Hepatoprotective Role of Endogenous Interleukin-13 in a Murine

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Chem. Res. Toxicol. 2007, 20, 734-744

Hepatoprotective Role of Endogenous Interleukin-13 in a Murine Model of Acetaminophen-Induced Liver Disease Steven B. Yee,* Mohammed Bourdi, Mary Jane Masson, 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, 9000 RockVille Pike, Building 10, Room 8N110, Bethesda, Maryland 20892 ReceiVed December 6, 2006

Recent evidence suggests that a deficiency in one or more hepatoprotective regulatory mechanisms may contribute to determining susceptibility in drug-induced liver disease. In the present study, we investigated the role of interleukin (IL)-13 in acetaminophen (APAP)-induced liver disease (AILD). Following APAP (200 mg/kg) administration to male C57BL/6 wild-type (WT) mice, hepatotoxicity developed up to 24 h post-APAP, with a concomitant increase in serum IL-13 concentration. Pretreatment of these mice with an IL-13-neutralizing antibody exacerbated liver injury, as did APAP administration to IL-13 knockout (KO) mice in comparison to WT mice. No difference was observed in either overall APAP-protein adduct formation or liver glutathione levels between KO and WT mice following APAP administration, suggesting that the increased susceptibility of IL-13 KO mice to AILD was not due to enhanced APAP bioactivation but rather injurious downstream events. In this regard, multiplex antibody arrays were used to identify potential IL-13-regulated biomarkers, including various cytokines and chemokines, as well as nitric oxide (NO), associated with AILD that were present at higher concentrations in the sera of APAP-treated IL-13 KO mice than in WT mice. Subsequent inhibition studies determined interferon-γ, NO, neutrophils, natural killer cells, and natural killer cells with T-cell receptors had pathologic roles in AILD in IL-13 KO mice. Taken together, these results suggest that IL-13 is a critical hepatoprotective factor modulating the susceptibility to AILD and may provide hepatoprotection, in part, by down-regulating protoxicant factors and cells associated with the innate immune system. Introduction (DILD)1

Drug-induced liver disease is a significant health risk, often leading to morbidity and mortality (1). Because of its idiosyncratic nature, DILD is one of the most frequent causes for the failure of new drugs to obtain regulatory approval and for drugs to acquire postmarketing warnings or to be withdrawn from the marketplace (2). The relatively low incidence, inconsistent temporal patterns, and insufficient understanding of the underlying mechanism suggest that susceptibility to DILD may be the result of multiple, intersecting factors (3). Consequently, DILD remains difficult to predict, representing both an unacceptable health and safety risk to patients and an undesirable financial risk to the pharmaceutical industry (3). Recent animal model studies, particularly those involving acetaminophen (APAP) in mice, have suggested that susceptibility to DILD may be the result, in part, of an alteration in the normal regulatory mechanisms balancing protoxicant and hepatoprotective mediators to favor injury (4-8). Indeed, a number of protoxicant mediators including macrophage migration * To whom correspondence should be addressed. Tel: 301-496-4841. Fax: 301-480-4852. E-mail: [email protected]. 1 Abbreviations: IL, interleukin; APAP, acetaminophen; AILD, APAPinduced liver disease; WT, wild type; KO, knockout; NO, nitric oxide; NK, natural killer cell; NKT, natural killer cell with T-cell receptors; DILD, drug-induced liver disease; IFN, interferon; OPN, osteopontin; PMN, polymorphonuclear leukocyte; GSH, glutathione; SAL, saline vehicle; NAb, neutralizing antibody; CAb, control antibody; TNF, tumor necrosis factor; Gr, granulocyte; iNOS, inducible nitric oxide synthase; AG, aminoguanidine; ALT, alanine aminotransferase; H&E, hematoxylin and eosin; HPF, highpowered field; KC, keratinocyte-derived chemokine (murine homolog of IL-8); MIP, macrophage inflammatory protein; GM-CSF, granulocyte macrophage-colony stimulating factor; CYP, cytochrome P450; NAPQI, N-acetyl-p-aminoquinone imine; R, receptor; CV, central vein.

inhibitory factor (4), interferon (IFN)-γ (9), lipopolysaccharidebinding protein (10), osteopontin (OPN) (11), natural killer (NK) cells and natural killer cells with T-cell receptors (NKT) (12), and neutrophils (polymorphonuclear leukocytes, PMN) (13, 14) appear to have roles in determining APAP-induced liver disease (AILD) susceptibility in mice. Moreover, the loss of critical hepatoprotective mediators such as interleukin (IL)-10 (5), IL-6 (6), cyclooxygenase-2 (7), C-C chemokine receptor 2 (15), and nuclear factor-erythroid 2-related factor 2 (16) also results in increased susceptibility to AILD. In the present study, we found that IL-13 deficiency in mice, produced either through a neutralizing antibody or through genetic manipulation, increased the susceptibility to AILD. Exacerbated liver injury was likely the result of events downstream of APAP metabolic activation largely mediated by proinflammatory cytokines, chemokines, and innate immune cells. Although further mechanistic studies are needed, these findings implicate endogenous IL-13 as a critical hepatoprotective factor in liver injury caused by APAP, and possibly other drugs, through the down-regulation of protoxicant factors and cells associated with the innate immune system.

