Endothelial cell injury and coagulation system activation during synergistic hepatotoxicity from monocrotaline and bacterial lipopolysaccharide coexposure.

A small, noninjurious dose of bacterial lipopolysaccharide (LPS; 7.4 x 106 EU/kg) administered 4 h after a small, nontoxic dose of monocrotaline (MCT; 100 mg/kg) produces synergistic hepatotoxicity in rats within 6 to 12 h after MCT exposure. The resulting centrilobular (CL) and midzonal (MZ) liver lesions are characterized by hepatic parenchymal cell (HPC) necrosis. Pronounced hemorrhage, disruption of sinusoidal architecture, and loss of central vein intima suggest that an additional component to injury may be the liver vasculature. In the present investigation, the hypothesis that sinusoidal endothelial cell (SEC) injury and coagulation system activation occur in this model was tested. Plasma hyaluronic acid (HA) concentration, a biomarker for SEC injury, was significantly increased in cotreated animals before the onset of HPC injury and remained elevated through the time of maximal HPC injury (i.e., 18 h). SEC injury was confirmed by immunohistochemistry and electron microscopy. Pyrrolic metabolites were produced from MCT by SECs in vitro, which suggests that MCT may injure SECs directly through the formation of its toxic metabolite, monocrotaline pyrrole. Inasmuch as SEC activation and injury can promote hemostasis, activation of the coagulation system was evaluated. Coagulation system activation, as marked by a decrease in plasma fibrinogen, occurred before the onset of HPC injury. Furthermore, extensive fibrin deposition was observed immunohistochemically within CL and MZ regions after MCT/LPS cotreatment. Taken together, these results suggest that SEC injury and coagulation system activation are components of the synergistic liver injury resulting from MCT and LPS coexposure.     

Role of neutrophils in the synergistic liver injury from monocrotaline and bacterial lipopolysaccharide exposure.

Synergistic liver injury develops in Sprague-Dawley rats from administration of a small, noninjurious dose (7.4 x 10(6) EU/kg) of bacterial lipopolysaccharide (LPS) given 4 h after a nontoxic dose (100 mg/kg) of the pyrrolizidine alkaloid, monocrotaline (MCT). Previous studies demonstrated that liver injury is mediated through inflammatory factors, such as Kupffer cells and tumor necrosis factor alpha (TNF-alpha), rather than through simple interaction between MCT and LPS. In the present study, the hypothesis that neutrophils (polymorphonuclear leukocytes or PMNs) are causally involved in this injury model is tested, and the interdependence between PMNs and other inflammatory components is explored. Hepatic PMN accumulation and the appearance of cytokine-induced neutrophil chemoattractant-1 in plasma preceded the onset of liver injury, suggesting that PMNs contribute to toxicity. Hepatic PMN accumulation was partially dependent on TNF-alpha. Prior depletion of PMNs in MCT/LPS-cotreated animals resulted in attenuation of both hepatic parenchymal cell (HPC) and sinusoidal endothelial cell (SEC) injury at 18 h. PMN depletion did not, however, protect against early SEC injury that occurred before the onset of HPC injury at 6 h. This observation suggests that SEC injury is not entirely dependent on PMNs in this model. In vitro, MCT caused PMNs to degranulate in a concentration-dependent manner. These results provide evidence that PMNs are critical to the HPC injury caused by MCT/LPS cotreatment and contribute to the progression of SEC injury.     

Endothelial cell injury and fibrin deposition in rat liver after monocrotaline exposure.

