The Role of Kupffer Cells and TNF-{alpha} in Monocrotaline and Bacterial Lipopolysaccharide-Induced Liver Injury

Steven B. Yee, Patricia E. Ganey and Robert A. Roth1

Department of Pharmacology and Toxicology, National Food Safety and Toxicology Center and Institute for Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824

Received July 30, 2002; accepted October 10, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Coexposure to small, noninjurious doses of the pyrrolizidine alkaloid phytotoxin monocrotaline (MCT) and bacterial lipopolysaccharide (LPS) results in synergistic hepatotoxicity. Both centrilobular and midzonal liver lesions occur and are similar to those seen from large, toxic doses of MCT and LPS, respectively. The nature of the lesions in vivo and results from studies in vitro suggest that injury is mediated indirectly rather than from a simple interaction of MCT and LPS with hepatic parenchymal cells. Accordingly, the role of inflammatory factors, such as Kupffer cells and TNF-{alpha}, in the development of MCT/LPS-induced liver injury was investigated. In Sprague-Dawley rats, MCT (100 mg/kg, ip) was administered 4 h before LPS (7.4 x 106 EU/kg, iv). Pretreatment of these animals with gadolinium chloride, an inhibitor of Kupffer cell function, attenuated liver injury 18 h after MCT administration. An increase in plasma TNF-{alpha} preceded the onset of hepatic parenchymal cell injury, raising the possibility that this inflammatory cytokine contributes to toxicity. Either pentoxifylline, an inhibitor of cellular TNF-{alpha} synthesis, or anti-TNF-{alpha} serum coadministered to MCT/LPS-treated animals significantly attenuated liver injury. These results suggest that Kupffer cells and TNF-{alpha} are important mediators in the synergistic hepatotoxicity resulting from MCT and LPS coexposure.

Key Words: liver; endotoxin; monocrotaline; Kupffer cell; TNF-{alpha}; inflammation; gadolinium chloride; pentoxifylline; COX-2; NS-398.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocrotaline (MCT) is a pyrrolizidine alkaloid phytotoxin that is well known for producing hepatic toxicity in both humans and animals (Mattocks, 1986Go; Stegelmeier et al., 1999Go). MCT must be bioactivated to monocrotaline pyrrole (MCTP) for liver injury to occur (Stegelmeier et al., 1999Go; White and Mattocks, 1972Go). At acutely toxic doses, MCT-induced liver lesions are characterized by centrilobular hepatocellular necrosis, dilated and congested sinusoids, hemorrhage, and injured central venous and sinusoidal endothelial cells (SECs) (DeLeve et al., 1999Go; Schoental and Head, 1955Go; Yee et al., 2000Go).

Bacterial lipopolysaccharide (LPS), a constituent of the outer cell wall of Gram-negative bacteria, is a potent inflammagen (Holst et al., 1996Go). In rats, liver injury from a large, toxic dose of LPS is characterized by neutrophil infiltration with hepatocellular degeneration and coagulative necrosis in midzonal regions of liver lobules (Hewett et al., 1992Go; Yee et al., 2000Go). The mechanism for LPS-induced liver injury is complex and involves the interaction of numerous inflammatory cells and soluble mediators, including Kupffer cells (KCs) and tumor necrosis factor-{alpha} (TNF-{alpha}) (Brouwer et al., 1995Go; Brown et al., 1997Go; Ganey and Roth, 2001Go; Hewett et al., 1992Go, 1993Go).

Exposure to a smaller LPS dose results in a modest, noninjurious inflammatory response (Ganey and Roth, 2001Go; Michie, 1988Go; Spitzer and Mayer, 1993Go). It has been hypothesized that the release of inflammatory mediators has the potential to alter tissue homeostasis and increase the susceptibility of tissues to chemical-induced injury (Ganey and Roth, 2001Go). Yee et al. (2000)Go recently demonstrated that synergistic liver injury resulted when a noninjurious dose of LPS was administered 4 h after a small, nonhepatotoxic dose of MCT. The resulting liver lesions were both centrilobular and midzonal, exhibiting characteristics similar to lesions associated with larger, toxic doses of MCT or LPS, respectively. Failure to reproduce synergistic injury in isolated hepatic parenchymal cells (HPCs) in vitro suggested that the enhanced toxicity resulted not from direct interaction of MCT and LPS with HPCs, but rather from an indirect mechanism (Yee et al., 2000Go). Inasmuch as LPS is an inflammagen, the present study was designed to identify inflammatory factors critical to the synergistic hepatotoxicity in this model.