Experimental Procedures Animals. Eight to 11 week old (20-25 g) male C57BL/6 wildtype (WT) mice from Jackson Laboratories (Bar Harbor, ME) and IL-13 knockout (KO) (C57BL/6 background) mice from Taconic Farms (Terrytown, NY; courtesy of Dr. Thomas A Wynn, NIAID/ NIH) were used in all studies. Animals were housed in autoclaved microisolator cages and were acclimated to a 12 h light/dark cycle in a temperature- and humidity-controlled, specific pathogen-free environment for at least 1 week before use. Mice were allowed autoclaved food (Quality Lab Products, Inc., Elkridge, MD) and

10.1021/tx600349f CCC: $37.00 © 2007 American Chemical Society Published on Web 04/18/2007

IL-13 Attenuates APAP-Induced LiVer Disease water ad libitum. All procedures on animals conformed to the guidelines for humane treatment set by the Association for Assessment and Accreditation for Laboratory Animal Care International’s Guide for the Care and Use of Laboratory Animals and by the National Institutes of Health. Animal Treatment Protocol. Before each study, mice were fasted overnight (14-16 h; free access to water) to uniformly deplete hepatic glutathione (GSH) stores (17). Food supplies were restored after i.p. administration of APAP (200 mg/kg in warm saline; Sigma-Aldrich, Inc., St. Louis, MO) or saline vehicle (SAL). All antibodies were dissolved in PBS (pH 7.4; Invitrogen Corp., Carlsbad, CA) for the following neutralization studies. 1. IL-13 Neutralization. Monoclonal IL-13 neutralizing antibody (NAb, 125 µg) or IgG2b isotype control antibody (CAb, 125 µg) (R&D Systems, Inc., Minneapolis, MN) was administered i.p. to WT mice 2 h before APAP or SAL treatment. 2. Tumor Necrosis Factor (TNF)-r Neutralization. IL-13 KO mice were pretreated i.p. with single or multiple doses of TNF-R NAb or IgG isotype CAb (gifts from Dr. David J. Shealy, Centocor, Inc., Malvern, PA) (18) ranging from 500 µg to 1.7 mg between 2 and 24 h before administration of APAP or SAL. 3. IFN-γ Neutralization. IL-13 KO mice were pretreated i.p. with 500 µg of IFN-γ NAb (9) or IgG1 isotype CAb (BioLegend, San Diego, CA) 2 h before APAP or SAL treatment. 4. IL-6 Neutralization. IL-13 KO mice were pretreated i.p. with 250 µg of IL-6 NAb or IgG1 isotype CAb (R&D Systems) 1 h before administration of APAP or SAL. 5. NK and NKT Cell Neutralization. IL-13 KO mice were pretreated intravenously with 150 µg of NK 1.1 NAb (12) or IgG2a isotype CAb (BioLegend) 42 h before APAP or SAL administration. 6. PMN Depletion. IL-13 KO mice were pretreated i.p. with 250 µg of PMN NAb [anti-granulocyte (Gr-1) antibody, clone RB68C5] (13, 14) or IgG2b isotype CAb (BioLegend) 48 h before APAP or SAL administration. An equivalent dose of anti-PMN NAb was found to significantly attenuate PMN accumulation for up to 5 days post-administration (13, 19). 7. Inducible Nitric Oxide Synthase (iNOS) Inhibition. To inhibit nitric oxide (NO) levels, the iNOS inhibitor, aminoguanidine (AG; 100 mg/kg in PBS; Sigma-Aldrich) (20), or SAL was administered i.p. to IL-13 KO mice 2 h after administration of APAP or SAL. Sera and Tissue Collection. Blood was collected by retro-orbital puncture or from the inferior vena cava. Blood samples were allowed to clot in microtainer serum separator tubes (Becton Dickinson and Co., Franklin Lakes, NJ) for approximately 1 h at room temperature and then centrifuged. Serum was separated for alanine aminotransferase (ALT) measurement, and the remaining serum was snap-frozen and stored at -80 °C until further analysis. A portion of the left and right lateral liver lobes were fixed in 10% buffered formalin (Fischer Scientific, Fair Lawn, NJ) with the remainder snap-frozen and stored at -80 °C for further study. Fixed tissue was embedded in paraffin, processed by standard histological techniques, and stained with hematoxylin and eosin (H&E; American Histolabs, Gaithersburg, MD). Assessment of Liver Injury. Hepatic parenchymal cell injury was evaluated by measuring serum ALT activity using a microtiter plate adaptation of a commercially available kit (Teco Diagnostics, Anaheim, CA) and through histopathological examination of H&Estained liver sections under light microscopy. Quantitative evaluation of liver injury in H&E-stained sections was analyzed morphometrically via light microscopy using Axiovision software (v4.5; Carl Zeiss Microimaging, Inc., Thornwood, NY) and a 64point grid (21). Approximately 10% of the total area of each liver section was examined. All slides were coded and randomized before evaluation. Immunohistochemical Analysis of Hepatic PMNs. Unstained formalin-fixed liver sections were deparaffinized, and endogenous peroxidase was quenched with 0.3% hydrogen peroxide in methanol for 30 min. Slides were blocked with normal rabbit serum (“blocking solution”; 1:100 in PBS; Vector Laboratories, Burlingame, CA) for 20 min, rinsed with PBS, and incubated with rat

