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Published online before print October 13, 2003
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* Division of Molecular Bioregulation, Cancer Research Institute, and
Department of Forensic & Social Environmental Medicine, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan;
Department of Forensic Medicine, Wakayama Medical University, Japan; and
Department of Pathology, Toyama Medical and Pharmaceutical University Hospital, Japan
1Correspondence: Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-0934, Ishikawa, Japan. E-mail: naofumim{at}kenroku.kanazawa-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-). However, it still remains elusive regarding the roles of TNF- in APAP-induced liver injury. Hence, we examined pathogenic roles of the TNF-–TNF receptor with a molecular weight of 55 kDa (TNF-Rp55) axis in APAP-induced hepatotoxicity using TNF-Rp55-deficient [TNF-Rp55-knockout (KO)] mice. When wild-type (WT) BALB/c and TNF-Rp55-KO mice were intraperitoneally injected with a lethal dose of APAP (750 mg/kg), the mortality of TNF-Rp55-KO mice was marginally but significantly reduced compared with WT mice. Upon treatment with a nonlethal dose (600 mg/kg), WT mice exhibited an increase in serum transaminase levels. Histopathologically, centrilobular hepatic necrosis with leukocyte infiltration was evident at 10 and 24 h after APAP challenge. Moreover, mRNA expression of adhesion molecules, several chemokines, interferon-
(IFN-), and inducible nitric oxide synthase (iNOS) was enhanced in the liver. On the contrary, serum transaminase elevation and histopathological changes were attenuated in TNF-Rp55-KO mice injected with APAP (600 mg/kg). The gene expression of all molecules except for IFN- and iNOS was significantly attenuated in TNF-Rp55-KO mice. Moreover, anti-TNF- neutralizing antibodies alleviated liver injury when administered at 2 or 8 h after but not at 1 h before APAP challenge to WT mice. Collectively, the TNF-–TNF-Rp55 axis has pathogenic roles in APAP-induced liver failure.
Key Words: drug-induced hepatotoxicity • TNF- • cytokines • chemokines • inducible nitric oxide synthase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN-)-deficient mice were highly resistant to APAP-induced hepatotoxicity with attenuated intrahepatic leukocyte infiltration [5
]. Thus, inflammatory reactions may have roles in the establishment of APAP-induced liver injury.
Tumor necrosis factor (TNF-) is a pleiotropic cytokine produced by a variety of cell types including macrophages, T cells, mast cells, and keratinocytes [6
]. TNF- has two distinct receptors: TNF receptor with a molecular weight of 55 kDa (TNF-Rp55) and 75 kDa (TNF-Rp75) [7
,8
]. There is the homology of 30% between these two receptors at the amino acid level in their extracellular, cysteine-rich, and ligand-binding regions. TNF-Rp55-deficient [TNF-Rp55-knockout (KO)] mice were resistant to lipopolysaccharide (LPS)- or staphylococcal enterotoxin-induced shock but were highly susceptible to infection Listeria monocytegenes [9
,10
], whereas TNF-Rp75-deficient mice were less sensitive to these treatments. These results suggest that TNF-Rp55 may have more profound roles in acute inflammation. Moreover, TNF-Rp55-KO mice exhibited less leukocyte infiltration in skin wound-healing [11
], acute liver failure induced by the combination of Propionibacterium acnes and bacterial LPS [12
], and liver fibrosis by dimethylnitrosamine [13
], suggesting the involvement of TNF-Rp55 in leukocyte infiltration.