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) plant toxin that produces hepatotoxicity in people and animals. Human exposure to PAs occurs through consumption of contaminated grains and herbal remedies. Injection (ip) of MCT in rats produced dose-dependent hepatic parenchymal cell injury that was significant at 200 mg/kg. Injection of 300 mg/kg MCT produced time-dependent hepatotoxicity with significant injury beginning by 12 h after treatment. Histopathologic examination of liver sections revealed coagulative hepatocellular necrosis, widening of sinusoids and hemorrhage in centrilobular regions. MCT-induced damage to central venular endothelial cells (CVECs) and sinusoidal endothelial cells (SECs) in the liver was quantified using immunohistochemical staining and by increased plasma hyaluronic acid concentration. MCT damaged CVECs and SECs in the liver by 8 h after treatment. Extensive endothelial cell injury was restricted to centrilobular regions. To determine if damage to endothelial cells in the liver stimulated activation of the coagulation system, fibrin deposition was quantified using immunohistochemistry. Extensive fibrin deposition occurred in the liver after MCT treatment and was restricted to centrilobular regions. Interestingly, both endothelial cell damage and fibrin deposition preceded the onset of hepatic parenchymal cell injury. These results suggest that endothelial cell damage and fibrin deposition in centrilobular regions of the liver are prominent features of MCT-induced liver injury.     

The role of Kupffer cells and TNF-alpha in monocrotaline and bacterial lipopolysaccharide-induced liver injury.

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, i.p.) was administered 4 h before LPS (7.4 x 10(6) EU/kg, i.v.). 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.     

The role of tumor necrosis factor alpha in lipopolysaccharide/ranitidine-induced inflammatory liver injury.

Exposure to a nontoxic dose of bacterial lipopolysaccharide (LPS) increases the hepatotoxicity of the histamine-2 (H2) receptor antagonist, ranitidine (RAN). Because some of the pathophysiologic effects associated with LPS are mediated through the expression and release of inflammatory mediators such as tumor necrosis factor alpha (TNF), this study was designed to gain insights into the role of TNF in LPS/RAN hepatotoxicity. To determine whether RAN affects LPS-induced TNF release at a time near the onset of liver injury, male Sprague-Dawley rats were treated with 2.5 x 10(6) endotoxin units (EU)/kg LPS or its saline vehicle (iv) and 2 h later with either 30 mg/kg RAN or sterile phosphate-buffered saline vehicle (iv). LPS administration caused an increase in circulating TNF concentration. RAN cotreatment enhanced the LPS-induced TNF increase before the onset of hepatocellular injury, an effect that was not produced by famotidine, a H2-receptor antagonist without idiosyncrasy liability. Similar effects were observed for serum interleukin (IL)-1beta, IL-6, and IL-10. To determine if TNF plays a causal role in LPS/RAN-induced hepatotoxicity, rats were given either pentoxifylline (PTX; 100 mg/kg, iv) to inhibit the synthesis of TNF or etanercept (Etan; 8 mg/kg, sc) to impede the ability of TNF to reach cellular receptors, and then they were treated with LPS and RAN. Hepatocellular injury, the release of inflammatory mediators, hepatic neutrophil (PMN) accumulation, and biomarkers of coagulation and fibrinolysis were assessed. Pretreatment with either PTX or Etan resulted in the attenuation of liver injury and diminished circulating concentrations of TNF, IL-1beta, IL-6, macrophage inflammatory protein-2, and coagulation/fibrinolysis biomarkers in LPS/RAN-cotreated animals. Neither PTX nor Etan pretreatments altered hepatic PMN accumulation. These results suggest that TNF contributes to LPS/RAN-induced liver injury by enhancing inflammatory cytokine production and hemostasis.     

Anticoagulants prevent monocrotaline-induced hepatic parenchymal cell injury but not endothelial cell injury in the rat

Monocrotaline (MCT) is a pyrrolizidine alkaloid plant toxin that produces hepatotoxicity in humans and animals. Human exposure to MCT occurs through consumption of contaminated grains and herbal medicines. Administration of MCT to rats stimulates activation of the coagulation system and fibrin deposition in the liver. Fibrin deposition occurs simultaneously with endothelial cell damage and prior to hepatic parenchymal cell injury. Accordingly, the hypothesis that activation of the coagulation system is required for MCT-induced liver injury was tested. Treatment of rats with either heparin or warfarin significantly reduced MCT-induced activation of the coagulation system and the increase in alanine aminotransferase activity in the plasma, a biomarker of hepatic parenchymal cell injury. Histopathological examination of liver sections revealed that heparin decreased parenchymal cell necrosis but did not affect central venular endothelial cell damage, congestion and dilation of the sinusoids, or hemorrhage in the liver. Morphometric analysis revealed that 28% of the area of livers from MCT-treated rats contained regions of coagulative necrosis, whereas less than 5% of the area of livers from rats treated with MCT and heparin contained these regions. By contrast, neither heparin nor warfarin prevented MCT-induced damage to endothelial cells in the liver as estimated by increased plasma hyaluronic acid concentration. These results suggest that activation of the coagulation system is required for MCT-induced parenchymal cell injury but not endothelial cell injury in the liver.     

