT3 signal transduction pathway (23, 28). Herein, we now report that IL-6 can also be protoxicant in a model AILD when it is overexpressed as a result of impaired regulation because of a deficiency in expression of both IL-10 and IL-4.

Experimental Procedures Reagents. Reagents were purchased from the following commercial sources: APAP and aminoguanidine (AG) (Sigma, St. Louis, MO); alanine aminotransferase (ALT) kit (Teco Diagnostics, Anaheim, CA); peroxidase-conjugated antirabbit IgG (Roche, Indianapolis, IN); ready-to-go reverse transcriptase-polymerase chain reaction (RT-PCR) beads (Amersham Biosciences, Little, Chalfont Buckinghamshire, England); colorimetric, nonenzymatic nitric oxide (NO) assay kit (Oxford Biomedical Research, Oxford, MI); RNeasy Midi kit (Qiagen, Valencia, CA); complete protease inhibitor cocktail tablets (Roche Molecular Biochemicals, Indianapolis, IN); rat antimouse IL-6 neutralizing antibody clone MP520F3, rat IgG1 isotype control antibody, and osteopontin enzyme-linked immunoabsorbent assay (ELISA) kit (R&D systems, Minneapolis, MN); anti-signal transducer and activator of transcription-3 (STAT3), anti-phosphorylated (Tyr705 and Tyr727) STAT3 (pSTAT3) (Cell Signaling, Beverly, MA); anti-arginase 1 (BD Biosciences, San Jose, CA); RNA 6000 Nano assay kit, DNA 1000 assay kit, and 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA); and anti-APAP-protein adduct serum that was kindly provided by Drs. Jack Hinson and Neil Pumford (University of Arkansas, Little Rock, AR). Animals. Wild type (WT), IL-10-/-, and IL-6-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME), while IL-4-/- and IL-10/4-/- mice were kindly provided by Dr. Thomas A. Wynn (NIAID, NIH, Bethesda, MD) (31). All animals were on a C57Bl/6 background. Mice deficient in IL-10, IL-4, and IL-6 (IL10/4/6-/-) were produced by breeding IL-10/4-/- with IL-6-/- mice. The genotype of the IL-10/4/6-/- mice was confirmed by PCR analysis using primer sequences and reaction conditions outlined in the Genotyping Protocols of The Jackson Laboratory. All animals were acclimated for at least 6-7 days to a 12-h light/dark cycle in a humidity and temperature-controlled, specific-pathogen-free environment in microisolator autoclaved cages according to National Institutes of Health standards. Mice were allowed free access to autoclaved food and water until experimental use. APAP Treatment and Tissue Collection. Male mice that were 7-9 weeks old were fasted overnight for approximately 15-16 h before treatment with APAP or saline by intraperitoneal injection (routinely 8:30-9:30 a.m.). At times thereafter, blood was collected by retro-orbital puncture and the livers were removed. Blood samples were allowed to clot for approximately 2 h at room