KCs are the resident macrophages of the liver. When activated by LPS, they produce and release numerous mediators, including cytokines (TNF-{alpha}, interleukin-1, and interleukin-6) and reactive oxygen species (Holst et al., 1996Go; Michie et al., 1988Go). Cyclooxygenase-2 (COX-2) is also induced, leading to the enhanced synthesis of prostaglandins (PGs) that have proinflammatory actions (Brouwer et al., 1995Go; Dieter et al., 1999Go). Many of these mediators further activate or modulate the effects of nearby cells involved in the inflammatory process and can contribute to the development of injury (Hewett et al., 1993Go; Holst et al., 1996Go). Inactivation of KCs with gadolinium chloride (GdCl3) in rats given a large, hepatotoxic dose of LPS significantly attenuates hepatocellular necrosis, suggesting that KCs are critical to the pathogenesis of LPS-induced injury (Brown et al., 1997Go).

TNF-{alpha} is a potent inflammatory cytokine produced by activated macrophages and to a lesser degree by other cell types (Bradham et al., 1998Go; Vassalli, 1992Go). This cytokine can exert a variety of effects on cells ranging from mitochondrial damage and oncotic or apoptotic necrosis to cell proliferation (Bradham et al., 1998Go). TNF-{alpha} may also prompt the accumulation of neutrophils (PMNs) by activating endothelial cells (Bradham et al., 1998Go; Vassalli, 1992Go). It can indirectly promote toxicity by priming PMNs to release reactive oxygen and nitrogen species and proteases that damage nearby cells (Nagaki et al., 1991Go; Vasselli, 1992). Inhibition of TNF-{alpha} synthesis or activity attenuates injury caused by a hepatotoxic dose of LPS, indicating that TNF-{alpha} is a critical factor in the pathogenesis (Hewett et al., 1993Go).

The purpose of this study was to investigate the role of inflammatory factors in the synergistic liver injury that occurs following coexposure to small doses of MCT and LPS. Specifically, the hypothesis that KCs and TNF-{alpha} play causal roles in the hepatotoxicity was tested. In addition, the importance of COX-2 products for the development of injury was evaluated.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
LPS (Escherichia coli, serotype 0128:B12, 1.7 x 106 endotoxin units (EUs)/mg), pentoxifylline, and sodium citrate were purchased from Sigma Chemical Company (St. Louis, MO). Serum directed against TNF-{alpha} (antirat TNF-{alpha} serum; ATS) was produced in New Zealand White rabbits (Hewett et al., 1993Go). GdCl3-6H2O was purchased from Aldrich Chemical Company (St. Louis, MO). The selective COX-2 inhibitor N-(2-cyclohexyloxy-4-nitro-phenyl)methane sulfonamide (NS-398) was acquired from Cayman Chemical Co. (Ann Arbor, MI). MCT was obtained from Trans World Chemicals (Rockville, MD). Sterile saline was acquired from Abbott Laboratories (North Chicago, IL). Formalin fixative was obtained from Surgipath Medical Industries, Inc. (Richmond, IL). Diagnostic kits 58 UV and 59 UV for the determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, respectively, were also purchased from Sigma (St. Louis, MO). The ELISA kit for hyaluronic acid was acquired from Corgenix, Inc. (Westminster, CO). The ELISA kit for rat TNF-{alpha} was purchased from Biosource International, Inc. (Camarillo, CA).

Animals.
Male Sprague-Dawley rats (Crl:CD (SD)IGS BR, Charles River, Portage, MI) weighing 200–300 g were used in all studies. Animals were allowed food (Rodent Chow/Tek 8640, Harlan Teklad, Madison, WI) and water ad libitum. They were housed no more than three to a cage on Aspen chip bedding (Northeastern Products Company, Warrenburg, NY) and were maintained on a 12-h light/dark cycle in a controlled temperature (18–21°C) and humidity (55 ± 5%) environment for a period of 1 week before use. All procedures on animals followed the guidelines for humane treatment set by the American Association of Laboratory Animal Sciences and the University Laboratory Animal Research Unit at Michigan State University.