Modes of cell death in rat liver after monocrotaline exposure.

Monocrotaline (MCT) is a pyrrolizidine alkaloid (PA) plant toxin that produces sinusoidal endothelial cell (SEC) injury, hemorrhage, fibrin deposition, and coagulative hepatic parenchymal cell (HPC) oncosis in centrilobular regions of rat livers. Cells with apoptotic morphology have been observed in the livers of animals exposed to other PAs. Whether apoptosis occurs in the livers of MCT-treated animals and whether it is required for full manifestation of pathological changes is not known. To determine this, rats were treated with 300 mg MCT/kg, and apoptosis was detected by transmission electron microscopy and the TUNEL (TdT-mediated dUTP nick end labeling) assay. MCT produced significant apoptosis in the liver by 4 h after treatment. To determine if MCT kills cultured HPCs by apoptosis, HPCs were isolated from the livers of rats and exposed to MCT. MCT caused a concentration-dependent release of alanine aminotransferase (ALT), a marker of HPC injury. Furthermore, caspase 3 was activated and TUNEL staining increased in MCT-treated HPCs. MCT-induced TUNEL staining and release of ALT into the medium were completely prevented by the pancaspase inhibitors z-VAD.fmk and IDN-7314, suggesting that MCT kills cultured HPCs by apoptosis. To determine if caspase inhibition prevents MCT-induced apoptosis in the liver, rats were cotreated with MCT and IDN-7314. IDN-7314 reduced MCT-induced TUNEL staining in the liver and release of ALT into the plasma. Morphometric analysis confirmed that IDN-7314 reduced HPC oncosis in the liver by approximately 50%. Inasmuch as HPC hypoxia occurred in the livers of MCT-treated animals, upregulation of the hypoxia-regulated cell-death factor, BNIP3 (Bcl2/adenovirus EIB 19kD-interacting protein 3), was examined. BNIP3 was increased in the livers of mice treated 24 h earlier with MCT. Results from these studies show that MCT kills cultured HPCs by apoptosis but causes both oncosis and apoptosis in the liver in vivo. Furthermore, caspase inhibition reduces both apoptosis and HPC oncosis in the liver after MCT exposure.     

Bacterial lipopolysaccharide exposure augments aflatoxin B(1)-induced liver injury.

Bacterial endotoxin (lipopolysaccharide; LPS) given to animals in large doses results in pronounced, midzonal liver injury. Exposure to smaller, non-injurious doses of LPS augments the toxicity of certain hepatotoxicants. This study was conducted to delineate the development of injury in a rat model of augmentation of aflatoxin B(1) (AFB(1)) hepatotoxicity by LPS. At large doses (i.e., > 1 mg/kg, ip), AFB(1) administration resulted in pronounced injury to the periportal regions of the liver. Male, Sprague-Dawley rats (250-350 g) were treated with 1 mg AFB(1)/kg, ip or its vehicle (0.5% DMSO/saline) and 4 h later with either E. coli LPS (7.4 x 106 EU/kg, iv) or its saline vehicle. Liver injury was assessed 6, 12, 24, 48, 72, or 96 h after AFB(1) administration. Hepatic parenchymal cell injury was evaluated as increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum and from histologic examination of liver sections. Biliary tract alterations were evaluated as increased concentration of serum bile acids and activities of gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), and 5'-nucleotidase (5'-ND) in serum. At all times and for all markers, injury in rats treated with either AFB(1) or LPS alone was absent or modest. In the AFB(1)/LPS cotreated group, hepatic parenchymal cell injury was pronounced by 24 h and had returned to control values by 72 h. The injury began in the periportal region and spread midzonally with time. Furthermore, changes in serum markers indicative of biliary tract alterations were evident by 12 h and had returned to control values by 72 h. Thus, the nature of the hepatic lesions suggested that LPS potentiated the effects of AFB(1) on both parenchymal and bile duct epithelial cells.     