Chem. Res. Toxicol., Vol. 20, No. 2, 2007 209 temperature and then overnight at 4 °C. Aliquots of sera were separated for measurement of ALT activity and the remainders of the sera were snap-frozen and stored at -80 °C for further use. Upon removal, a portion of each liver was fixed in buffered formalin and then was embedded in paraffin, was mounted onto glass slides, and was stained with hematoxylin and eosin (American Histolabs, Gaithersburg, MD). The remainder of the liver tissue was snapfrozen and stored at -80 °C for subsequent mRNA and protein analyses. In other hepatotoxicity experiments, mice were posttreated with aminoguanidine (AG, 100 mg/kg in saline) or saline 2 h after APAP treatment. Reverse Transcription-Polymerase Chain Reaction. Total RNA was isolated from frozen liver tissues of individual mice using RNeasy Midi Kits. RNA concentration and purity were determined by measuring the absorbance at 260 nm and the 260 nm/280 nm absorbance ratio, respectively. The concentration and integrity of RNA were also assessed using 6000 Nano assay kits and a 2100 Bioanalyzer. APAP-induced alterations in liver mRNA expression were determined semiquantitatively from purified samples of total RNA (1-2 µg) in conjunction with ready-to-go reverse transcription-polymerase chain reaction (RT-PCR) beads and gene-specific primer sets for IL-6 and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as reported previously (19). Following amplification, PCR products were detected and quantified using DNA 1000 assay kits and a 2100 Bioanalyzer, and the results were normalized to the corresponding G3PDH expression within a given sample. Assessment of Hepatotoxicity. Liver injury was determined by measuring ALT activity in the sera using a commercially available kit and by examining histological changes in liver tissue sections. Cytokines and Chemokines Protein Assays. Serum concentrations of IL-6, TNF-R, IFN-γ, MIP-2, MIP-1R, and MCP-1 were measured commercially with the use of SearchLight Proteome Arrays (multiplex antibody arrays; Pierce Biotechnology, Woburn, MA) while serum levels of osteopontin were determined using an ELISA kit. Other Methods. SDS-PAGE and immunoblotting were performed as previously described (19). Total sera nitrite/nitrate levels, a measure of nitric oxide (NO) production, were determined by use of colorimetric nonenzymatic nitric oxide assay kit. Hepatic glutathione (GSH) levels were measured as previously described (32). Total protein concentrations were determined by Bradford Method using Coomassie Plus Protein Assay Kit (Pierce, Rockford, IL). Statistical Analyses. Experimental groups were compared using unpaired Student’s t-test and analysis of variance where appropriate. Differences were considered significant when p < 0.05. Grubbs’ test (GraphPad Software, Inc, San Diego, CA) was used to exclude outliers.

Results High Susceptibility of IL-10/4-/- Mice to APAP-Induced Liver Injury and Death. We previously reported elevated levels of IL-10 in the serum of WT mice treated with APAP and found that IL-10-/- mice were more susceptible than WT mice to APAP, indicating a hepatoprotective role of IL-10 against AILD (19). Because IL-4 was also elevated in the serum of WT mice treated with APAP (19), we investigated here whether IL-4 is also hepatoprotective in this model and whether it has a potential additive or even synergistic roles with IL-10 in AILD. IL-10/ 4-/- mice were found to be highly susceptible to liver injury following a dose of 120 mg/kg of APAP. Serum ALT activities rose rapidly (Figure 1A) and within 24 h, 75% of the mice had died; at 48 h, only 8% of the mice remained alive (Figure 1B). In contrast, IL-10-/- mice showed moderate liver injury only at 24 h post-APAP (120 mg/kg) treatment with no deaths, while IL-4-/- and WT mice were almost completely resistant to liver injury. IL-10/4-/- mice treated with 120 mg/kg of APAP were also more susceptible to liver injury and death than WT mice

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Figure 1. IL-10/4-/- mice are more susceptible to hepatic injury and death than WT, IL-4-/-, and IL-10-/- mice following APAP treatment. Mice were treated with APAP (120 mg/kg; WT also treated with 300 mg/kg). (A) After 2, 8, and 24 h, liver injury was assessed by measuring ALT activities. Results represent the mean ( SEM of four to six animals per group. All data are from the same experiment except for IL-10/ 4-/- mice 24 h after APAP treatment where data represent a pool from several experiments because of high dead animal number at this time point. *, Significant from all other groups of mice; #, significant from WT and IL-4-/- mice treated with 120 mg/kg of APAP, and WT mice treated with 300 mg/kg of APAP. (B) Comparison of survival rates of WT, IL-4-/-, IL-10-/-, and IL-10/4-/- mice 24 and 48 h following APAP treatment (N ) 18-24 animals per group).