Chem. Res. Toxicol., Vol. 20, No. 5, 2007 735 anti-mouse PMN Gr-1 antibody (clone RB6-8C5, 1:500 in blocking solution; Biolegend) or rat anti-mouse IgG2b negative CAb (1: 500 in blocking solution; Biolegend) at room temperature for 30 min. Following PBS rinse, the secondary antibody (anti-rat IgG biotinylated antibody, dilution per kit instructions; Vector Laboratories) was applied at room temperature for 30 min. Avidinbiotin horseradish peroxidase (Vector Laboratories) was used for antigen localization and diaminobenzidine tetrahydrochloride (Vector Laboratories) as the chromagen. Slides were counter-stained with 0.1% hematoxylin (Sigma-Aldrich, Inc.). PMNs were identified by positive staining and cell morphology. Hepatic PMN accumulation was assessed by averaging the number of PMNs enumerated in 10 randomly selected, high-powered fields (HPFs, original magnification 40×) per liver section (22). This represented approximately 20% of the total area for each liver section. Measurement of Serum Biomarkers. IL-13 serum levels were measured with an enzyme-linked immunosorbent assay (Biosource International, Camarillo, CA), while IL-6, IL-10, IL-12p70 (IL12), IL-18, keratinocyte-derived chemokine (KC; i.e., murine homolog of IL-8), TNF-R, IFN-γ, macrophage inflammatory protein (MIP)-2, eotaxin-1 (eotaxin), granulocyte macrophage-colony stimulating factor (GM-CSF), and OPN were measured commercially using SearchLight Proteome Arrays (multiplex antibody arrays; Pierce Biotechnology, Woburn, MA). Total serum nitritenitrate concentration, a measure of NO production (5), was determined colorimetrically with a nonenzymatic NO assay kit (Oxford Biomedical Research, Oxford, MI). Immunoblot Analysis. Liver tissue was homogenized (20 s; FastPrep FP120 homogenizer; Q-Biogene, Irvine, CA) in a Matrix D Lysing tube (Q-Biogene) containing 10 mM Tris (pH 7.4) with 250 mM sucrose and 1 mM EDTA, complete protease inhibitor cocktail (Roche Diagnostic Corp., Indianapolis, IN), and 1 mM phenylmethanesulfonyl fluoride solution (Sigma-Aldrich). Proteins in liver homogenates were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and immunoblotted as previously reported (23). Membranes were immunochemically probed with a specific antisera directed against APAP-protein adducts (a gift from Drs. Neil R. Pumford and Jack A. Hinson, University of Arkansas, Little Rock, AR) or anti-cytochrome P450 2e1 (CYP2e1; gift from Dr. Dennis R. Koop, Oregon Health Science University, Portland, OR). After incubation with the appropriate peroxidase-conjugated secondary antibody (Chemicon International; Temecula, CA), protein signals were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) using a Kodak Station 2000 RT Imager and software (Eastman Kodak, Rochester, NY). Analysis of Protein Concentration and Liver GSH Levels. Protein concentration was determined via a Bradford assay (Coomassie Plus Protein Assay Reagent; Pierce). A commercial GSH Assay Kit (Cayman Chemicals, Ann Arbor, MI) was used to measure total GSH levels in liver homogenates. Flow Cytometry for NK and NKT Cell Depletion. Briefly, saline-perfused livers were pressed through cell strainers, and the resulting pellet was centrifuged at 800g for 15 min in 35% Percoll (Sigma-Aldrich) in RPMI (Invitrogen). Following Fc receptor blocking, hepatic mononuclear cells were stained with anti-mouse NK1.1 PE and anti-mouse CD3 (BD Pharmingen; San Diego, CA). NK and NKT cell (NK1.1+CD3- and NK1.1+CD3+ cells, respectively) percentages were determined using a CyAnTM LX Flow Cytometer (DakoCytomation, Carpintaria, CA). Statistical Analysis. Results are expressed as mean ( SEM. Data for single comparisons were analyzed by Student’s t test. Multiple comparisons of homogeneous data were analyzed by one-way or two-way analysis ANOVA, as appropriate, and group means were compared using Student-Newman-Keuls posthoc test. The criterion for significance was p e 0.05 for all comparisons.