Bacterial lipopolysaccharide exposure alters aflatoxin B(1) hepatotoxicity: benchmark dose analysis for markers of liver injury.

Aflatoxin B(1) (AFB(1)) is a fungal toxin that causes both acute hepatotoxicity and hepatocellular carcinoma in humans and experimental animals. Previous studies demonstrated that a small, noninjurious dose of bacterial lipopolysaccharide (LPS) augments the hepatotoxicity of AFB(1) through activation of inflammatory cells and production of soluble inflammatory mediators (Barton et al., 2000b, 2001). This study was conducted to examine the effect of LPS on the dose-response relationship for AFB(1)-induced liver injury. Male Sprague-Dawley rats (250-350g) were treated with AFB(1) (0.1 mg/kg-6.3 mg/kg, ip) and 4 h later with a noninjurious dose of E. coli LPS (7.4 x 10(6) EU/kg, iv). Twenty-four h after AFB(1) administration, hepatic parenchymal cell injury was estimated from elevations in serum alanine aminotransferase and aspartate aminotransferase activities. Injury to intrahepatic bile ducts was evaluated from increased serum gamma-glutamyl transferase and alkaline phosphatase activities. Based on benchmark dose (BMD) analysis, the AFB(1) BMD for parenchymal cell injury was decreased 10-fold by LPS cotreatment, whereas AFB(1) BMDs for bile duct injury were decreased nearly 20-fold. The data suggest that concurrent inflammation renders the liver considerably more sensitive to the hepatotoxic effects of AFB(1).     

Augmentation of aflatoxin B1 hepatotoxicity by endotoxin: involvement of endothelium and the coagulation system.

Aflatoxin B(1) (AFB(1)) is a fungal toxin that causes both acute hepatotoxicity and liver carcinoma in exposed humans and animals. Previous studies have shown that exposure of rats to nontoxic doses of bacterial lipopolysaccharide (LPS) augments AFB(1) acute hepatotoxicity, resulting in enhanced injury to hepatic parenchymal cells and bile ducts. At larger doses, LPS causes damage to sinusoidal endothelial cells (SECs) and activation of the coagulation system. Accordingly, we tested the hypothesis that treatment of rats with AFB(1) and LPS damages SECs and activates the coagulation system, which is critical for potentiation of AFB(1) hepatotoxicity by LPS. Male, Sprague-Dawley rats were given 1 mg/kg AFB(1) (ip), then 4 hours later 7.4 x 10(6) EU/kg LPS was administered (iv). A time-dependent injury to SECs and parenchymal cells was observed in AFB(1)/LPS-cotreated animals that became significant by 12 h, as estimated by increases in plasma hyaluronic acid (HA) and alanine aminotransferase (ALT) activities, respectively. Immunohistochemical analysis revealed that endothelial cell immunostaining was decreased in both centrilobular and periportal regions after AFB(1)/LPS treatment. Immunohistochemical evidence of fibrin deposition was found in both centrilobular and periportal regions by 12 h, but these deposits persisted only in periportal regions by 24 h. Administration of the anticoagulant heparin to AFB(1)/LPS-cotreated animals markedly attenuated increases in markers of hepatic parenchymal cell injury but provided only minimal amelioration of bile duct injury. These results suggest that AFB(1)/LPS coexposure results in SEC injury and activation of the coagulation system, and that the coagulation system is required for the development of hepatic parenchymal cell injury but not bile duct injury in this model.     

Enhancement of allyl alcohol hepatotoxicity by endotoxin requires extrahepatic factors.

