Department of Pharmacology and Toxicology, Institute for Environmental Toxicology and National Food Safety and Toxicology Center, 214 Food Safety and Toxicology Building, Michigan State University, East Lansing, Michigan 48824
Received February 25, 2002; accepted July 10, 2002
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ABSTRACT |
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Key Words: lipopolysaccharide; thrombin; coagulation; liver; potentiation; inflammation; perfusion; heparin.
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INTRODUCTION |
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The agents for which toxicity is increased by LPS are diverse in chemical structure and mechanism of toxicity. Allyl alcohol is a three-carbon, unsaturated alcohol commonly used as an intermediate in the synthesis of many industrial compounds, and has been used in food flavorings. It causes a periportal-specific hepatocellular lesion in rat livers. Liver injury is dependent upon allyl alcohols metabolism to the reactive aldehyde acrolein via alcohol dehydrogenase (Patel et al., 1980; Reid, 1972
; Serafini-Cessi et al., 1972
). Humans can be exposed to acrolein through the degradation of a variety of compounds including cigarette smoke, automobile exhaust, and burning oils. In addition, acrolein is a metabolite of the anticancer drug cyclophosphamide (Sladek, 1971
).
The mechanisms underlying the enhancement of allyl alcohol-induced injury by LPS are unknown. Although LPS can alter the hepatic metabolism of some compounds through changes in cytochromes P450 expression (Morgan, 2001), LPS did not affect the rate of allyl alcohol metabolism (Sneed et al., 1997
). There is evidence to suggest that the LPS enhancement of allyl alcohol toxicity involves components of the inflammatory system. For example, hepatocytes (HCs) exposed to LPS were not more sensitive to allyl alcohol than naïve HCs, indicating that LPS does not increase toxicity by directly altering HCs (Sneed et al., 1997
). In addition, inhibition of activity of Kupffer cells (KCs), the resident macrophages of the liver, significantly decreased the potentiation of allyl alcohol hepatotoxicity by LPS, indicating that KCs play a critical role in this response (Sneed et al., 1997
). Inhibition of KCs can attenuate the toxicity of many xenobiotics including carbon tetrachloride (Badger et al., 1996
; Pereira et al., 1997
; Wueweera et al., 1996
), vinylidene chloride (Wueweera et al., 1996
), acetaminophen (Michael et al., 1999
), D-galactosamine (Stachlewitz et al., 1999
), 1,2-dichlorobenzene (Hoglen et al., 1998
), LPS (Fujita et al., 1995
; Iimuro et al., 1994
; Sarphie et al., 1996
), ethanol (Adachi et al., 1994
; Bautista and Spitzeret, 1999
), and diethyldithiocarbamate (Ishiyama et al., 1995
). Because KCs are thought to play a critical role in the development of liver injury from many compounds, it was of interest to understand better the involvement of KCs in the enhancement of allyl alcohol hepatotoxicity by LPS. Results presented here demonstrate that the model is more complex than a simple interaction among KCs, LPS, and HCs.
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MATERIALS AND METHODS |
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Animals.
Male, Sprague-Dawley rats (CD-Crl:CD-(SD)BR VAF/Plus; Charles River, Portage, MI) weighing 175225 g were allowed food (Wayne Lab-Blox, Allied Mills, Chicago, IL) and water ad libitum. They were housed under conditions of controlled temperature and humidity and 12-hr light and dark cycle. All procedures on animals were carried out according to the humane guidelines of the American Association for Laboratory Animal Science and the University Laboratory Animal Research Unit at Michigan State University.
Kupffer cell isolation.