Treatment protocol.
MCT was dissolved in sterile saline minimally acidified by 0.2 M HCl. The pH was brought to 7 by addition of 2 M NaOH, and the volume adjusted with sterile saline to the appropriate final concentration. Rats were given MCT (100 mg/kg) or an equivalent volume of sterile saline, intraperitoneally, followed 4 h later by LPS (7.4 x 106 EU/kg) or its saline vehicle via tail vein injection. LPS was administered 4 h after MCT to minimize interference with MCT bioactivation (Allen et al., 1972Go). As previously reported, these doses of MCT and LPS resulted in minimal to no injury by themselves. However, when they were administered together, synergistic liver injury resulted, with maximal injury occurring by 18 h (Yee et al., 2000Go).

At the times indicated in figure and table legends, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip). A midline abdominal incision was made, blood was collected from the inferior vena cava into a syringe containing sodium citrate (0.38% final concentration), and animals were euthanized by exsanguination. Livers were removed intact and fixed in 10% neutral buffered formalin for at least 3 days before being processed for histologic analysis.

Treatment with gadolinium chloride.
Rats were treated with 10 mg GdCl3-6H2O/kg (pH 3.5) or saline vehicle intravenously 24 h before the administration of LPS or its vehicle. This treatment protocol has been shown to inactivate KC function (Brown et al., 1997Go).

Treatment with pentoxifylline.
Rats received 100 mg PTX/kg or its saline vehicle intravenously 1 h before LPS treatment. This treatment regimen prevents the LPS-induced increase in plasma TNF-{alpha} activity (Barton et al., 2001Go).

Treatment with anti-TNF-{alpha} serum.
Rats were treated intravenously with rabbit ATS (1 ml) or control rabbit serum (CS; 1 ml) 1 h before LPS administration. This treatment protocol has been shown to prevent LPS-induced increase in plasma TNF-{alpha} activity (Barton et al., 2001Go; Hewett et al., 1993Go).

Treatment with NS-398.
NS-398 is a selective inhibitor of COX-2 (Futaki et al., 1994Go, 1997Go). Rats were given 5 mg NS-398/kg or olive oil intraperitoneally 1 h before LPS administration.

Assessment of liver injury, plasma TNF-{alpha}, and plasma hyaluronic acid.
HPC injury was estimated by increases in the activities of plasma ALT and AST. An ELISA was used to measure plasma hyaluronic acid (HA), a marker of hepatic SEC injury (Copple et al., 2002Go; Deaciuc et al., 1993Go, 1994Go). Plasma TNF-{alpha} concentration was determined via a rat TNF-{alpha} ELISA kit.

Histopathologic evaluation and morphometry.
Serial transverse sections from the left lateral liver lobe were processed for light microscopy. Paraffin-embedded sections were cut at 4 µm, stained with hematoxylin and eosin, and evaluated for lesion size and severity. Tissue sections were analyzed histologically without knowledge of the treatment group.

Digitized color images of hematoxylin- and eosin-stained liver sections were visualized with an Olympus AX-80T light microscope (Olympus Corp., Lake Success, NY) interfaced with a high-resolution CCD color camera (OLY-750, Olympus-America, Inc., Melville, NY) to quantify treatment-induced changes in liver morphology. Images were evaluated with Scion Image software (Scion Corporation, Frederick, MD) employing a 64-point lattice grid to determine (1) the total area of liver analyzed, (2) the area of centrilobular lesion, (3) the area of midzonal lesion, (4) the area of normal parenchyma, and (5) the area of nonparenchymal space. A lesion was defined as hepatic parenchymal cells with either swollen, eosinophilic cytoplasm and karyolytic or pyknotic nuclei (i.e., oncosis), or cells with shrunken cytoplasm and karyorrhexic nuclei or apoptotic bodies (i.e., apoptosis; Levin et al., 1999Go; Majno and Joris, 1995Go). Nonparenchymal space was defined as nonparenchymal tissue, vessel lumen, and regions outside the perimeter of the liver section. The area of each object (category) of interest (i.e., lesion) was calculated from the following expression (Cruz-Orive, 1982Go):



Distance between points was 55 µm. Accordingly, the area represented by each point was 3025 µm2. One section from the liver of each animal in a treatment group was systematically scanned using adjacent, nonoverlapping microscopic fields. The first image field analyzed in each section was chosen using a random number table (i.e., any image field between 1 and 10). Thereafter, every 10th field containing hepatic parenchymal cells was evaluated (minimum of 20 fields measured/section). The measured fields represented approximately 10% of the total area of each liver section. Eight animals per group were analyzed. Percentage lesion area was estimated based on the following formula:


Statistical analysis.
Results are expressed as mean ± SEM. Data expressed as percentages were transformed by arc sine square root prior to analysis. Data for single comparisons were analyzed by Student's t-test or, when appropriate, Fisher's exact test (Steele et al., 1997Go). Homogeneous data were analyzed by one- or two-way analysis of variance (ANOVA), as appropriate, and group means were compared using Student-Newman-Keuls post hoc test (Steele et al., 1997Go). When variances were not homogeneous, data were analyzed using the Kruskal-Wallis nonparametric ANOVA, and Dunn's Multiple Comparison test was used to compare group means (Steele et al., 1997Go). The criterion for significance was p <= 0.05 for all comparisons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of GdCl3 on MCT/LPS-Induced Liver Injury
To test the hypothesis that KCs are important for the development of liver injury in the MCT/LPS model, rats were pretreated with the KC inactivator GdCl3 at a dose that inhibits KC-mediated phagocytosis (Brown et al., 1997Go). As further verification of GdCl3 efficacy, plasma TNF-{alpha} concentration was monitored (Fig. 1AGo). Plasma TNF-{alpha} concentration was elevated in the MCT/LPS-cotreated animals, and this increase was reduced with GdCl3 pretreatment. Plasma TNF-{alpha} concentration was not elevated in animals receiving either vehicles or GdCl3 alone.



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FIG. 1. GdCl3 protects against MCT/LPS-induced liver injury. LPS (7.4 x 106 EU/kg) or saline vehicle (Veh) was administered, iv, to rats 4 h after ip administration of MCT (100 mg/kg) or saline vehicle. Rats were pretreated with 10 mg GdCl3-6H2O/kg or saline vehicle, iv, 24 h before LPS administration. TNF-{alpha} concentration (A), ALT (B) and AST (C) activities, and HA concentration (D) were evaluated in plasma 18 h after MCT administration. Data are expressed as mean ± SEM; n = 4 for Veh/Veh/Veh, 4 for GdCl3/Veh/Veh, 14 for Veh/MCT/LPS, and 13 for GdCl3/MCT/LPS. (a) Significantly different from respective value in the absence of MCT/LPS; (b) significantly different from Veh/MCT/LPS group.

 
Plasma ALT activity was elevated in MCT/LPS-cotreated animals (Fig. 1BGo). GdCl3 pretreatment markedly attenuated this increase. Similar results were observed with plasma AST activity (Fig. 1CGo). Plasma HA concentration was also elevated in animals that received MCT/LPS cotreatment. This increase was attenuated slightly but significantly in GdCl3-pretreated rats (Fig. 1DGo). No elevated plasma ALT or AST activity or plasma HA concentration was observed in control animals that received vehicles or GdCl3 alone.

MCT/LPS-cotreated control animals exhibited centrilobular and midzonal liver lesions as previously described by Yee et al. (2000)Go. Centrilobular lesions consisted of moderate to marked hepatocellular apoptotic and oncotic necrosis, degeneration, hemorrhage, and vascular injury. Midzonal lesions in the MCT/LPS-cotreated group comprised marked but more frequent and well-defined areas of hepatocellular coagulative necrosis accompanied by neutrophil infiltration with congestion and hemorrhage. Liver lesions from animals treated with GdCl3/MCT/LPS exhibited qualitatively similar centrilobular and midzonal lesions; however, these lesions were both smaller in size and less frequent. The GdCl3-induced reduction in lesion size and frequency was more pronounced in the midzonal regions. Morphometric analysis of liver lesions supported the observation that GdCl3 pretreatment reduced the liver area affected by centrilobular and midzonal lesions (Table 1Go). Furthermore, the number of hepatic PMNs was smaller in MCT/LPS-treated animals that received GdCl3 pretreatment compared to MCT/LPS-treated animals that did not. Histologically, no evidence of liver injury was observed in animals treated with vehicles or GdCl3 alone.