Noninjurious doses of bacterial endotoxin (lipopolysaccharide; LPS) enhance allyl alcohol-induced liver damage in rats in a Kupffer cell (KC)-dependent fashion. To investigate the mechanism by which KCs contribute to liver injury in this model, isolated KCs and hepatocytes (HCs) were cocultured. Addition of LPS to the cocultured cells did not enhance allyl alcohol-induced cytotoxicity. In addition, recirculating perfusion of isolated livers from na?ve rats with LPS for 2 h did not significantly enhance allyl alcohol-induced toxicity as measured by release of alanine aminotransferase (ALT). These results suggest an extrahepatic factor is required for LPS potentiation of allyl alcohol hepatotoxicity. To examine whether the coagulation cascade contributes to injury in this model, rats were given either warfarin at 42 and 18 h before LPS, or heparin at 1 h before LPS, and were treated with allyl alcohol 2 h after LPS. Warfarin and heparin each significantly blocked the decrease in plasma fibrinogen levels and attenuated the increase in plasma ALT activity in rats treated with LPS and allyl alcohol. To assess the role of thrombin in this injury, isolated livers from rats pretreated with LPS were perfused with thrombin or vehicle and allyl alcohol. Though LPS pretreatment enhanced the toxicity of allyl alcohol compared with livers from na?ve rats, perfusion with thrombin did not increase sensitivity to allyl alcohol. In summary, LPS augments the hepatotoxicity of allyl alcohol through a mechanism involving extrahepatic factors, one of which may be a component of the coagulation cascade.     

Gene expression analysis points to hemostasis in livers of rats cotreated with lipopolysaccharide and ranitidine.

Studies in rats have demonstrated that modest underlying inflammation can precipitate idiosyncratic-like liver injury from the histamine 2-receptor antagonist, ranitidine (RAN). Coadministration to rats of nonhepatotoxic doses of RAN and the inflammagen, bacterial lipopolysaccharide (LPS), results in hepatocellular injury. We tested the hypothesis that hepatic gene expression changes could be distinguished among vehicle-, LPS-, RAN- and LPS/RAN-treated rats before the onset of significant liver injury in the LPS/RAN-treated rats (i.e., 3 h post-treatment). Rats were treated with LPS (44 x 10(6) EU/kg, i.v.) or its vehicle, then two hours later with RAN (30 mg/kg, i.v.) or its vehicle. They were killed 3 h after RAN treatment, and liver samples were taken for evaluation of liver injury and RNA isolation. Hepatic parenchymal cell injury, as estimated by increases in serum alanine aminotransferase (ALT) activity, was not significant at this time. Hierarchal clustering of gene expression data from Affymetrix U34A rat genome array grouped animals according to treatment. Relative to treatment with vehicle alone, treatment with RAN and/or LPS altered hepatic expression of numerous genes, including ones encoding products involved in inflammation, hypoxia, and cell death. Some were enhanced synergistically by LPS/RAN cotreatment. Real-time PCR confirmed robust changes in expression of B-cell translocation gene 2, early growth response-1, and plasminogen-activator inhibitor-1 (PAI-1) in cotreated rats. The increase in PAI-1 mRNA was reflected in an increase in serum PAI-1 protein concentration in LPS/RAN-treated rats. Consistent with the antifibrinolytic activity of PAI-1, significant fibrin deposition occurred only in livers of LPS/RAN-treated rats. The results suggest the possibility that expression of PAI-1 promotes fibrin deposition in liver sinusoids of LPS/RAN-treated rats and are consistent with the development of local ischemia and consequent tissue hypoxia.     

Lipopolysaccharide augments aflatoxin B(1)-induced liver injury through neutrophil-dependent and -independent mechanisms.