KCs were isolated using a procedure based on the method of Knook and Sleyster, 1977. Rats were anesthetized with sodium pentobarbital (50 mg/kg). The portal vein was cannulated and the liver was perfused with Ca2+/Mg2+-free Hanks balanced salt solution to clear the blood from the vasculature. The liver was perfused with 200 ml collagenase type II (0.4 mg/ml) in Geys balanced salt solution (GBS). Pronase (0.2% in GBS with 2 µg/ml DNase) was then perfused in a recirculating manner for 1015 min. The liver was removed and gently combed to loosen the cells. The cells were filtered through gauze and centrifuged (50 x g, 2 min) to remove HCs, and the supernatant fractions were collected from two centrifugations of the cells. The cells in the supernatant fluids were then pooled via centrifugation (600 x g, 5 min), resuspended in GBS containing 2 µg/ml DNase, and loaded into a centrifugal elutriator (Beckman J6-MI centrifuge with JE-6B elutriator rotor). GBS was pumped through the rotor at a rate of 12 ml/min. Flow rate was then increased to 24 ml/min for 150 ml to remove endothelial cells. Flow was increased again to 42 ml/min for 150 ml and collected. This fraction, containing a population of cells enriched with KCs, was spun in a centrifuge (600 x g, 5 min), and the pellet was resuspended in RPMI culture medium supplemented with 1% Medium NCTC-109 and 15% fetal bovine serum. The KCs were plated in 12-well Falcon Primaria culture plates at 1 x 106 cells/well (37°C, 7.5% CO2, 92.5% air). KC purity was greater than 90% via peroxidase staining and latex bead phagocytosis. KC responsiveness was assessed by measuring TNF- concentration in the medium 90 min after stimulation with LPS (105 EU/ml). TNF-
concentration was determined using a commercial, rat TNF-
ELISA kit.
Hepatocyte isolation.
Hepatic parenchymal cells (HCs) were isolated using the method described by Seglen, 1973. Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip). The portal vein was cannulated, and the liver was perfused with Ca2+/Mg2+-free Hanks balanced salt solution to remove red blood cells and digested by perfusion for 30 min with a solution containing 60 mg collagenase type II in 250 ml Williams Medium-E. The digested liver was gently combed, filtered through sterile gauze, and centrifuged (50 x g, 2 min) to pellet the HCs. The cells were washed twice with Williams Medium-E and resuspended in Williams Medium-E containing 1% gentamicin and 10% fetal bovine serum. Viability was assessed via trypan blue exclusion. If cells were greater than 80% viable, they were plated in 12-well Falcon Primaria culture plates at a density of 2.5 x 105 cells/well. They were allowed to incubate (37°C, 7.5% CO2, 92.5% air) for 4 h before they were washed and used in an experiment.
In vitro coculture experiments.
KCs were isolated and plated as described above. After 20 h of incubation, medium was removed, and HCs isolated as described above were plated with the KCs. After an additional 4 h of incubation, cocultures were treated with LPS and allyl alcohol. Medium was collected 1.5 h after addition of allyl alcohol, and cells were lysed with 1 ml of 1% Triton X. Cytotoxicity was assessed from release of alanine aminotransferase (ALT) into the medium. Activity of ALT was determined in cell-free supernatant fluids using Sigma Diagnostics Kit No. 52. Percent total ALT was calculated by dividing ALT activity in medium by the total ALT activity found in medium and cell lysates. ALT release from lysed KCs did not significantly contribute to total ALT activity in cocultures. Total ALT activity in the medium plus cell lysates did not change with any culture treatment.
Isolation and perfusion of rat livers.
Rats were weighed and anesthetized with sodium pentobarbital (50 mg/kg, ip), and the portal vein was exposed. The portal vein was cannulated with polyethylene tubing (PE 190, Clay Adams, Parsippany, NJ). The perfusion medium was Krebs-Henseleit bicarbonate buffer supplemented with 2% bovine serum albumin and saturated with 95% O2 and 5% CO2 gas. Flow was constant at a rate of 0.14 ml/min/g body weight. The thoracic portion of the inferior vena cava was cannulated (PE 240 Clay Adams, Parsippany, NJ) for outflow. The liver was placed in a temperature-controlled cabinet maintained at 37°C as described previously (Moulin et al., 1996). The livers were allowed to stabilize for 10 min with single-pass perfusion. A sample of the perfusion medium was then taken (time = 0), and the system was switched to recirculating perfusion with medium containing LPS (1.2 x 106 EU/kg body weight). Samples (500 µl) of the perfusion medium were taken every 30 min for 2 h. At 2 h, allyl alcohol (30, 25, or 20 mg/kg body weight) was added to the perfusion medium, and samples were taken every 15 min for another 2 h. ALT activity in the samples was determined as described above. Previous experience has demonstrated that perfusion of naïve livers with this dose of LPS does not produce injury. Accordingly, livers for which ALT activity in the perfused medium was greater than 200 U/l at 2 h were considered damaged during the isolation procedure and were not included in the experiment.