Results Enhanced AILD in IL-13 Deficient Mice. To elucidate the role of IL-13 in AILD, WT mice were pretreated with either

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Figure 1. Increased susceptibility of IL-13 deficient mice to AILD. (A) Serum IL-13 levels and (B) ALT activities were determined at various times in WT mice pretreated with IL-13 NAb (125 µg) or IgG CAb (125 µg) 2 h prior to APAP (200 mg/kg) or SAL treatment. Serum IL-13 concentrations and ALT activities for control CAb/SAL- and IL-13 NAb/SAL-treated groups averaged 18 and 12 pg/mL and 14 and 21 U/L, respectively. (C) Serum ALT activities were determined at various times in WT and IL-13 KO mice following treatment with APAP (200 mg/kg) or SAL. Serum ALT activities for control SALtreated WT and KO mice averaged 24 and 25 U/L, respectively. Results represent the mean ( SEM of 4-8 animals per group for A and B and 7-31 animals per group for C. (a) Significantly different from respective SAL-treated group at the same time. (b) Significantly different from CAb/APAP group at the same time. (c) Significantly different from APAP-treated WT group at the same time.

CAb or IL-13 NAb 2 h before APAP (200 mg/kg) or SAL administration. Similar to earlier findings (5), serum IL-13 levels increased following APAP administration (Figure 1A). This increase was significantly attenuated up to 8 h post-APAP with IL-13 NAb pretreatment. Liver injury, as determined by elevated serum ALT activity, was significantly exacerbated in APAPtreated WT mice receiving IL-13 NAb pretreatment in comparison to animals pretreated with CAb (Figure 1B). The hepatoprotective nature of endogenous IL-13 was further confirmed when IL-13 KO mice were found to be more susceptible to AILD than WT, as determined both biochemically (Figure 1C) and histopathologically (Figure 2 and Table 1). While no animal deaths occurred in the IL-13 neutralization study, minimal mortality (about 3%, one animal out of 31) was observed at 24 h in APAP-treated IL-13 KO mice and progressed to approximately 22% (four animals out of 18) by

Figure 2. Representative photomicrographs of H&E liver sections from WT and IL-13 KO mice 8 h after treatment with APAP (200 mg/kg) or SAL. (A) Liver from a SAL-treated KO mice exhibited normal liver architecture with no histopathological changes or inflammatory infiltrate. Similarly, a liver from a SAL-treated WT mouse exhibited no histopathological changes (photomicrograph not shown). (B) Liver from an APAP-treated WT mouse. Moderate centrilobular coagulative necrosis was observed along with disruption of sinusoidal architecture, mild to moderate hemorrhage at the periphery, and mild to moderate PMN infiltration (black arrow) in the lesion area. (C) Liver from an APAP-treated IL-13 KO mouse. Marked to severe centrilobular coagulative necrosis, sometimes bridging, was observed, along with marked disruption of sinusoidal architecture, moderate hemorrhage, and moderate PMN infiltration (black arrow) in the lesion area. CV, central vein; bar ) 50 µm.

IL-13 Attenuates APAP-Induced LiVer Disease

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Table 1. Morphometric Analysis of Liver Lesions from APAP-Treated WT and IL-13 KO Micea percent lesion area (time after APAP administration) treatment

4h

8h

24 h

WT APAP KO APAP

12.6 ( 1.6 30.3 ( 5.6b

17.6 ( 2.5 34.0 ( 4.7b

22.0 ( 1.5 40.3 ( 3.0b

a APAP (200 mg/kg) was administered to WT or IL-13 KO mice. Livers were removed from mice at various times after APAP treatment, fixed in formalin, and processed for light microscopy. Morphometric analysis was performed as described in the Experimental Procedures. Results represent mean ( SEM of 4-6 animals per group. b Significantly different from the APAP-treated WT group.

Figure 3. Equivalent levels of APAP-protein adducts in livers from APAP-treated WT and IL-13 KO mice. Two hours after APAP (200 mg/kg) or SAL treatment of WT and IL-13 KO mice, liver homogenates were immunoblotted with antisera directed against APAP-protein adducts. (A) Representative immunoblot. (B) Scanned immunoblot normalized to β-actin levels. Results represent the mean ( SEM of four mice per group.

36 h post-APAP. In contrast, WT mice treated at the same time as IL-13 KO mice were resistant to APAP-induced death. APAP-Protein Adducts and GSH Levels in Livers from APAP-Treated WT and IL-13 KO Mice. APAP-protein adducts and GSH levels were measured in liver homogenates to evaluate whether exacerbated AILD in IL-13 KO mice was