Exposure to small, noninjurious doses of the inflammagen, bacterial endotoxin (lipopolysaccharide, LPS) augments the toxicity of certain hepatotoxicants including aflatoxin B(1) (AFB(1)). Mediators of inflammation, in particular neutrophils (PMNs), are responsible for tissue injury in a variety of animal models. This study was conducted to examine the role of PMNs in the pathogenesis of hepatic injury after AFB(1)/LPS cotreatment. Male, Sprague-Dawley rats (250-350 g) were treated with either 1 mg AFB(1)/kg, ip or its vehicle (0.5% DMSO/saline), and 4 h later with either E. coli LPS (7. 4 x 10(6) EU/kg, iv) or its saline vehicle. Over a course of 6 to 96 h after AFB(1) administration, rats were killed and livers were stained immunohistochemically for PMNs. LPS resulted in an increase in PMN accumulation in the liver that preceded the onset of liver injury. To assess if PMNs contributed to the pathogenesis, an anti-PMN antibody was administered to reduce PMN numbers in blood and liver, and injury was evaluated. Hepatic parenchymal cell injury was evaluated as increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in serum and from histologic examination of liver sections. Biliary tract alterations were evaluated as increased concentration of serum bile acids and activities of gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), and 5'-nucleotidase (5'-ND) in serum. Neutrophil depletion protected against hepatic parenchymal cell injury caused by AFB(1)/LPS cotreatment but not against markers of biliary tract injury. This suggests that LPS augments AFB(1) hepatotoxicity through two mechanisms: one of which is PMN-dependent, and another that is not.     

Metallothionein-null mice are more sensitive than wild-type mice to liver injury induced by repeated exposure to cadmium.

Liver is a major target organ of cadmium (Cd) toxicity following acute and chronic exposure. Metallothionein (MT), a low-molecular-weight, cysteine-rich, metal-binding protein has been shown to play an important role in protection against acute Cd-induced liver injury. This study investigates the role of MT in liver injury induced by repeated exposure to Cd. Wild-type and MT-I/II knockout (MT I/II-null) mice were injected sc with a wide range of CdCl(2) doses, 6 times/week, for up to 10 weeks, and their hepatic Cd content, hepatic MT concentration, and liver injury were examined. Repeated administration of CdCl(2) produced acute and nonspecific chronic inflammation in the parenchyma and portal tracts and around central veins. Higher doses produced granulomatous inflammation and proliferating nodules in liver parenchyma. Apoptosis and mitosis occurred concomitantly in liver following repeated Cd exposure, whereas necrosis was mild. As a result, significant elevation of serum enzyme levels was not observed. In wild-type mice, hepatic Cd concentration increased in a dose- and time-dependent manner, reaching 400 microgram/g liver, along with 150-fold increases in hepatic MT concentrations, the latter reaching 1200 microgram/g liver. In contrast, in MT I/II-null mice, hepatic Cd concentrations were about 10 microgram/g liver. Despite the lower accumulation of Cd in livers of MT I/II-null mice, the maximum tolerated dose of Cd was one-eighth lower than that for wild-type mice at 10 weeks, and liver injury was more pronounced in the MT I/II-null mice, as evidenced by increases in liver/body weight ratios and histopathological analyses. In conclusion, these data indicate that (1) nonspecific chronic inflammation, granulomatous inflammation, apoptosis, liver cell regeneration, and presumably, preneoplastic proliferating nodules are major features of liver injury induced by repeated Cd exposure, and (2) intracellular MT is an important protein protecting against this Cd-induced liver injury.     

The role of the hemostatic system in murine liver injury induced by coexposure to lipopolysaccharide and trovafloxacin, a drug with idiosyncratic liability