To assess whether LPS stimulates liver cells during perfusion with buffer, liver donor rats were anesthetized, and the liver was removed and perfused for 4 h in a recirculating manner as described above. LPS (96 x 106 EU/kg of donor rat weight) or its saline vehicle was introduced into the perfusion buffer 10 min after the start of recirculation. Aliquots of perfusate were taken 4 h after the addition of LPS and analyzed for MIP-2 protein using a commercially available ELISA.
In a separate experiment, rats were weighed and then treated with LPS (1.2 x 106 EU/kg body weight, iv) or an equivalent volume of saline as control. After 2 h, the rat livers were isolated and perfused as described above. Thrombin (70 U/rat) or vehicle was added to the perfusion medium before the addition of allyl alcohol (0, 20, 25, 30, 32.5, or 35 mg/kg body weight) or its vehicle. Medium samples (350 µl) were taken every 15 min for 2 h. ALT activity in the samples was determined as described above.
Experiments with heparin in vivo.
Rats were treated with heparin (2000 U/kg body weight, iv) or an equivalent volume of saline as control 1 h before administration of LPS. Rats were treated with LPS (1.2 x 106 EU/kg body weight, iv), and 2 h later they were treated with allyl alcohol (30 mg/kg body weight, ip). At 2, 3, or 8 h after LPS administration, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip) for removal of blood (in 0.38% sodium citrate) and liver samples. Plasma samples were analyzed for ALT, aspartate aminotransferase (AST), and alkaline phosphatase activities as a measure of liver injury using Sigma Diagnostic Kits No. 52, 51, and 245, respectively. The extent of coagulation activation was assessed via measurement of plasma fibrinogen concentration using a BBL fibrometer (Becton, Dickinson and Company, Hunt Valley, MD). LPS is known to induce hepatic synthesis of fibrinogen, which could complicate interpretation of effects on coagulation when using plasma fibrinogen values as a marker of activation. However, plasma fibrinogen values did not change during the first 8 h after treatment of rats with LPS (203 ± 25 vs. 190 ± 8 mg/dl for vehicle- and LPS-treated, respectively; J. Luyendyk, unpublished data), confirming the utility of this marker of coagulation in this model. Livers were weighed, and a portion of the liver was frozen immediately in liquid nitrogen and stored at 80°C for preparation of RNA (see below). The remaining liver sample was fixed in formalin and processed for histological evaluation. Liver sections were stained for neutrophils (PMNs) as described previously (Pearson et al., 1995) using an anti-PMN antibody prepared as described by Hewett et al.(1992)
. Stained cells were counted under light microscopy in 20 random fields at 400x magnification.
In a separate experiment to examine the effect of heparin on hepatotoxicity from a larger dose of allyl alcohol, rats were treated with heparin (2000 Units/kg body weight, iv) or an equivalent volume of saline as control 3 h before treatment with allyl alcohol (50 mg/kg body weight, ip). At 9 h after heparin administration, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip) for removal of blood (in 0.38% sodium citrate). Blood samples were analyzed for ALT and fibrinogen as described above.
Determination of CINC-1 and COX-2 mRNA expression.