the result of enhanced APAP bioactivation. Although some minor alterations were observed in the band patterns and intensities of APAP-protein adducts in liver homogenates between WT and IL-13 KO mice 2 h after APAP treatment, the overall quantitative nature of the APAP-protein adducts remained the same (Figure 3). Likewise, equivalent amounts of CYP2e1, a major form of cytochrome P450 responsible for metabolizing APAP to N-acetyl-p-aminoquinone imine (NAPQI) (24), were observed in liver homogenates from APAP-treated WT and IL13 KO mice 2 h after APAP administration (data not shown). Because liver GSH is depleted as a result of conjugation with NAPQI (the toxic, reactive metabolite of APAP) (24, 25), GSH depletion serves as an additional, albeit indirect, measure of bioactivation. Fasting reduced liver GSH levels equivalently (approximately 51%) in both naı¨ve WT and IL-13 KO mice (Figure 4A). Similarly, hepatic GSH levels were reduced equally (about 90%) in APAP-treated WT and IL-13 KO mice 2 h after APAP administration (Figure 4B) and returned to SAL-treated control levels by 8 h (Figure 4C and D). Serum Biomarker Profile of APAP-Treated IL-13 KO Mice. To evaluate other underlying mechanisms responsible for the enhanced susceptibility of IL-13 deficient animals to AILD, several cytokines, chemokines, and other factors were measured in the sera of WT and IL-13 KO mice before and at various times after APAP treatment (Figure 5). In most cases, serum biomarker concentrations increased significantly in APAPtreated WT and IL-13 KO mice as compared to SAL treatment. For example, TNF-R (A), IL-6 (B), KC (C), MIP-2 (D), and OPN (E) levels were higher in APAP-treated WT and IL-13 KO mice at all time points evaluated. Similarly, IFN-γ (F), IL18 (G), IL-10 (H), and nitrite-nitrate (I) levels were higher in APAP-treated IL-13 KO mice as compared to their SAL-treated controls at all times, while they were only higher for a portion of the time or not at all in APAP-treated WT mice in comparison to SAL-treated WT mice. Moreover, APAP-treated IL-13 KO mice also had higher levels of IL-12 (J), eotaxin (K), and GMCSF (L) than SAL-treated KO mice at either 4 or 4 and 8 h

Figure 4. Equivalent hepatic GSH levels in APAP-treated WT and IL13 KO mice. GSH levels were determined from liver homogenates of WT and IL-13 KO mice, before (A) and at 2 (B), 8 (C), and 24 (D) h after APAP (200 mg/kg) or SAL treatment. Results represent the mean ( SEM of 4-10 animals per group. (a) Significantly different from respective groups fed ad libitum. (b) Significantly different from respective SALtreated mice.


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Figure 5. Profile of serum biomarkers from APAP-treated WT and IL-13 KO mice. Biomarker levels were measured from the sera of WT and IL-13 KO mice at 4, 8, and 24 h after APAP (200 mg/kg) or SAL treatment. Mean values of each biomarker investigated for SAL-treated WT and IL-13 KO mice, respectively, are enclosed in brackets: (A) TNF-R [3, 2], (B) IL-6 [17, 5], (C) KC [13, 7], (D) MIP-2 [6, 6], (E) OPN [202, 126], (F) IFN-γ [10, 6], (G) IL-18 [110, 30], (H) IL-10 [4, 5], (I) NO [59, 80], (J) IL-12 [0.6, 0.5], (K) eotaxin [26, 21], and (L) GM-CSF [5, 4]. Results represent the mean ( SEM of 4-12 animals per group. (a) Significantly different from respective SAL-treated groups at the same time. (b) Significantly different from APAP-treated WT group at the same time.

post-APAP administration, whereas an increase in these biomarker levels was not observed in APAP-treated WT mice as compared to SAL-treated WT mice. Significantly higher concentrations of several factors were found in the sera of IL-13 KO mice as compared to WT mice at various times following APAP treatment (Figure 5). IL-12 (J), IL-18 (G), and MIP-2 (D) were significant elevated at 4 h in APAP-treated KO mice as compared to WT, while IL-6 (B), IL-10 (H), eotaxin (K), and GM-CSF (L) were significantly elevated at both 4 and 8 h after APAP treatment. Likewise, IFN-γ (F) at 8 and 24 h and TNF-R (A), KC (C), OPN (E), and

nitrite-nitrate (I) at all times post-APAP treatment were higher in the sera of APAP-treated KO mice. Role of TNF-r, IFN-γ, NO, and IL-6 in AILD in IL-13 KO Mice. To investigate whether increased serum levels of TNF-R, IFN-γ, NO, and/or IL-6 may have a causal role in the enhanced susceptibility of IL-13 KO mice to AILD, the levels of these factors were reduced either with neutralizing antibodies or, in the case of NO, biochemically with an inhibitor of its synthesis. While pretreatment with TNF-R NAb 4 h before APAP administration significantly reduced serum TNF-R concentration in APAP-treated IL-13 KO mice at 8 h (Figure



Figure 6. Role of TNF-R, IFN-γ, and IL-6 in APAP-treated IL-13 KO mice. TNF-R NAb (500 µg) or CAb was administered 4 h before administration of APAP (200 mg/kg) or SAL. Serum TNF-R (A) and ALT (B) levels were evaluated 8 and 24 h after APAP treatment. Serum TNF-R concentrations and ALT activities for Cab/SAL- and TNF-R NAb/SAL-treated groups were 5 and 6 pg/mL and 11 and 9 U/L, respectively. IFN-γ NAb (500 µg) or CAb was administered 2 h before administration of APAP (200 mg/kg) or SAL to IL-13 KO mice. Serum IFN-γ (C) levels and ALT (D) activities were evaluated 8 and 24 h post-APAP treatment. Serum IFN-γ concentrations and ALT activities for Cab/SAL- and IFN-γ NAb/SALtreated groups were 7 and 8 pg/mL and 18 and 16 U/L, respectively. IL-6 NAb (250 µg) or CAb was administered 1 h before administration of APAP (200 mg/kg) or SAL to IL-13 KO mice. Liver injury (E) was observed at 4 and 8 h following APAP treatment. Serum ALT activities for Cab/SAL- and IL-6 NAb/SAL-treated groups were 38 and 34 U/L, respectively. Results represent the mean ( SEM of 4-6 animals per group for (A and B) and 3-7 animals per group for (C-E). (a) Significantly different from respective SAL-treated groups at the same time. (b) Significantly different from CAb/APAP group at the same time.