Several lines of observations demonstrated that TNF- was produced after APAP challenge [14
,15
]. However, it remains elusive regarding its roles in the establishment of APAP-induced liver injury. Blazka et al. [14
,15
] demonstrated that TNF- was released in APAP intoxication and was responsible for pathological manifestations of APAP-induced liver injury. On the contrary, Simpson and colleagues [16
] demonstrated that anti-TNF- antibodies were ineffective to alleviate APAP-induced liver injury when they were administered 1 h before APAP challenge. Moreover, Boess and colleagues [17
] observed that there was no difference in the severity of APAP-induced liver injury between TNF-/lymphotoxin (LT)- double-KO and control mice. Hence, we herein examined the roles of TNF-, particularly focusing on TNF-Rp55 in APAP-induced liver injury, by using TNF-Rp55-KO mice. We demonstrated that TNF-Rp55-KO mice were resistant to liver injury induced by APAP.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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pAb, goat anti-mouse TNF-Rp55 pAb (Santa Cruz Biotechnology, Santa Cruz, CA), cyanine dye (cy)3-conjugated donkey anti-rabbit immunoglobulin G (IgG) pAb, cy3-conjugated donkey anti-rat IgG pAb, and fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG pAb (Jackson Immunoresearch Laboratories, West Grove, PA). For immunoneutralization, rabbit anti-mouse TNF- IgG was prepared as described previously [18
]. IgG (1 µg) completely neutralized the biological activities of 1 ng mouse TNF- on the fibroblast cytotoxicity assay (data not shown).
Mice
Pathogen-free, 8-week-old male BALB/c mice were obtained from Sankyo Laboratories (Tokyo, Japan) and were designated as wild-type (WT) mice in the present experiments. TNF-Rp55-KO mice were backcrossed to BALB/c mice for eight to 10 generations [10
] and bred under specific pathogen-free conditions at the Animal Research Center of Kanazawa University (Japan). Eight-week-old male TNF-Rp55-KO mice were used for the experiments. All animal procedures in this study complied with the Guidelines for the Care and Use of Laboratory Animals on the Takara-machi Campus of Kanazawa University.
APAP-induced liver injury
APAP solution was made immediately before each experiment by dissolving the compound in phosphate-buffered saline (PBS; pH 7.2) and was warmed to 37°C. In all experiments, mice were allowed free access to water but not food for 10 h before an intraperitoneal (i.p.) injection with APAP at a dose of 600 (a nonlethal dose) or 750 mg/kg (a lethal dose). In another series of experiments, WT mice received 250 µg neutralizing anti-TNF- Ab or control rabbit IgG i.p. at 1 h before or at 2 or 8 h after the administration of APAP (600 mg/kg). Every experiment was repeated at least three times, and each group consisted of at least six mice.
Determination of serum alanine aminotransferase (ALT) levels
Whole blood samples were collected at the indicated time intervals after APAP injection (600 mg/kg) to determine the serum ALT levels with a Fuji DRI-CHEM 5500V as instructed by the manufacturer (Fuji Medical System, Tokyo, Japan).
Histological and immunohistochemical analysis
To obtain liver tissues, the mice were killed at the indicated time intervals after 600 mg/kg APAP challenge. Liver tissues were fixed in 4% formaldehyde buffered with PBS (pH 7.2) and then embedded with paraffin. Sections (6-µm thick) were stained with hematoxylin and eosin. Immunohistochemical analysis was performed using anti-MPO, anti-F4/80, and anti-CD3 Ab to detect neutrophils, macrophages, and T cells, respectively, as described previously [11
,19
]. Deparaffinized sections were immersed in 0.3% H2O2 in methanol for 30 min to eliminate endogenous peroxidase activities. The sections were further incubated with PBS containing 1% normal serum derived from the same species as those used for preparation of the secondary Ab and 1% bovine serum albumin (BSA) to reduce nonspecific reactions. The sections were incubated with anti-MPO, anti-F4/80, or anti-CD3 Ab at a concentration of 1 µg/mL at 4°C overnight. After the incubation with biotinylated secondary Ab (2.5 µg/mL) at room temperature for 30 min, immune complexes were visualized using a catalyzed signal amplification system (Dako, Kyoto, Japan) according to the manufacturer’s instructions. The exclusion of the first antibodies did not give rise to any positive reaction (our unpublished results) indicating the specificities of the reactions.