Total RNA was isolated from frozen liver tissue using Tri Reagent (Molecular Research Center, Cincinnati, OH) as described by Chomczynski et al. (Chomczynski, 1993; Chomczynski and Mackey, 1995
). The concentration and purity of RNA was determined from absorbance at 260 and 280 nm. RNA was adjusted to 50 µg/ml with RNase-free water. First-strand cDNA was synthesized using the RETROscript protocol. Semiquantitative polymerase chain reaction (PCR) was performed using the Quantum RNA kit; 18S rRNA was used as the internal control. Samples were denatured for 90 s at 94°C, followed by 35 cycles of 30 s at 94°C, 45 s at 60°C, and 45 s at 72°C, with a final 7-min extension step at 72°C. PCR products were separated via electrophoresis on a 1.5% agarose gel containing ethidium bromide. Band intensity was quantified using Quantity One quantitation software (version 4, BIO-RAD). Band intensities for COX-2 and CINC-1 were normalized to the band intensity of the 18S rRNA internal standard.
Experiments with warfarin in vivo.
Rats were treated with warfarin (20 mg/kg body weight, po) or vehicle twice, 24 h apart. Eighteen hours after the last warfarin treatment, rats were treated with LPS (1.2 x 106 EU/kg, iv), and 2 h after LPS they were treated with allyl alcohol (30 mg/kg, ip). Eight hours after LPS administration, rats were anesthetized with sodium pentobarbital (50 mg/kg, ip) for removal of blood (in 0.38% sodium citrate) and liver samples. Plasma samples were analyzed for ALT activity and fibrinogen concentration as described above.
Statistical analysis.
Data are expressed as mean ± standard error of the mean (SEM). For all results presented, n represents the number of individual experiments. Data with nonhomogeneous variances were square root- or log-transformed prior to analysis. Data represented as percents were arcsine-transformed prior to analysis. Data were analyzed using repeated measures analysis of variance for in vitro experiments and general linear model analysis of variance for in vivo and isolated, perfused liver experiments (provided in the statistics program NCSS 2000). Comparisons among groups were performed using Fishers Least Squares Difference test. The criterion for statistical significance was p 0.05.
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RESULTS |
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In vivo, the ratio of KCs to HCs is about 1:10 (Altin and Bygrave, 1988); however, toxic responses observed in KC/HC cocultures require a larger ratio of KCs to HCs (Kausalya et al., 1993
; Lysz et al., 1990
). Cocultures with a greater ratio of KCs:HCs produce less PGE2 upon LPS stimulation than cocultures with lower ratios (Billiar et al., 1990
). Because previous experiments have shown that prostaglandins can increase the toxicity of allyl alcohol in HCs (Ganey et al., 2001
), a decreased production of prostaglandins might influence the response in cocultures. To examine whether the lack of effect of LPS was related to the high ratio of KCs to HCs, cocultures were plated using different ratios of these two cell types. Allyl alcohol caused a concentration-dependent cytotoxicity, and LPS did not increase the sensitivity of HCs to allyl alcohol at any of the KC:HC ratios tested (Fig. 2
). To examine whether LPS was accelerating the rate of injury but not affecting maximal injury measured at 90 min, ALT release was assessed after 30 and 60 min of exposure to allyl alcohol. Under these conditions, LPS did not affect allyl alcohol-induced cytotoxicity. In addition, varying the time of incubation of cells with LPS (0, 0.5, 2, or 8 h) did not lead to either an increased sensitivity of HCs to allyl alcohol or an increased rate of HC injury in the presence of LPS-stimulated KCs (data not shown).
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The plasma fibrinogen concentration of animals treated only with LPS 3 h earlier did not decrease, whereas that of animals treated with allyl alcohol alone tended to decrease; however, the change was not significant (Table 1). Plasma fibrinogen concentrations in animals cotreated with LPS and allyl alcohol were significantly decreased at this time. Pretreatment with heparin prevented the decrease. Plasma ALT activity was not elevated in any of the treatment groups, indicating that significant liver injury had not developed by 3 h after LPS administration (Table 1
). Plasma AST activity was slightly but significantly elevated in animals receiving LPS and allyl alcohol cotreatment compared with animals receiving saline only.