6A), AILD was unaffected (Figure 6B) at 8 and 24 h postAPAP. Additional studies exploring a variety of NAb doses (up to 1.7 mg) as well as different dosing regimens (from 2 to 24 h prior to APAP treatment) failed to result in a change in AILD susceptibility (data not shown). In contrast, administration of IFN-γ NAb 2 h before APAP treatment attenuated both serum IFN-γ concentration (Figure 6C) and AILD (Figure 6D) up to 24 h post-APAP administration. Similarly, when the iNOS inhibitor AG (20) was administered to IL-13 KO mice 2 h postAPAP treatment, to avoid interfering with APAP bioactivation (26), serum nitrite-nitrate concentration (Figure 7A) and the severity of AILD (Figure 7B) diminished at both 4 and 8 h after APAP treatment. IL-6 NAb administered 1 h before APAP treatment, however, increased AILD at 4 and 8 h after APAP treatment (Figure 6E). In all cases, the severity of the liver lesion observed histologically in these studies correlated with the biochemical measurements of liver injury (data not shown). Role NK and NKT Cells and PMNs in AILD in IL-13 KO Mice. Because NK and NKT cells are major sources for IFN-γ (12), the role of these immune cells was investigated (Figure 6D). NK 1.1 NAb was administered to IL-13 KO mice

42 h before APAP treatment to deplete NK and NKT cells, which both have the NK 1.1 marker on their surface (12, 27). Flow cytometry confirmed that the dosing regimen of the NK 1.1 NAb decreased both NK and NKT cells in the liver by 90 and 98%, respectively (data not shown). The depletion of these cells attenuated AILD at 8 and 24 h post-APAP administration (Figure 8A) and abrogated serum IFN-γ levels at 24 h postAPAP in comparison to IL-13 KO mice treated with a control antibody prior to APAP administration (Figure 8B). Greater hepatic PMN accumulation was observed both histologically and immunohistochemically in liver sections from APAP-treated IL-13 KO mice in comparison to WT (Figures 2C and 9C and Figures 2B and 9B, respectively) at 8 h postAPAP treatment. Quantitative analysis of immunostained liver sections revealed PMN accumulation was significantly elevated in APAP-treated KO mice (28.5 ( 2.2 PMNs/HPF) as compared to APAP-treated WT mice (17.2 ( 2.5 PMNs/HPF). To determine whether these innate immune cells contributed to the increased susceptibility of IL-13 KO mice to AILD, animals were pretreated with a PMN NAb 48 h before APAP administration to decrease PMNs. PMN depletion (compare Figure


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Discussion

Figure 7. Role of NO in APAP-treated IL-13 KO mice. AG (100 mg/ kg) or SAL was administered to IL-13 KO mice 2 h after treatment with APAP (200 mg/kg) or SAL. Serum nitrite-nitrate (A) levels and ALT (B) activities were determined at 4 and 8 h after APAP administration. Serum nitrite-nitrate levels and ALT activities for SAL/ SAL- and SAL/AG-treated groups were 60 and 46 µM and 23 and 7 U/L, respectively. Results represent the mean ( SEM of 3-9 animals per group. (a) Significantly different from respective SAL-treated groups at the same time. (b) Significantly different from APAP/SAL group at the same time.

Figure 8. Role of NK and NKT cells in APAP-treated IL-13 KO mice. NK 1.1 NAb (150 µg) or CAb was administered 42 h before administration of APAP (200 mg/kg) or SAL to IL-13 KO mice. Serum ALT (A) and IFN-γ (B) levels were evaluated up to 24 h after APAP treatment. Serum ALT activities and IFN-γ concentrations for Cab/ SAL- and NK 1.1 NAb/SAL-treated groups were 8 and 8 U/L and 7 and 8 pg/mL, respectively. Results represent the mean ( SEM of 3-7 animals per group. (a) Significantly different from respective SALtreated groups at the same time. (b) Significantly different from CAb/ APAP group at the same time.

9D and E) significantly decreased AILD at 8 and 24 h after APAP administration (Figure 9F).