Double-color immunofluorescence analysis
A double-color immunofluorescence analysis was also performed to determine the types of TNF- or TNF-Rp55-expressing cells in the liver of mice. Deparaffinized sections were incubated with PBS containing 1% normal donkey serum and 1% BSA to reduce nonspecific reactions. Thereafter, the sections were further incubated in pairs of anti-TNF- and anti-F4/80, anti-TNF- and anti-MPO, anti-TNF-Rp55 and anti-F4/80, or anti-TNF-Rp55 and anti-MPO Ab at a concentration of 1 µg/mL at 4°C overnight. After incubation with fluorochrome-conjugated secondary Ab (15 µg/mL) at room temperature for 1 h, the sections were observed under a fluorescence microscopy. The exclusion of the first antibodies did not give rise to any fluorescence (our unpublished results), indicating the specificities of the reactions.
Measurements of leukocyte infiltration
The numbers of neutrophils, macrophages, and T cells in the liver were enumerated by a pathologist (K. Tsuneyama), without a prior knowledge about the experimental procedures, on 10 randomly chosen visual fields (original magnification, x400) of the sections stained with anti-MPO, anti-F4/80, and anti-CD3 Ab, respectively. Then, the average of 10 selected microscopic fields was calculated as described previously [5
].
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from liver homogenates using Isogen (Nippon Gene, Toyama, Japan), according to the manufacturer’s instructions. Total RNA (5 µg) was then reverse-transcribed into cDNA using the Superscript II RT (Invitrogen Life Technologies, Tokyo, Japan) with oligo (dT) primers (Invitrogen Life Technologies). Serially two-fold-diluted cDNA products were amplified for ß-actin with Taq polymerase (Nippon Gene) using specific sets of primers and experimental conditions (Table 1
) to evaluate the amount of the transcribed cDNAs. Thereafter, equal amounts of cDNAs were amplified, using specific primers and experimental conditions as shown in Table 1
. Nonradioisotopic, a semiquantitative RT-PCR, was performed as described previously [11
,19
]. To determine the optimal cycle number and annealing temperature, we performed PCR on each molecule by increasing PCR cycle numbers from 20 to 40 by two at different annealing temperatures. The PCR products were then fractionated on 2% agarose gels containing 0.3% ethidium bromide, and the band intensity was measured using a charge-coupled device imaging system (GelDoc 2000, Bio-Rad, Hercules, CA) and NIH Image Analysis Software Ver. 1.61 (National Institutes of Health, Bethesda, MD). The band intensities were plotted against cycle numbers on semilogarithmic graphs. The number of PCR cycles, where fluorescence intensity of PCR products increased exponentially, was determined as the optimal cycle for each molecule. The intensities of the bands were determined with the use of NIH image analysis software, and the ratios to ß-actin were determined. To standardize the condition of gel staining, a constant amount of control DNA marker was electrophoresed every time. The PCR procedure was performed at least three times for each sample.
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and MIP-1 levels were measured with commercial ELISA kits (TNF-, BioSource, Inc., Camarillo, CA; MIP-1, Quantikine M, R&D Systems, Minneapolis, MN), according to the manufacturer’s recommendation. MCP-1 and MIP-2 levels were measured by ELISA as described previously [20
]. The detection limits in each method were as follows: TNF- > 5 pg/mL; MIP-1 > 1.5 pg/mL; MCP-1 > 10 pg/mL; and MIP-2 > 10 pg/mL. Total protein in the supernatant was measured with a commercial kit (BCA protein assay kit, Pierce, Rockford, IL) using BSA as a standard. The data were expressed as the target molecule (pg/mL)/total protein (mg/mL) for each sample.