The use of the fluoroquinolone antibiotic trovafloxacin (TVX) was severely restricted in 1999 due to its association with idiosyncratic hepatotoxicity. Previously, we reported that a nontoxic dose of TVX interacts with a nontoxic dose of lipopolysaccharide (LPS) to cause robust hepatocellular injury in mice. This interaction with LPS was not seen in mice treated with levofloxacin (LVX), a fluoroquinolone not associated with hepatotoxicity in people. TVX/LPS-coexposure caused an increase in plasma alanine aminotransferase (ALT) activity as early as 4.5 h after LPS administration which progressed through 15 h. We examined the role of the hemostatic system in TVX/LPS-induced liver injury. At the onset of liver injury, coexposure to TVX/LPS, but not exposure to TVX, LVX, LPS or LVX/LPS, caused increased plasma concentration of thrombin-antithrombin dimers and decreased plasma circulating fibrinogen. LPS treatment induced a small increase in plasma plasminogen activator inhibitor-1 (PAI-1) concentration, and TVX pretreatment enhanced this effect. TVX/LPS coexposure also resulted in hepatic fibrin deposition. Anticoagulant heparin administration reduced TVX/LPS-induced hepatic fibrin deposition and liver injury. PAI-1^−/− mice treated with TVX/LPS exhibited similar fibrin deposition to wild-type mice but had significantly reduced hepatocellular injury. PAI-1^−/− mice, but not heparin-treated mice, had reduced plasma concentrations of several cytokines compared to TVX/LPS-treated controls. In summary, TVX/LPS-coexposure caused an imbalance in the hemostatic system, resulting in thrombin activation increased, plasma concentration of PAI-1 and hepatic fibrin deposition. Both thrombin activation and PAI-1 play critical roles in the progression of TVX/LPS-induced liver injury, but through different modes of action.     

The role of interleukin-6 in pulmonary inflammation and injury induced by exposure to environmental air pollutants.

This study was designed to examine the role of the cytokine interleukin-6 (IL-6) in environmental air pollutant-induced pulmonary inflammation, injury, and repair. IL-6 knockout (KO) mice and wild-type (WT) mice were exposed to filtered air; aged and diluted cigarette smoke (ADSS), a surrogate for environmental tobacco smoke; ozone; or ADSS followed by ozone (ADSS/ozone). The proportion of monocytes and neutrophils recovered by bronchoalveolar lavage (BAL) as well as the level of total protein in BAL fluid were significantly increased in both IL-6 KO and WT mice following exposure to ozone or to ADSS/ozone. However, bromodeoxyuridine (BrdU) labeling within terminal bronchiolar epithelium and proximal alveolar regions in IL-6 KO mice exposed to ozone or to ADSS/ozone was significantly reduced compared with IL-6 sufficient mice (WT). WT mice treated with IL-6 antibodies also demonstrated a reduction in BrdU cell labeling similar to that observed in IL-6 KO mice following exposure to ozone or ADSS/ozone. Clara cell secretory protein (CCSP) abundance, a marker of Clara cell maturation and function, was markedly reduced in the terminal bronchiolar epithelium of WT mice following exposure to ADSS and/or ozone, whereas CCSP abundance was unchanged in IL-6 KO mice. We conclude that endogenous IL-6 in mice plays a critical role in the progress of lung inflammation/injury, but CCSP may also play a role to protect the lungs of mice exposed to toxic air pollutants. Data from this study further suggest that IL-6 antibody treatment modalities may be a means to attenuate pulmonary inflammation and injury.     

The endocannabinoid system as a key mediator during liver diseases: new insights and therapeutic openings

Chronic liver diseases represent a major health problem due to cirrhosis and its complications. During the last decade, endocannabinoids and their receptors have emerged as major regulators of several pathophysiological aspects associated with chronic liver disease progression. Hence, hepatic cannabinoid receptor 2 (CB(2)) receptors display beneficial effects on alcoholic fatty liver, hepatic inflammation, liver injury, regeneration and fibrosis. Cannabinoid receptor 1 (CB(1)) receptors have been implicated in the pathogenesis of several lesions such as alcoholic and metabolic steatosis, liver fibrogenesis, or circulatory failure associated with cirrhosis. Although the development of CB(1) antagonists has recently been suspended due to the high incidence of central side effects, preliminary preclinical data obtained with peripherally restricted CB(1) antagonists give real hopes in the development of active CB(1) molecules devoid of central adverse effects. CB(2) -selective molecules may also offer novel perspectives for the treatment of liver diseases, and their clinical development is clearly awaited. Whether combined treatment with a peripherally restricted CB(1) antagonist and a CB(2) agonist might result in an increased therapeutic potential will warrant further investigation.