IL-13 is an important immunoregulatory cytokine produced by a variety of cell types including NK and NKT cells, eosinophils, basophils, mast cells, and Kupffer cells (28-31). This cytokine mediates its actions through the IL-13 receptor (R), a heterodimer composed of IL-4RR and IL-13RR1 (30). The IL-13R is expressed on B cells, monocytes/macrophages, T-lymphocytes, eosinophils, basophils, hepatic parenchymal cells, and endothelial cells (30, 32, 33). Exhibiting a wide range of functions (30, 34), IL-13 serves as a protoxicant in some disease models while functioning as a protectant in others. Accordingly, IL-13 overexpression has been shown to evoke allergic asthma through airway hyperresponsiveness and mucus hypersecretion (35) and acts as a key proinflammatory cytokine in Schistosomiasis manisomi infection-induced liver fibrosis (36). However, IL-13 is also protective to both hepatic parenchymal cells and sinusoidal endothelial cells in liver ischemia/reperfusion injury (37) and moderates inflammatory reactions and diseases, such as in chronic Leishmania major infections and other nematode infections (38). Hence, the function of IL-13 is both diverse and complex. In the present study, we demonstrate that endogenous IL-13 protects the liver from AILD. Following a hepatotoxic dose of APAP, IL-13 serum levels in WT mice rose in apparent response to liver injury (Figure 1A) (5). When APAP was administered to WT mice pretreated with an IL-13 NAb or to IL-13 KO mice, the liver injury was more severe as compared to corresponding control mice (Figures 1B, C, and 2), suggesting that the increased IL-13 levels controlled the magnitude of AILD. The increased susceptibility of IL-13 KO mice to AILD, however, did not appear to be exclusively the result of enhanced APAP bioactivation, since total APAP-protein adduct levels in liver homogenates from IL-13 KO mice at 2 h post-APAP administration did not differ quantitatively from those observed in WT mice (Figure 3B), and only minor qualitative differences in APAP-protein adducts were observed between the two groups of mice (Figure 3A). Consistent with this observation, hepatic levels of GSH, which detoxifies NAPQI and prevents oxidative injury to the liver (24), did not differ between fasted WT and IL-13 KO mice before and after APAP treatment (Figure 4). Taken together, these findings suggest that pathologic events downstream of APAP bioactivation, or possibly independent of APAP bioactivation, play a role in the enhanced susceptibility of IL-13 KO mice to AILD. IL-13 is known to down-regulate IFN-γ-inducing proinflammatory cytokines IL-12 and indirectly affect IL-18 (30, 39, 40). Early increases in these cytokines (Figure 5G and J) may be responsible for the observed increased serum levels of IFN-γ in APAP-treated IL-13 KO mice relative to control mice (Figure 5F). IFN-γ has been shown to play a critical, injurious role in AILD in WT mice, as a result of proinflammatory activities, which can lead to, among others, increased hepatic expression of chemokines, adhesion molecules, Fas-ligand, and NO, as well as infiltration of hepatotoxic neutrophils (9, 12). In the present study, IFN-γ also appeared to play a protoxic role in APAPtreated IL-13 KO mice because IFN-γ NAb partially protected IL-13 KO mice from AILD (Figure 6D). Moreover, because liver NK and NKT cells are major sources for hepatic IFN-γ (12) (Figure 8B) and contribute to the pathology of AILD in both WT (9, 12) and IL-13 KO mice (Figure 8A), these findings suggest, but do not definitively demonstrate, that IL-13 may modulate IFN-γ synthesis in these cells and thereby regulate susceptibility to AILD.



Figure 9. Role of PMNs in APAP-treated IL-13 KO mice. Representative photomicrographs of liver sections immunochemically stained for PMNs. APAP (200 mg/kg) or SAL was administered to either WT or IL-13 KO mice. Livers were removed from mice 8 h after APAP or SAL, fixed in formalin, and processed for PMN immunohistochemistry. PMN presence was determined through positive immunochemical staining associated with the morphological features of PMNs. (A) Liver from a SAL-treated IL-13 KO mice exhibited normal liver architecture with no evidence of inflammatory infiltrates. Similar histology was observed in livers from SAL-treated WT mice (photomicrograph not shown). (B) Liver from an APAP-treated WT mouse. Moderate centrilobular coagulative necrosis with mild to moderate PMN accumulation (black arrow) was observed. (C) Liver from an APAP-treated IL-13 KO mouse. Marked to severe centrilobular coagulative necrosis along with moderate PMN accumulation was apparent in the lesion area. In a separate study, PMN NAb (250 µg) or CAb was administered 48 h before administration of APAP (200 mg/kg) or SAL to IL-13 KO mice. Livers were removed from mice 24 h after APAP or SAL, fixed in formalin, and processed for PMN immunohistochemistry. (D) Liver from a CAb/APAP-treated IL-13 KO mouse exhibited severe centrilobular coagulative necrosis along with moderate to marked PMN accumulation in the lesion area. (E) PMN accumulation was greatly reduced or absent in the APAP-induced liver lesion from a NAb/APAP-treated IL-13 KO mouse, which exhibited moderate to marked centrilobular coagulative necrosis along with moderate hydropathy at the peripheral of the liver lesion. CV, central vein; bar ) 50 µm. (F) PMN NAb protected IL-13 KO from liver injury at 8 and 24 h after APAP treatment. Serum ALT activities for Cab/SAL- and PMN NAb/SAL-treated groups were 17 and 15 U/L, respectively. Results represent the mean ( SEM of 3-9 animals per group. (a) Significantly different from respective SAL-treated groups at the same time. (b) Significantly different from CAb/APAP group at the same time.