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TABLE 1 Morphometric Analysis of Liver Lesions from MCT/LPS-Cotreated Rats following Administration of GdCl3, PTX, or ATS  GdCl3/MCT/LPSCentrilobular0.0 ± 0.0*
 
Plasma TNF-{alpha} Concentration
Plasma TNF-{alpha} concentration was assessed at various times after the administration of MCT or its saline vehicle (Table 2Go). It did not increase after Veh/Veh or MCT/Veh cotreatments at any of the times evaluated but was elevated 2 h after LPS administration, irrespective of MCT-treatment. Whereas plasma TNF-{alpha} concentration in Veh/LPS-cotreated animals returned to baseline within the next 8 h, plasma TNF-{alpha} concentration in MCT/LPS-cotreated animals remained modestly but significantly elevated 14 h after LPS administration.


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TABLE 2 Plasma TNF-{alpha} Concentration after MCT/LPS Administration in Sprague-Dawley Rats Time after MCT or vehiclePlasma TNF-{alpha} concentration (pg/ml)
 
Effect of PTX on Hepatotoxicity from MCT/LPS Cotreatment
PTX decreases the synthesis of TNF-{alpha} at the mRNA level (Dezube et al., 1993Go) and was used to determine whether TNF-{alpha} has a causal role in the development of liver injury. Control animals treated with vehicles or PTX alone did not have detectable plasma TNF-{alpha} concentrations at 18 h (Fig. 2AGo). The rise in plasma TNF-{alpha} concentration in MCT/LPS-treated control animals was markedly attenuated by PTX treatment.



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FIG. 2. Effect of PTX on MCT/LPS-induced liver injury. LPS (7.4 x 106 EU/kg) or its saline vehicle was administered, iv, to rats 4 h after ip administration of MCT (100 mg/kg) or its vehicle. PTX (100 mg/kg) or saline vehicle was given, iv, to rats 1 h before LPS. Plasma TNF-{alpha} concentration (A), ALT (B) and AST (C) activities, and HA concentration (D) were evaluated 18 h after MCT administration. Data are expressed as mean ± SEM; n = 4 for Veh/Veh/Veh, 4 for Veh/PTX/Veh, 13 for MCT/Veh/LPS, and 8 for MCT/PTX/LPS. (a) Significantly different from respective value in the absence of MCT/LPS; (b) significantly different from MCT/Veh/LPS group.

 
As determined by plasma ALT (Fig. 2BGo) and AST (Fig. 2CGo) activities, HPC injury was significantly reduced in animals that received PTX. Plasma HA concentration, however, was not significantly affected by PTX in MCT/LPS-treated animals (Fig. 2DGo). No elevated plasma ALT or AST activity or plasma HA concentration was observed in control animals that received vehicles or PTX alone.

Histological examination of the livers from animals that received the MCT/PTX/LPS treatment revealed lesions qualitatively similar to those observed in animals that received MCT/LPS alone, but they were smaller in size and less frequent. Midzonal lesions exhibited the greatest reduction in size and frequency and demonstrated less congestion and hemorrhage. These observations were consistent with the mophometric analysis of lesions in this study (Table 1Go). Hepatic PMN accumulation was also reduced by PTX treatment. No lesions were observed in livers from animals that were given vehicles or PTX alone.

Effect of Anti-TNF-{alpha} Serum on Liver Injury from MCT/LPS Cotreatment
MCT/LPS-cotreated animals were given ATS before LPS treatment to confirm the causal relationship between TNF-{alpha} and MCT/LPS-induced liver injury. ATS given to MCT/LPS-treated animals prevented the increase in plasma TNF-{alpha} concentration at 18 h (Fig. 3AGo).



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FIG. 3. Anti-TNF-{alpha} serum protects against hepatic injury from MCT/LPS-cotreatment. LPS (7.4 x 106 EU/kg) or saline vehicle was administered, iv, to rats 4 h after ip administration of MCT (100 mg/kg) or saline vehicle. A 1-ml dose of either ATS or CS was administered, iv, 1 h before LPS. Plasma TNF-{alpha} concentration (A), ALT (B) and AST (C) activities, and HA concentration (D) were evaluated 18 h after MCT administration. Data are expressed as mean ± SEM; n = 4 for Veh/CS/Veh, 4 for Veh/ATS/Veh, 8 for MCT/CS/LPS, and 5 for MCT/ATS/LPS. (a) Significantly different from respective value in the absence of MCT/LPS; (b) significantly different from MCT/Veh/LPS group.