treated with 300 mg/kg of APAP (Figure 1). At this higher dose of APAP, IL-4-/- mice similarly appeared to be more susceptible than WT mice to AILD because their serum ALT activities 8 h after APAP treatment were higher than those of WT mice (ALTs of 7239 ( 523.5 IU/L and 2445 ( 251.1 IU/L for IL4-/- and WT mice, respectively). The apparent decrease in ALT activities of the IL-10/4-/- mice 24 h after APAP (Figure 1A) is misleading because most of the mice in this group had already died of liver injury prior to assessments of ALT activity. Role of IL-6 in High Susceptibility of IL-10/4-/- Mice to AILD. In evaluating the underlying mechanisms responsible for the high susceptibility of IL-10/4-/- mice to AILD and death, hepatic mRNA expression levels of IL-6 at 2 h post-APAP were significantly elevated above those of untreated IL-10/4-/- mice and were higher than any other genotypic group studied, including WT mice treated with 300 mg/kg of APAP (Figure 2A). It seemed reasonable that the elevated levels of IL-6 in the liver of IL-10/4-/- mice might be a hepatoprotective response, because we previously found that IL-6-/- mice were more susceptible than WT mice to AILD (17). This hypothesis was tested by pretreating IL-10/4-/- mice with IL-6 neutralizing antibody (250 µg/mouse) 1 h prior to APAP treatment. Surprisingly, the anti-IL-6 neutralizing antibody partially protected these mice from liver injury at 4 and 8 h following APAP treatment (Figure 2B), suggesting that in IL-10/4-/- mice, IL-6 was a hepatotoxicant. This latter hypothesis was confirmed when serum ALT activities of the genetic crosses of IL-10/4-/- and

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Figure 2. IL-6 has a role in determining the high susceptibility of IL-10/4-/- mice to AILD. (A) IL-6 mRNA expression as determined by RT-PCR analysis in livers of WT, IL-4-/-, IL-10-/-, and IL-10/ 4-/- mice 2 h following treatment with saline or APAP (120 mg/kg; WT also treated with 300 mg/kg). Results represent the expression levels relative to those of G3PDH and are the mean ( SEM of three to four mice per group. *, Significant from all other groups of mice. (B) Effect of anti-IL-6 antibody pretreatment on APAP-induced liver injury in IL-10/4-/- mice. Mice were pretreated with 250 µg of anti-IL-6 or isotype control antibody 1 h before treatment with 120 mg/kg of APAP. At 4 and 8 h following APAP treatment, liver injury was assessed by measuring serum ALT activities. The results represent the mean ( SEM of five to seven mice per group. #, Significant from control groups of mice.

IL-6-/- mice (IL-10/4/6-/- mice) were found to be as low as WT mice treated with 120 mg/kg of APAP (Figure 3A). Similarly, liver sections from WT and IL-10/4/6-/- mice treated 8 h earlier with 120 mg/kg of APAP revealed comparable signs of histological liver injury with the WT mice showing panlobular hepatocellular vacuolization and the IL-4/10/6-/mice showing midzonal to periportal regions of hepatocellular vacuolization and nearly normal perivenous hepatocytes (Figure 3B). Both groups exhibited minimal isolated necrotic hepatocytes. In contrast, sections of IL-10/4-/- mice treated with the same dose of APAP contained widespread regions of hemorrhagic necrosis and mixed infiltrates, which also differed histopathologically from sections of WT mice treated with 300 mg/kg of APAP that displayed less extensive regions of perivenous necrosis and only minor infiltrates. Since IL-6 signaling can be mediated downstream by activated, phosphorylated forms of STAT3, p-STAT3 expression was measured by immunoblotting liver homogenates of WT, IL-10/4-/-, and IL-10/4/6-/- mice. pSTAT3 (Tyr705) was detected in livers of IL-10/4-/- mice at 2 h following APAP treatment and was even more highly expressed at 8 h posttreatment (Figure 4). p-STAT3 (Ser727) was also detected at relatively high levels in the livers of IL-10/4-/- mice at 8 h following APAP treatment. In contrast, p-STAT3 was barely detectable in WT and IL-10/4/6-/- mice 2 h after APAP treatment, and although increased expression was observed at 8 h, expression was much lower than that of IL-10/4-/- mice even in WT mice treated with 300 mg/kg of APAP (Figure 4).

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Figure 3. IL-10/4/6-/- mice are less susceptible than IL-10/4-/- mice to AILD. (A) Liver injury was determined by measurement of serum ALT activities at 0 (before treatment), 2, 8, and 24 h after treatment of WT, IL-10/4-/-, and IL-10/4/6-/- mice with 120 mg/kg of APAP. Results represent the mean ( SEM of six to seven animals per group. *, Significant from all other groups of mice. (B) Liver injury was confirmed by histologic examination of hematoxylin and eosin stained liver sections 8 h after treatment of mice with 120 mg/kg (WT, IL-10/4-/-, and IL-10/ 4/6-/- mice) or 300 mg/kg of APAP (WT mouse, WT300) (original magnification, 40×). The histological findings are representative of other mice of each strain treated in a similar manner.