Statistical analysis
The means and SEs were calculated for all parameters determined in this study. Statistical significance was evaluated using ANOVA or Mann-Whitney’s U test. P< 0.05 was accepted as statistically significant. The survival curve by the Kaplan-Meier procedure was analyzed by a log-rank test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, TNF-Rp55, and -Rp75 in the liver after APAP challenge mRNA expression was also faintly detected in liver in WT and TNF-Rp55-KO mice. APAP (600 mg/kg) administration significantly enhanced the gene expression of TNF- at 10 and 24 h in WT and TNF-Rp55-KO mice, although the increases were more prominent in WT mice than KO mice (Fig. 2a
). Concomitantly, intrahepatic TNF- protein contents began to increase in WT mice later than 4 h, reached the maximum level at 10 h, and still remained at similar levels at 24 h after APAP challenge (Fig. 2b)
. However, the enhanced TNF- expression was significantly attenuated at the protein as well as mRNA levels in TNF-Rp55-KO mice (Fig . 2a
and 2b)
. A double-color immunofluorescence analysis revealed that neutrophils and hepatic macrophages were cellular sources of TNF- (Fig. 2c)
. These observations suggest that APAP induced TNF- production in the liver and that the TNF-–TNF-Rp55 axis may have an important role by an autocrine and/or paracrine manner in APAP-induced liver injury.
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-KO mice treated even with a lethal dose of APAP [5
].
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, IL-1ß, IL-6, IFN-, monocyte MCP-1, MIP-1, MIP-2, KC, and iNOS was faintly detected in the liver of untreated WT and TNF-Rp55-KO mice (Fig. 5a
). In WT mice, gene expression of all these molecules was enhanced in the liver tissue after APAP challenge. On the contrary, in TNF-Rp55-KO mice, the enhanced gene expression of these adhesion molecules, cytokines, and chemokines except for IFN- and iNOS was significantly attenuated at 10 and/or 24 h after APAP challenge, compared with WT mice (Fig . 5b
5c
5d
5e
5f
5g
5h
5i
5j
5k)
. The gene expression of IFN- and iNOS was enhanced in the liver of TNF-Rp55-KO mice to a similar extent as WT mice after APAP administration, suggesting that the expression of both genes can be enhanced independently of TNF-Rp55 (Fig . 5l
and 5m)
.
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, and MIP-2 between untreated WT and TNF-Rp55-KO mice (Fig. 6
). After APAP administration, these protein levels were markedly increased in the liver of WT mice. Consistent with results obtained with RT-PCR, intrahepatic protein levels of MCP-1, MIP-1, and MIP-2 were increased but to a lesser degree in TNF-Rp55-KO mice at 10 and 24 h after APAP challenge, when compared with WT mice (Fig. 6)
.
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antibodies in the APAP-induced liver injury antibodies on established APAP-induced liver injury. Administration of anti-TNF- antibodies at 1 h before APAP challenge failed to prevent a significant increase in serum ALT levels. On the contrary, when WT mice were injected with anti-TNF- antibodies at 2 or 8 h after APAP challenge, serum ALT levels and hepatic histopathological changes were significantly attenuated, compared with those treated with normal rabbit IgG (Fig. 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-KO mice exhibited a reduction in liver injury with a concomitant, reduced, intrahepatic leukocyte infiltration after APAP treatment [5
]. Accumulating evidence suggests that the TNF-–TNF-Rp55 axis has essential roles in leukocyte infiltration by enhancing the expression of chemokines and adhesion molecules [11
,25
]. Moreover, TNF-Rp55 deficiency resulted in diminished leukocyte infiltration in various liver disease models [12
,13
]. Also in APAP-induced liver injury, the lack of TNF-Rp55 resulted in reduced expression of chemokines and adhesion molecules, thereby reducing leukocyte infiltration and subsequent hepatic injury. Thus, TNF-Rp55 is presumed to have crucial roles in intrahepatic leukocyte infiltration even in this type of hepatic injury.
As TNF-Rp55 is a receptor for TNF- and LT-, our present observations suggest the involvement of these cytokines in APAP-induced hepatotoxicity. However, Boess and colleagues [17
] reported that TNF-/LT- double-KO mice on a C57BL/6 background exhibited APAP-induced hepatic injury to a similar extent as WT mice. We observed that TNF-Rp55-KO mice on a BALB/c background were more resistant to APAP-induced liver injury than WT mice. Moreover, Boess and his colleagues [17
] administered a lower dose of APAP in their experiments, compared with ours. Thus, the discrepancy may be explained by the differences in the APAP doses or the used mouse strain. In addition, we observed that serum ALT levels were maximal in WT mice at 10 h after APAP treatment. On the contrary, Boess [17
] and his colleagues determined serum ALT levels and pathological changes only at 4 and 8 h after APAP challenge, when liver damage might not be maximal. Moreover, they observed discernible but insufficient differences in serum ALT levels and pathological changes between WT and TNF-/LT- double-KO mice even at these time points. Thus, it is more probable that the differences in the time points to evaluate liver injury may account for the discrepancy.