 
ATS treatment before LPS administration markedly attenuated liver injury in MCT/LPS-cotreated animals. Plasma ALT (Fig. 3BGo) and AST (Fig. 3CGo) activities were significantly less in MCT/ATS/LPS-treated animals compared to controls that received MCT/CS/LPS. ATS treatment caused a small but significant reduction in plasma HA concentration in MCT/LPS-treated rats (Fig. 3DGo). No elevation in plasma ALT or AST activity or plasma HA concentration was observed in control animals that received vehicles or ATS alone.

Animals treated with MCT/ATS/LPS had liver lesions qualitatively similar to those in rats given MCT/CS/LPS, although the lesions were reduced in size and frequency. Midzonal liver lesions from MCT/ATS/LPS-treated animals exhibited less congestion and hemorrhage than livers from MCT/CS/LPS animals. Morphometric analysis of liver lesions supports the observation that ATS treatment lessened the centrilobular, MCT-like and midzonal, LPS-like lesions (Table 1Go). Furthermore, hepatic PMN accumulation was reduced in MCT/ATS/LPS-treated animals compared to animals given MCT/CS/LPS. No injury was observed histologically in the livers of animals that received treatment with vehicles or ATS alone.

Effect of NS-398 on MCT/LPS-Induced Liver Injury
Among the various mediators released by LPS-activated KCs are metabolites of arachidonic acid, including PGE2 and PGD2 (Decker, 1990Go; Ganey et al., 2001Go). These PGs are produced via the inflammation-inducible COX-2 enzyme and have a variety of effects on other inflammatory cells and HPCs that can contribute to pathophysiological alterations in tissues (Casteleijn et al., 1988Go; Decker, 1990Go; Ganey et al., 2001Go).

NS-398, a selective COX-2 inhibitor, was administered one hour before LPS at a dose that reduces PG production in vivo (Ganey et al., 2001Go). NS-398 did not attenuate plasma ALT (Fig. 4AGo) or AST (Fig. 4BGo) activity in MCT/LPS-treated animals. Plasma ALT or AST activity was unaffected in animals receiving treatment with NS-398 alone. Histologically, liver lesions appeared the same in the MCT/LPS-cotreated animals given NS-398 or its vehicle. No liver lesions were observed in control animals that received vehicles or NS-398 alone.



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FIG. 4. NS-398 does not protect against MCT/LPS-induced liver injury. LPS (7.4 x 106 EU/kg) or its saline vehicle was administered, iv, to rats 4 h after ip administration of MCT (100 mg/kg) or its vehicle. Rats were given 5 mg NS-398/kg or olive oil vehicle, ip, 1 h before LPS administration. Plasma ALT (A) and AST (B) activities were evaluated 18 h after MCT administration. Data are expressed as mean ± SEM; n = 3 for Veh/Veh/Veh, 3 for Veh/NS-398/Veh, 7 for MCT/Veh/LPS, and 4 for MCT/NS-398/LPS. (a) Significantly different from respective value in the absence of MCT/LPS.

 
Mortality
Mortality ranged from 19 to 27% in MCT/LPS-cotreated control animals and from 0 to 33% in MCT/LPS-cotreated animals that received administration of a pharmacologic agent (i.e., GdCl3, PTX, ATS, or NS-398.) None of the pharmacologic agents significantly affected survival in MCT/LPS-cotreated animals. No animals died that received vehicles or any pharmacologic agent alone.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inasmuch as KCs have proven to be critical in the pathogenesis of some inflammatory tissue injuries (Adachi et al., 1994Go; Stachlewitz et al., 1999Go), the hypothesis that KCs are causally important in the synergistic hepatotoxicity from MCT/LPS cotreatment was tested. Inhibition of KC function by GdCl3 in MCT/LPS-cotreated animals resulted in pronounced attenuation of HPC injury (Figs. 1B and 1CGo), suggesting that KCs are causally involved in the pathogenesis. The dose of GdCl3 used in this study was effective in inactivating KCs (Brown et al., 1997Go) and inhibiting the increase in plasma TNF-{alpha} concentration (Fig. 1AGo).