Figure 4. p-STAT3 protein level is high in livers of IL-10/4-/- mice after treatment with APAP. STAT3 and p-STAT3 levels were determined in liver homogenates by immunoblot analysis at 2 and 8 h after APAP treatment with 120 mg/kg (WT, IL-10/4-/-, and IL-10/4/6-/-) or 300 mg/kg (WT) of APAP.

Low GSH and High APAP-Protein Adducts in Liver of IL-10/4-/- Mice. Because both hepatic GSH and APAP-protein adducts are apparently involved in the etiology of AILD (4, 5), we determined whether changes of these parameters could also explain, at least in part, the high susceptibility of IL-10/4-/mice to AILD. Hepatic levels of GSH were 35% lower in IL10/4-/- mice compared to other strains before APAP treatment (Figure 5). Two hours after 120 mg/kg of APAP, GSH was depleted almost completely in IL-10/4-/- mice and in WT mice treated with 300 mg/kg of APAP, whereas other strains except for the IL-4-/- mice had similarly depressed levels of GSH. By 8 h, the hepatic GSH levels of the IL-10/4-/- mice still

remained extremely low whereas those of WT, IL-4-/-, IL10-/-, and IL-10/4/6-/- mice had recovered close to baseline levels. Even the GSH levels of WT mice treated with 300 mg/ kg of APAP had recovered partially by 8 h. A major pathway for the depletion of GSH in the liver following APAP treatment is through the formation of a GSH conjugate with the reactive and toxic metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI) (33). As GSH becomes depleted, the competing reactions of NAPQI with liver proteins to form covalent adducts become more significant (4, 5, 34). In this regard, the levels of hepatic APAP-protein adducts were significantly higher in IL-10/4-/- mice than in all other strains of mice at 2 h following treatment with 120 mg/kg of APAP (Figure 6A and C). Whereas the APAP-protein adduct levels did not differ among WT, IL-10-/-, and IL-10/4/6-/mice, IL-4-/- mice did show significantly lower levels of APAPprotein adducts than all other groups. When WT mice were treated with 300 mg/kg of APAP, APAP-protein adducts levels were comparable to those of the IL-10/4-/- mice treated with 120 mg/kg of APAP (Figure 6A and C). Similar results were found at 1 h postdose, before the onset of widespread liver damage (Figure 6B). Moreover, before APAP treatment, the hepatic protein baseline level of cyp2e1, a major cytochrome P-450 involved in the metabolism of APAP to form NAPQI (35), was similar in all mice strains (data not shown).

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Figure 5. GSH levels are low in livers of IL-10/4-/- mice before and after treatment with APAP. GSH levels in liver homogenates were determined at 0 (before treatment), 2, and 8 h following treatment of mice with APAP (120 mg/kg; WT also treated with 300 mg/kg). Results represent the mean ( SEM of four mice per group. *, Significant from all other groups of mice; #, significant from WT, IL-4-/-, IL-10-/-, and IL-10/4/6-/- after 120 mg/kg APAP; †, significant from all groups of mice except WT mice treated with 120 mg/kg APAP.

High Serum Levels of Nitric Oxide (NO) and Low Expression of Liver Arginase Type 1 in IL-10/4-/- Mice. Since the depletion of GSH can result in increased NO-induced oxidative stress (4, 5) and because NO synthase 2-mediated biosynthesis of NO is negatively regulated by IL-4 (36) and IL-10 (37), we anticipated that high levels of NO might have a role in the enhanced susceptibility of IL-10/4-/- mice to AILD. Serum nitrite/nitrate levels, an indicator of NO production (4, 5), were similar in all mice before APAP treatment (Figure 7A). At 2 and 8 h posttreatment with 120 mg/kg of APAP, IL-10/ 4-/- mice showed significantly higher serum levels of nitrite/ nitrate than those seen in IL-10/4/6-/- and WT mice. Serum nitrite/nitrate levels in IL-10/4/6-/- mice were similar to those of WT mice at 2 h but were slightly higher at 8 h after APAP. We next wanted to find out whether depressed levels of hepatic arginase type 1, which reduces NO synthesis by hydrolyzing arginine into ornithine and urea (38), might have contributed to the high levels of nitrite/nitrate found in the serum of the IL-10/4-/- mice because this enzyme is normally induced by IL-10 and IL-4 (38). Indeed, before APAP treatment, levels of protein expression of arginase type 1 in IL-10/4-/- mice showed a trend toward reduction relative to those of WT and IL-10/4/6-/- mice. This difference became significant (p < 0.05) at 2 and 8 h after APAP dosing (Figure 7B). Support for the role of NO in the increased susceptibility of the IL-10/4-/- mice to AILD was suggested by the finding that treatment of mice with the NO synthase 2 (NOS-2) inhibitor (39) AG 2 h after APAP treatment, when APAP-protein adducts had reached maximum levels, (40) still protected IL-10/4-/mice from AILD at 4 h, with a trend at 8 h post-APAP treatment (Figure 7C). High Expression Levels of Inflammatory Factors in IL10/4-/- Mice. Since deficiencies in both IL-10 and IL-4 could