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Warfarin interferes with the coagulation cascade through inhibition of the synthesis of functional coagulation factors VII, IX, X, and II (prothrombin). As before, plasma fibrinogen concentration was significantly decreased in rats treated with both LPS and allyl alcohol, and pretreatment with warfarin blocked the LPS/allyl alcohol-induced decrease in plasma fibrinogen (Fig. 8A). Liver injury from LPS and allyl alcohol cotreatment was attenuated in rats pretreated with warfarin (Fig. 8B
). Warfarin treatment alone did not cause liver injury.
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The ability of thrombin to induce injury in the isolated liver is dependent on the presence of neutrophils (Moulin et al., 2001). To verify that there were neutrophils in livers upon liver isolation, the number of neutrophils was counted in liver sections from animals 2 h after treatment with LPS. Hepatic neutrophil numbers were also determined just after single-pass perfusion of isolated livers from LPS-treated rats and at the end of isolated, perfused liver experiments. Livers from LPS-treated rats at 2 h (i.e., the time livers are isolated) contained significantly more neutrophils than livers perfused single pass for 10 min (Table 2
). After 2 h of recirculating perfusion, the number of neutrophils decreased further, but remained significantly different from the number of neutrophils in livers from naïve rats.
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DISCUSSION |
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Several alterations were made to the KC/HC cocultures in an attempt to reproduce the LPS-induced enhancement of allyl alcohol hepatotoxicity observed in vivo. For example, increasing the concentration of LPS did not increase KC/HC coculture sensitivity to allyl alcohol. The ratio of KCs to HCs can influence the ability of KCs to produce inflammatory mediators (Billiar et al., 1990), an effect that may be due to factors produced by HCs which regulate KC function. However, LPS did not affect the cytotoxicity of allyl alcohol in HCs at any of the KC/HC ratios examined. In fact, under no condition examined did LPS-stimulated KCs increase the sensitivity of HCs to allyl alcohol toxicity, suggesting that the enhanced response observed in vivo requires additional factors.
Extrahepatic Factors Are Required for LPS Enhancement of Allyl Alcohol-Induced Hepatotoxicity
There are many other factors that may explain why KC/HC cocultures fail to mimic the LPS-induced enhancement of allyl alcohol toxicity observed in vivo. An obvious possibility is that the coculture system lacks one or more elements critical to the response. These may include KC-derived reactive oxygen species. KCs do not release reactive oxygen species upon LPS exposure in vitro, although they do in vivo (Ikejima et al., 1999). Additionally, the isolation process may remove critical receptors from KCs or HCs, or factors released from the KCs could be highly labile and depend on the close association of KCs and HCs found in vivo. To explore these possibilities, the isolated, perfused liver was used as a model to reproduce the response observed in vivo. Though perfusion of livers with LPS stimulated release of MIP-2, an inflammatory protein upregulated by LPS in the liver (Rovai et al., 1998
), LPS did not enhance allyl alcohol-induced liver injury, suggesting that even in this system a critical factor was missing.
The Coagulation Cascade Is Involved in the LPS Enhancement of Allyl Alcohol-Induced Heptatotoxicity
Injury from larger doses of allyl alcohol alone is not dependent on extrahepatic factors, as evidenced by the lack of protection offered by neutrophil depletion (Ganey and Schultze, 1995) or heparin (results presented here) and the ability of allyl alcohol to injure isolated HCs (Ohno et al., 1985
; Pang et al., 1997
; Sneed et al., 1997
). In contrast, LPS-induced liver injury from large doses of LPS is dependent on extrahepatic factors (Hewett et al., 1992
; Moulin et al., 1996
). One extrahepatic factor is the coagulation cascade, which can be activated by LPS. Both intrinsic and extrinsic pathways of coagulation are activated by LPS (Aasen et al., 1978
; Yamaguchi et al., 2000
). Within 3 h of a lethal dose of LPS to rats, circulating fibrinogen concentrations decrease by more than 90% (Margaretten et al., 1967
; Prager et al., 1979
). At the same time, fibrin clots appear in the microcirculation of the liver. Furthermore, inhibition of coagulation protects against liver injury from large doses of LPS (Hewett and Roth, 1995
; Moulin et al., 1996
; Pearson et al., 1996
). Accordingly, it was of interest to examine a possible role for the coagulation cascade in the LPS enhancement of allyl alcohol hepatotoxicity.