In this model of liver injury, plasma TNF-{alpha} concentration is elevated before the onset of HPC injury, raising the possibility that this cytokine might have a causal role in injury development (Table 2Go). Either inhibition of TNF-{alpha} synthesis by PTX (Figs. 2B and 2CGo) or neutralization of TNF-{alpha} activity by ATS (Figs. 3B and 3CGo) resulted in the attenuation of HPC injury. Both PTX and ATS decreased plasma TNF-{alpha} concentration in MCT/LPS cotreated animals, confirming that the agents were effective in reducing TNF-{alpha} (Figs. 2A and 3AGoGo). These results suggest that TNF-{alpha} is causally involved in the hepatocellular injury.

In rats treated with a small, noninjurious dose of LPS 4 h after a small, nontoxic dose of MCT, morphologic evidence of liver lesions develop within 12–18 h after MCT administration, consistent with elevated plasma ALT and AST activities (Yee et al., 2000Go). Both centrilobular and midzonal liver lesions result and are qualitatively similar to lesions that characterize larger, toxic doses of MCT and LPS, respectively. Centrilobular liver lesions comprise moderate to marked hepatocellular apoptotic and oncotic necrosis, degeneration, congestion, hemorrhage and vascular injury. Midzonal lesions consist of frequent, well-defined areas of marked hepatocellular coagulative necrosis, accompanied by neutrophil infiltration, congestion and hemorrhage (Yee et al., 2000Go). SEC injury is apparent in both lesions (Yee et al., 2002Go). The observation that both MCT-like and LPS-like lesions develop suggests that each agent enhances the effect of the other in this model (Yee et al., 2000Go).

Administration of GdCl3, PTX or ATS to MCT/LPS-treated animals reduced the size and frequency of liver lesions. Morphometric analysis of these lesions demonstrated that Kupffer cell inactivation or TNF-{alpha} depletion in MCT/LPS-cotreated animals significantly reduced the area of the centrilobular, MCT-like and midzonal, LPS-like lesions (Table 1Go). This suggests that pharmacologic manipulation ameliorated the interactive effect of these agents.

In this model of synergistic liver injury, LPS caused an expected, early rise in plasma TNF-{alpha} concentration. As seen previously (Barton et al., 2001Go), this increase was transient in rats treated with LPS alone. Unexpectedly, however, plasma TNF-{alpha} concentration remained significantly elevated for at least 12 h in rats treated with MCT/LPS (Table 2Go). This prolonged elevation contrasts with another model of LPS potentiation of hepatotoxicity in which the LPS-induced increase was not prolonged by aflatoxin B1 cotreatment (Barton et al., 2001Go). Even at a larger, hepatotoxic dose of LPS, the elevation in plasma TNF-{alpha} concentration remains transient (Iimuro et al., 1994Go; Pearson et al., 1996Go). It is noteworthy that MCT given by itself to rats, either at the nontoxic dose used in this study (Table 2Go) or at a larger, hepatotoxic dose (300 mg/kg; unpublished observation), does not increase plasma TNF-{alpha} concentration. Thus, the interaction of these two agents is required for this prolonged effect in MCT/LPS-treated rats.

Under normal conditions, approximately 90% of HA circulating in the blood is removed and degraded by SECs in the liver (Kobayashi et al., 1999Go). SEC injury impairs the ability of these cells to clear HA from the circulation, and this results in increased plasma HA concentration. Such increases have been used as a biomarker of SEC injury in vivo after exposure of animals to toxicants. An increase in plasma HA concentration correlates with histopathologic evidence of SEC injury and destruction in rats given a hepatotoxic dose of either MCT (Copple et al., 2002Go) or LPS (Deaciuc et al., 1994Go; Spapen et al., 1999Go). Similarly, MCT/LPS coexposure results in increased plasma HA concentration in rats, and this correlates with SEC injury (Yee et al., 2002Go). The elevation in plasma HA concentration was slightly but significantly smaller after coadministration of GdCl3 or ATS to MCT/LPS-treated animals (Figs. 1D and 3DGoGo). Administration of PTX to MCT/LPS-treated animals resulted in a trend toward a decrease that was not statistically significant (Fig. 2DGo). These results suggest that KC inactivation or TNF-{alpha} neutralization in MCT/LPS-treated animals significantly but incompletely attenuated SEC injury.