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Figure 6. APAP-protein adduct levels are high in the livers of IL10/4-/- mice after APAP treatment. Mice were treated with APAP (120 mg/kg; WT also treated with 300 mg/kg). After 1 or 2 h, APAP-protein adducts were detected in liver homogenates by immunoblot analysis with anti-APAP-protein adduct serum. (A) Immunoblot analysis 2 h after APAP treatment. (B) Immunoblot analysis 1 h after APAP treatment. (C) Quantification of APAP-protein adducts by densitometric analysis 2 h after APAP treatment. Results represent the mean ( SEM of four mice per group. *, Significant from IL-10/4-/- mice treated with APAP 120 mg/kg and WT mice treated with 300 mg/kg APAP; #, significant from IL-10-/-, IL-10/4-/-, and WT mice treated with 300 mg/kg APAP; †, significant from IL-4-/-, IL-10/4-/-, and WT mice treated with 300 mg/kg APAP; q, significant from WT, IL-4-/-, IL-10-/-, and IL-10/4/6-/- mice treated with 120 mg/kg APAP.

result in the dysregulation of inflammatory mediators (41) that might contribute to high sensitivity of the IL-10/4-/- mice to AILD, serum levels of several inflammatory factors were measured in mice prior to and 4 h after treatment with either 120 mg/kg (WT, IL-10/4-/-, and IL-10/4/6-/- mice) or with 300 mg/kg of APAP (WT) (Figure 8). Negligible levels of these mediators were found before APAP treatment. After treatment, IL-10/4-/- mice had very high serum levels of IL-6, while those of IL-10/4/6-/- and WT mice were undetectable or vastly lower than those observed in the IL-10/4-/- mice. Similarly, TNF-R, IFN-γ, MIP-2, and MIP-1R serum levels were also significantly higher in the IL-10/4-/- mice after APAP treatment, whereas MCP-1 serum levels in the IL-10/4-/- and IL-10/4/6-/- mice post-APAP treatment were not significantly different from each other but were higher than those of the WT mice. Osteopontin serum levels were also significantly higher in IL-10/4-/- mice at 8 h post-APAP treatment than in the serum of WT and IL10/4/6-/- mice (Figure 9).

Discussion The idiosyncratic nature of most cases of DILD may be related in part to a lack of more than one hepatoprotective mechanism secondary to genetic or environmental effects. This hypothesis and how it relates to IL-10 and IL-4 deficiency was tested here where we provide evidence indicating that this

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Figure 7. High nitric oxide levels may have a role in high susceptibility of IL-10/4-/- mice to AILD. (A) Nitrite/nitrate levels were measured in the serum at 0 (before treatment), 2, and 8 h following treatment of WT, IL-10/4-/-, and IL-10/4/6-/- mice with 120 mg/kg of APAP. Results represent the mean ( SEM of four to six mice per group. *, Significant from all other groups of mice; #, significant from WT mice 8 h after treatment with 120 mg/kg APAP. (B) Hepatic arginase type 1 protein levels were determined in liver homogenates of WT, IL-10/4-/-, and IL10/4/6-/- mice by immunoblot analysis at 0 (before treatment), 2, and 8 h following treatment of mice with 120 mg/kg of APAP. (C) AG (100 mg/kg, in saline) or saline was injected 2 h after treatment of mice with 120 mg/kg of APAP. Liver injury was determined by measuring ALT activities at 2, 4, and 8 h after APAP treatments. Results represent the mean ( SEM of four mice per group except for the 8-h AG group, which had three mice because one mouse died 4 h after APAP treatment. *, Significant from all other groups of mice except the 2-h time point (time just before AG injection); #, significant from all other groups of mice except the 8-h time point groups.