TNF- was produced in liver after APAP administration. However, Simpson and colleagues [16
] claimed that TNF- had little roles in the development of APAP-induced liver injury, based on their observation that pretreatment of blocking anti-TNF- antibodies failed to alleviate APAP-induced hepatic injury. We also observed that pretreatment with neutralizing anti-TNF- antibodies had few effects on APAP-induced liver injury. Unexpectedly, when mice were treated with anti-TNF- antibodies at 2 or 8 h after APAP challenge, the liver injury was attenuated significantly. We observed that TNF- protein expression was enhanced in liver later than 4 h after APAP treatment and reached a peak at 10 h. These results would indicate that TNF- was a mediator of the later phase of APAP hepatotoxicity. Thus, the post-treatment of the antibodies might be more effective for the prevention of the hepatic injury than its pretreatment. As N-acetyl cysteine, a single effective drug for APAP-induced liver injury, is effective only when it is administered after APAP ingestion, anti-TNF- antibodies may be an adjunctive drug for APAP-induced liver dysfunction.
NO is presumed to be one of the essential mediators in APAP-induced liver injury [26
]. Nakae and colleagues [27
] demonstrated that liposome-encapsulated superoxide dismutase prevented liver necrosis by APAP, suggesting the involvement of reactive oxygens including NO in APAP-induced liver injury. Moreover, we previously observed that APAP treatment failed to enhance iNOS expression in IFN--KO mice [5
]. Furthermore, a NO scavenger and a NOS inhibitor effectively prevented APAP-induced liver injury [5
,26
]. These observations suggest that NO generated by iNOS is a crucial mediator of APAP-induced liver injury. Several lines of evidence demonstrated that TNF- can induce iNOS expression in vitro in various types of cells including liver macrophages and eventually the generation of a massive amount of NO [28
,29
,30
]. However, TNF-Rp55-KO mice exhibited an enhanced iNOS expression to similar levels as WT mice. Moreover, IFN- gene expression was enhanced in TNF-Rp55-KO mice to a similar extent as WT mice. Collectively, IFN- but not TNF-Rp55 ligands may be a main regulator of iNOS expression in the liver during the course of APAP-induced liver injury, and the absence of IFN- but not TNF-Rp55 ligands abrogated iNOS expression and subsequent NO generation. Thus, these differences may explain why IFN--KO mice were more resistant to APAP than TNF-Rp55-KO mice, even at its lethal dose [5
].
On the contrary, anti-IFN- antibodies were less effective than anti-TNF- antibodies to reduce APAP hepatotoxicity when administered at 8 h after APAP administration. We previously observed that TNF- expression was not enhanced in APAP-treated IFN--KO mice [5
]. Moreover, IFN- mRNA expression was enhanced rapidly and returned to a basal level at 6 h after APAP administration, when TNF- mRNA expression reached a maximal level (our unpublished results). Thus, it is likely that IFN- is one of an initiating mediator(s) in APAP hepatotoxicity and induces effector molecules including TNF- and iNOS, which are involved in the subsequent development of hepatic injury. Therefore, the differences in time kinetics of expression may account for the different efficacy between anti-IFN- and anti-TNF- antibodies.
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ACKNOWLEDGEMENTS |
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Received April 15, 2003; revised September 12, 2003; accepted September 15, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the pathogenesis of acetaminophen-induced acute liver injury FASEB J 16,1227-1236 and tumor necrosis factor Toxicol. Pathol. 24,181-189[Medline]
gene knockout mice Hepatology 27,1021-1029[CrossRef][Medline]
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