The attenuation of HPC and SEC injury in this model was likely not the result of GdCl3, PTX, or ATS administration interfering with MCT bioactivation. In animals given a large, hepatotoxic dose of MCT (300 mg/kg), neither GdCl3 nor PTX pretreatment of rats altered liver injury (unpublished observation), suggesting that these agents do not interfere with MCT bioactivation. Badger et al. (1997)Go demonstrated that GdCl3 pretreatment of rats caused a modest decrease in hepatic cytochrome P450 (CYP). However, this reduction could be explained by a decrease in the CYP 2E1 isoform (Badger et al., 1997Go), and since MCT is bioactivated by a different isoform (i.e., CYP 3A family; Kasahara et al., 1997Go), it is unlikely that GdCl3 reduced injury in this model by decreasing CYP concentration. In the present study, PTX and ATS were given to MCT/LPS-cotreated animals at a time when most of the administered MCT has been metabolized (Allen et al., 1972Go). Neither GdCl3 (Mimura et al., 1995Go; Vollmar et al., 1996Go) nor PTX (Heller et al., 1999Go) interfere with LPS clearance. Accordingly, it is unlikely that the pharmacological agents used in this study reduced injury by interfering with MCT bioactivation or affecting LPS metabolism.

Rose et al. (2001)Go demonstrated that GdCl3 caused a transient activation of NF{kappa}B and enhanced hepatocellular proliferation in the liver. However, NF{kappa}B activation returned to baseline within 24 h after GdCl3 administration; therefore, it is unlikely that NF{kappa}B activation influenced the results in this study, since MCT/LPS-cotreatment commenced at this time. The possibility that GdCl3 had effects independent of its ability to inactivate Kupffer cells cannot be excluded; however, a similar degree of hepatotoxicity from a large, toxic dose of MCT (300 mg/kg) was unaffected by GdCl3 (unpublished observation), suggesting that it is unlikely to protect through nonselective modes of action such as enhancing cell proliferation.

TNF-{alpha} can promote liver injury in a number of ways. For example, in vitro TNF-{alpha} renders hepatocytes more susceptible to toxicity (Adamson and Billings, 1992Go; El-Sisi et al., 1993Go; Hoek and Pastorino, 2002Go). Likewise, HPCs altered homeostatically by the actions of hepatoxicants may be sensitive to TNF-{alpha}-induced cell killing (Jaeschke et al., 1998Go; Lawson et al., 1998Go). In addition, TNF-{alpha} can prime PMNs to release toxic products (i.e., ROS and proteases) that can damage nearby cells (Kushimoto et al., 1996Go; Nagaki et al., 1991Go; Vassalli, 1992Go). Further study will be required to understand how TNF-{alpha} acts to promote hepatotoxicity in this model.

In addition to TNF-{alpha}, other inflammatory mediators are released by KCs that may have deleterious effects on liver (Holst et al., 1996Go). For example, COX-2 products mediate liver injury in other models. NS-398, a selective COX-2 inhibitor, was administered to rats using a treatment protocol that inhibits COX-2 in vivo and affords protection from liver injury in a PG-dependent model (Ganey et al., 2001Go). The increases in plasma ALT and AST activities in MCT/LPS-cotreated animals were not attenuated by NS-398, which suggests that COX-2 products are not needed for HPC injury in this model (Figs. 4A and 4BGo). A similar result was observed in a model of LPS-potentiated aflatoxin B1 hepatotoxicity (Barton et al., 2001Go); in contrast, a significant protective effect occurred in rats treated with LPS and allyl alcohol (Ganey et al., 2001Go). These contrasting results suggest that the critical mediators of LPS-potentiated hepatotoxic responses differ with different hepatotoxicants.

In summary, GdCl3 administered to MCT/LPS-treated rats at a dose that inhibits KC function reduced HPC and SEC injury. Moreover, the administration of TNF-{alpha} depleting agents to MCT/LPS-cotreated rats also protected against HPC injury and caused a modest attenuation of SEC injury. Accordingly, KCs and TNF-{alpha} appear to play important roles in the synergistic hepatotoxicity from MCT/LPS exposure in rats.


    ACKNOWLEDGMENTS
 
The authors are grateful for the help and advice of Jack Harkema. The technical assistance of Zinah Hong, Yana Itkin, and Stacy Schwartz is gratefully acknowledged. This study was supported by NIH Grants ES 04139 and ES 08789. S.B.Y. was supported in part by training grant T32 ES 07255 from the NIEHS.


    NOTES
 
1 To whom correspondence should be addressed at B440 Life Sciences Building. Fax: (517) 353-8915. E-mail: rothr{at}msu.edu. Back


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