immune dysregulation leads to high levels of IL-6, which sensitizes mice to AILD. First, mice deficient in both IL-10 and IL-4, cytokines that down-regulate expression of IL-6 (42, 43), were highly susceptible to AILD (Figure 1) and had elevated expression levels of liver IL-6 mRNA (Figure 2A), serum IL-6 (Figure 8), and activated hepatic forms of IL-6 signaling mediator STAT3 (23) (Figure 4) as compared to other strains of mice. Second, the enhanced sensitivity of IL-10/4-/mice to AILD was diminished when IL-6 levels were reduced

with a neutralizing antibody (Figure 2B) or were abolished when the IL-10/4-/- mice were made genetically deficient in IL-6 (IL-10/4/6-/-) mice (Figure 3). In contrast, we previously found that relatively low levels of endogenous IL-6 can protect mice from AILD (17). Other studies have shown that endogenous IL-6 can have a dichotomous role in promoting or protecting against hepatotoxicity. In one study of ConA-induced liver injury, IL-6 serum levels induced in the early phase after ConA administration led

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Figure 8. Serum levels of IL-6 and other inflammatory factors are high in IL-10/4-/- mice after treatment with APAP. IL-6, TNF-R, IFN-γ, MIP2, MIP-1R, and MCP-1 protein levels were measured in the serum of mice with the use of a multiplex antibody arrays method before treatment (untreated) and 4 h after treatment with APAP (120 mg/kg; WT also treated with 300 mg/kg). Results represent the mean ( SEM of four mice per group. *, Significant from all other groups; #, significant from all other groups except the APAP-treated IL-10/4/6-/- mice; q, significant from all groups of untreated mice.

Figure 9. Serum levels of osteopontin are high in IL-10/4-/- mice after treatment with APAP. Osteopontin protein levels were measured in the serum of mice with the use of an ELISA kit before treatment (untreated) and 8 h after treatment with APAP (120 mg/kg; WT also treated with 300 mg/kg). Results represent the mean ( SEM of four mice per group. *, Significant from all other groups.

to hepatoprotection, while persistence of IL-6 production beyond this early phase enhanced liver injury (44). Similarly, IL-6 injection before ConA suppressed liver injury whereas administration of IL-6 at 6 h after ConA increased the extent of liver damage (27). Endogenous IL-6 has also been found to play a significant role in the induction of hepatic dysfunction and liver injury after trauma-hemorrhagic shock (45) and in lipopolysaccharides-mediated liver injury in mice overexpressing TGF-β1 in the liver (46). The high susceptibility of the IL-10/4-/- mice to AILD was associated with elevated levels of hepatic APAP-protein adducts (Figure 6). This appeared to be due in part to depressed levels

of hepatic GSH before APAP treatment (Figure 5), thus allowing NAPQI metabolite to react more extensively with hepatocyte proteins (4, 5, 33). The role of IL-6 in depressing hepatic GSH levels in IL-10/4-/- mice before APAP treatment (Figure 5) is not clear. Even though NAPQI and APAP-protein adducts are thought to have a direct causal role in AILD (4, 5, 8), they may not be the only factors contributing to the high susceptibility of IL-10/4-/- mice to AILD because the total levels and qualitative nature of APAP-protein adducts of IL-10/4-/- mice did not differ significantly from less susceptible WT mice treated with 300 mg/kg APAP (Figures 1 and 6). However, it remains possible that there are qualitative differences in APAP-protein adducts between the IL-10/4-/- and WT mice not detectable by immunoblotting that could contribute to the higher susceptibility of the IL-10/4-/- mice to AILD. Low hepatic levels of GSH might contribute to the increased susceptibility of IL-10/4-/- mice to AILD by IL-6 dependent mechanisms in addition to those involving enhanced APAPprotein adduct formation. For example, IL-6 has been shown to directly induce NOS-2 levels through a STAT3-dependent mechanism (49). This coupled with deficiencies of both IL-10 and IL-4, which negatively regulate NOS-2 (36, 37), and hepatic arginase type 1 (Figure 7B), which also reduces indirectly NO production (38), may have contributed to the high serum levels of nitrite/nitrate, a measure of NO formation, in IL-10/4-/- mice after APAP treatment as compared to WT and IL-10/4/6-/- mice (Figure 7A). The elevated production of NO, together with the