To begin to assess whether the coagulation cascade plays a role in LPS-enhanced allyl alcohol hepatotoxicity, heparin was used to inhibit thrombin activity. Pretreatment with heparin reduced liver injury from the cotreatment of allyl alcohol and LPS in vivo (Fig. 5). This effect is not likely to be caused by an inhibition of bioactivation of allyl alcohol by heparin, because heparin had no effect on the hepatotoxicity of a larger dose of allyl alcohol given alone. These data suggest that the coagulation cascade participates in the LPS enhancement of allyl alcohol-induced liver injury; however, heparin has actions in addition to inhibition of coagulation. For example, heparin inhibited the infiltration of PMNs into tissues and the production of superoxide anion and nitric oxide from stimulated neutrophils (Beltran et al., 1999
; Darien et al., 1998
; Downing et al., 1998
; Riesenberg et al., 1995
; Shin et al., 1997
). Heparin also inhibited the increase in hepatic levels of CINC messenger RNA in a model of ischemia/reperfusion injury (Hisama et al., 1996
). It is unlikely that heparin decreased injury in experiments presented here through direct inhibition of mediators of inflammation other than thrombin. This interpretation is based on several observations. First, heparin did not affect expression of CINC-1 (Fig. 7
), a chemokine released during inflammation that helps to bring neutrophils into liver (Zhang et al., 1995
). Second, neutrophil accumulation in livers was not diminished by treatment with heparin (Fig. 6
). Third, expression of COX-2, which is induced during inflammation (Ruetten and Thiemermann, 1997
) and plays a critical role in the potentiation of allyl alcohol hepatotoxicity by LPS (Ganey et al., 2001
), was increased in livers from rats cotreated with LPS and allyl alcohol, and this increase was unaffected by pretreatment with heparin (Fig. 7
).
Results from experiments using warfarin support the hypothesis that heparin protected against injury from LPS/allyl alcohol through inhibition of coagulation. Warfarin, an anticoagulant that works through a completely different mechanism to inhibit the coagulation cascade, also afforded protection from injury. Warfarin inhibits the regeneration of reduced vitamin K, which is critical for the carboxylation of glutamate to -carboxyglutamate residues on precursor protein factors VII, IX, X, and II. Collectively these data suggest that the coagulation cascade plays a critical role in the enhancement of allyl alcohol hepatotoxicity by LPS.
It Does Not Appear That Thrombin Contributes to the LPS Enhancement of Allyl Alcohol-Induced Hepatotoxicity through a Specific Receptor in the Liver
A component of the coagulation cascade that plays a critical role in liver injury from a large dose of LPS is thrombin. Infusion of thrombin into the portal vein leads to morphological changes in the liver similar to portal venous infusion of LPS. These changes include fibrin deposition, neutrophil accumulation, and hepatic injury (Hewett and Roth, 1993; Shibayama, 1987
). The anticoagulants heparin and warfarin and the thrombin-specific inhibitor hirudin protect against LPS-induced liver injury (Moulin et al., 1996
; Pearson et al., 1996
; Pernerstorfer et al., 1999
), underlining the critical role played by thrombin. However, ancrod, which depletes circulating fibrinogen and thereby prevents formation of fibrin clots, does not protect against LPS-induced liver injury (Hewett and Roth, 1995
; Moulin et al., 1996
). This result suggests that thrombin contributes to injury by a mechanism independent of clot formation. This interpretation was supported by the observation that perfusion of livers isolated from LPS-pretreated rats with medium containing thrombin produced injury, whereas no injury was observed in the absence of thrombin (Moulin et al., 1996
). Thrombin functions not only to cleave fibrinogen proteolytically, but also to initiate changes in cells through activation of specific receptors known as protease-activated receptors (PARs). Perfusion of livers isolated from LPS-treated rats with a peptide that activates PAR-1 produced injury similar to that seen upon perfusion with thrombin, suggesting that the mechanism by which thrombin contributes to LPS-induced injury involves PAR-1 in the liver (Copple et al., 2000
).