Elevated levels of various chemokines known to attract and activate PMNs were observed in greater concentration in the sera of IL-13 KO mice in comparison to WT mice after APAP treatment. These chemokines include KC (Figure 5C), MIP-2 (Figure 5D), OPN (Figure 5E), eotaxin (Figure 5K), and GMCSF (Figure 5L) (41-45), which have all been reported to be negatively regulated by IL-13, except for eotaxin (34, 46, 47). In this regard, PMNs accumulated to a greater extent in the livers of APAP-treated IL-13 KO mice than WT mice 8 h postAPAP administration (Figure 9C and B, respectively). Moreover, when APAP-treated IL-13 KO mice were pretreated with a PMN NAb, hepatic PMN accumulation was greatly attenuated and susceptibility to AILD decreased (Figure 9D-F). Similar results have been previously reported in APAP-treated WT mice (13, 14), although the exact role of PMNs in AILD in WT mice is controversial (48, 49). While additional PMN studies are needed in APAP-treated IL-13 KO mice, overall, these findings suggest that IL-13 may have a regulatory role in modulating the infiltration of cytotoxic PMNs into the liver following AILD. Uncontrolled NO production can cause cellular injury and dysfunction largely through the direct nitrososation of tissue proteins and other macromolecules or indirectly by reacting with superoxide to form injurious peroxynitrite, a powerful oxidizing agent, which, for example, can nitrate protein tyrosine residues (50, 51). IL-13 regulates NO levels by inhibiting iNOS expression and activity (52) and by up-regulating arginase type 1, which metabolizes L-arginine into urea and ornithine, thereby preventing the conversion of arginine into NO by iNOS (53, 54). Consequently, the elevated levels of serum nitrite-nitrate in APAP-treated IL-13 KO mice (Figure 5I) may be accounted for by the lack of IL-13 negative regulation of NO synthesis (53, 54) and/or by elevated levels of iNOS-inducing cytokines, particularly IFN-γ (Figure 5F) (9), which showed a trend toward an increase in serum as early as 4 h and was significant by 8 h after APAP treatment in IL-13 KO mice. Furthermore, because AG attenuated both serum nitrite-nitrate levels (Figure 7A) and liver injury (Figure 7B), these results suggest that NO may play a contributory role in the increased susceptibility of IL-13 KO mice to AILD. A similar pathologic role for NO in AILD was previously reported in studies with mice deficient in IL-10, a cytokine that also negatively regulates NO levels, but not in APAP-treated WT mice wherein IL-10 was expressed (5). TNF-R, which is negatively regulated by IL-13 (30), is known to have diverse injurious effects, including priming Kupffer cells and PMNs and sensitizing hepatic parenchymal cells to injury (55, 56). In the present study, however, TNF-R NAb failed to attenuate liver injury in APAP-treated IL-13 KO mice (Figure 6B). This result is consistent with similar studies involving either TNF-R KO mice or TNF-R NAb (57, 58), wherein it was concluded that TNF-R does not play a significant role in potentiating AILD in WT mice. Elevated serum levels of IL-10 (Figure 5H) and IL-6 (Figure 5B) in APAP-treated IL-13 KO mice are possibly compensatory responses to enhanced AILD, since IL-10 (5) and IL-6 (6) both appear to protect WT mice from AILD, as did IL-6 in APAPtreated IL-13 KO mice (Figure 6E). Increased levels of these cytokines, however, were unable to completely counteract the loss of hepatoprotective IL-13 in IL-13 KO mice, suggesting that IL-13 may have a nonredundant role in protecting the liver from AILD. In summary, endogenous IL-13 is a critical hepatoprotective factor in AILD. Our findings suggest that endogenous IL-13 may protect against AILD by negatively regulating hepatic protoxicants including cytokines, chemokines, NO, cytotoxic

Yee et al.

NK and NKT cells, and PMNs. Additional studies, however, will be needed to further elucidate this mechanism. Moreover, it remains to be determined whether IL-13 deficiency, perhaps as a consequence of IL-13 polymorphisms (59, 60) and/or antiIL-13 immunotherapy in asthma or other disease processes (6163), may be a risk factor in humans for AILD and possibly DILD caused by other drugs. Acknowledgment. We thank Drs. Neil R. Pumford and Jack A. Hinson (University of Arkansas) for providing anti-serum for the detection of APAP-protein adducts, Dr. Dennis R. Koop (Oregon Health Science University) for the anti-CYP2e1, Dr. David J. Shealy (Centocor, Inc.) for the anti-TNF-R, and Dr. Thomas A. Wynn (NIAID, NIH, Bethesda, MD) for the generous gift of IL-13-/- mice. We are also grateful to John W. George and Jose Flores (NHLBI, NIH, Bethesda, MD) for expert animal care and technical assistance. This research was supported by the Intramural Research Program of the NIH and the NHLBI.

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Hepatoprotective Role of Endogenous Interleukin-13 in a Murine

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