High Susceptibility of IL-10/4-/- Mice to APAP

possible increase of reactive oxygen species production secondary to low hepatic GSH (Figure 5), might lead to the generation of the highly toxic metabolite, peroxynitrite anion (4, 5), that could directly contribute to liver injury (4, 5, 50). This reactive species in turn may have played a role in the sustained depletion of GSH in the IL-10/4-/- mice (34) following APAP treatment (Figure 5). It seems possible that these reactions could have sensitized the IL-10/4-/- mice to AILD given that AG posttreatment partially inhibited AILD (Figure 7C) and that the enhanced susceptibility of IL-10-/- mice to AILD compared to WT mice reported earlier appeared to be due in large part to overproduction of NO (19). IL-10 and IL-4 synergistically inhibit macrophage cytotoxic activity (51) and enhance human monocyte apoptosis (52). They also down-regulate monocytes/macrophages inflammatory function including TNF-R, IFN-γ, and IL-6 secretion (51, 53, 54). Accordingly, these cytokines were detected at higher levels in the sera of IL-10/4-/- mice following APAP treatment relative to WT and IL-10/4/6-/- mice (Figure 8). TNF-R and IFN-γ can induce the expression of several inflammatory chemokines (38, 55-57) including MIP-2, MIP-1R, and OPN, which were more highly expressed in IL-10/4-/- mice than WT and IL-10/ 4/6-/- mice after APAP dosing (Figures 8 and 9). These chemokines could contribute to the accumulation of inflammatory cells in the liver of IL-10/4-/- mice (Figure 3B) and to the amplification of hepatic levels of IL-6 and those of other toxic inflammatory factors (38, 55, 57-60). Although the role of these inflammatory mediators and cells in enhancing the susceptibility of IL-10/4-/- mice to AILD remains to be determined, other studies of AILD have shown that IFN-γ (11), OPN (12), macrophage migration inhibitory factor (10), and neutrophils (15, 16) can contribute to the severity of AILD. In conclusion, our findings clearly indicate that IL-6 can enhance the susceptibility of mice to AILD when it is overexpressed because of deficiencies in IL-10 and IL-4. Although it is not clear yet how IL-6 sensitizes mice to AILD, dysregulation of GSH homeostasis appears to be involved, which can lead to elevated levels of APAP-protein adducts and possibly to toxic levels of reactive oxygen and nitrogen species including NO and other protoxicants. The lack of IL-4 and IL-10 can also lead to the enhanced production of several proinflammatory factors that may contribute to enhanced secondary toxic mechanisms of liver injury caused by APAP. Perhaps polymorphisms in genes encoding for IL-6-regulatory factors, such as IL-10 and IL-4 (61), may also lead to the overexpression of IL-6 in humans. Similarly, environmental factors including restraint, endotoxin, infection, and a variety of inflammatory diseases can lead to elevated plasma levels of IL-6 (24, 6265). It is conceivable that one or more of these conditions leading to elevated levels of IL-6 may predispose patients to the hepatotoxic effect of not only APAP, a leading cause of acute liver failure (2), but to other DILDs. In this regard, studies are being carried out to determine whether the IL-10/4-/- mice can be used to study the molecular basis of other hepatotoxic drugs that so far have not been amenable to model studies in animals. Acknowledgment. Supported in part by the Intramural Research Program of the National Heart, Lung, and Blood Institute, National Institutes of Health. The authors thank Dr. Thomas Wynn (NIAID, NIH, Bethesda, MD) for providing IL4-/- and IL-10/4-/- mice and Drs. Neil Pumford and Jack Hinson (University of Arkansas, Little Rock, AR) for providing anti-APAP serum.

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