To examine the possibility that thrombin acts through a receptor-mediated mechanism in the LPS-induced potentiation of allyl alcohol, isolated livers from LPS-treated rats were perfused with allyl alcohol and thrombin. Addition of thrombin to the medium did not enhance the hepatotoxicity of allyl alcohol. The possibility that this lack of effect was due to inactivation of thrombin receptors in vivo prior to liver isolation was examined. Although thrombin had been activated by the time livers were isolated, pretreatment of rats with heparin, which would prevent receptor activation and internalization, did not restore sensitivity of their isolated livers to an effect of thrombin. Taken together, the data suggest that LPS-mediated enhancement of allyl alcohol-induced liver injury is dependent on thrombin activation of blood components or on some other factor(s) of the coagulation cascade. One potential blood component is the neutrophil.
There was a greater number of neutrophils in livers isolated from LPS-treated rats compared with livers from naïve rats, but it is possible that the number was not sufficient to induce injury upon thrombin stimulation. In previous studies using perfused livers isolated from rats given a hepatotoxic dose of LPS, neutrophils were necessary for thrombin-induced injury (Moulin et al., 2001). After 2 h of perfusion of these livers, about 41 neutrophils per 20x field remained in the tissue (Copple, B. L., Moulin, F., Ganey, P. E., and Roth, R. A., unpublished observations). This number is four times greater than the number of neutrophils in the liver at the beginning of experiments presented here (Table 2
). A 3-fold larger dose of LPS was used in the previous studies compared with the dose used in experiments presented here, and this may explain the larger number of neutrophils present in the livers. This observation raises the possibility that a minimal number of neutrophils is needed to obtain thrombin-mediated injury, and that the number present in the livers for studies presented here did not reach this minimum.
Livers from LPS-treated rats were more sensitive to allyl alcohol hepatotoxicity, suggesting that there were enough neutrophils present to develop injury under conditions in which the coagulation cascade had been activated in vivo. In addition, plasma fibrinogen concentrations were significantly decreased 2 h after LPS administration, indicating that thrombin was activated prior to isolation of the liver. It is possible that thrombin did not increase injury in isolated livers because this activation of thrombin may have led to activation of PAR-1 and consequent downregulation of PAR-1 before the experiments began (Ishii et al., 1993). In this scenario, treatment of rats with heparin before exposure to LPS and isolation of livers should have preserved hepatic PMN accumulation (Fig. 6
) and PAR-1 receptors, allowing thrombin to enhance injury upon perfusion of livers with allyl alcohol. This was not the case, however, suggesting that the inability of thrombin to enhance the toxicity of allyl alcohol in isolated livers is due to the absence of other critical factors of the coagulation cascade or other thrombin-activated factors present in the blood.
In summary, although KCs play a critical role in the enhancement of allyl alcohol hepatotoxicity by LPS in vivo, they are not sufficient to enhance the sensitivity of HCs to allyl alcohol. Another critical factor required for the enhancement of allyl alcohol toxicity by LPS is the coagulation cascade, as evidenced by the protection afforded by heparin and warfarin. Thrombin does not appear to play a role in the potentiation of allyl alcohol-induced liver injury by LPS through a protease-activated receptor in the liver. Thus, the role of thrombin appears to be dependent on other coagulation factors or components of the blood.
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ACKNOWLEDGMENTS |
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NOTES |
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