Provocation of massive hepatic necrosis by endotoxin after partial hepatectomy in rats

When rats received endotoxin 48 hours after two-thirds liver resection, 50% of them died within 12 hours with massive hepatic necrosis at a dose that did not affect sham-operated rats. In the hepatic sinusoids, fibrin deposition and endothelial cell destruction occurred 5 hours after endotoxin administration. When antithrombin III concentrate was infused concomitantly with endotoxin administration, all rats survived 12 hours, and the extent of hepatic necrosis and the deranged serum glutamic pyruvic transaminase values were significantly attenuated at 5 hours compared with those in the control rats. Similar improvements in the incidence of mortality and liver injury were observed after treatment with gum arabic before hepatectomy. The stimulatory state of Kupffer cells based on the ability to produce superoxide anions estimated by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was increased between 24 and 72 hours after operation. This increase disappeared after gum arabic treatment. It is concluded that massive hepatic necrosis can occur as a result of sinusoidal fibrin deposition provoked by endotoxin in partially hepatectomized rats. Activated Kupffer cells may contribute to this provocation.     

Gut-derived substances in activation of hepatic macrophages after partial hepatectomy in rats.

When liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was performed in rats 24 h after two-thirds liver resection, there were marked deposits of formazan converted from nitro blue tetrazolium in hepatic macrophages throughout the liver, indicating macrophage activity. The extent of the deposits was significantly reduced when perfusion was performed following oral administration of polymyxin B sulfate, a non-absorbable bacteriocidal agent against gram-negative bacilli which can also bind endotoxin lipopolysaccharides. Polymyxin B sulfate administration also attenuated the derangements of SGPT and the histological liver injury provoked by endotoxin administration after partial hepatectomy. These results suggests that gut-derived substances sensitive to polymyxin B sulfate may contribute to activation of hepatic macrophages after partial hepatectomy in rats.     

Induction of ischemic tolerance in rat liver via reduced nicotinamide adenine dinucleotide phosphate oxidase in Kupffer cells

AIM To elucidate the mechanisms of hepatocyte preconditioning by H2O2 to better understand the pathophysiology of ischemic preconditioning.METHODS The in vitro effect of H2O2 pretreatment was investigated in rat isolated hepatocytes subjected to anoxia/reoxygenation. Cell viability was assessed with propidium iodide fluorometry. In other experiments, rat livers were excised and subjected to warm ischemia/reperfusion in an isolated perfused liver system to determine leakage of liver enzymes. Preconditioning was performed by H2O2 perfusion, or by stopping the perfusion for 10 min followed by 10 min of reperfusion.To inhibit Kupffer cell function or reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,gadolinium chloride was injected prior to liver excision, or diphenyleneiodonium, an inhibitor of NADPH oxidase, was added to the perfusate, respectively. Histological detection of o~gen radical formation in Kupffer cells was performed by perfusion with nitro blue tetrazolium.RESULTS Anoxia/reoxygenation decreased hepatocyte viability compared to the controls. Pretreatment with H2O2 did not improve such hepatocyte injury. In liver perfusion experiments, however, H2O2 preconditioning reduced warm ischemia/reperfusion injury, which was reversed by inhibition of Kupffer cell function or NADPH oxidase. Histological examination revealed that H2O2 preconditioning induced oxygen radical formation in Kupffer cells. NADPH oxidase inhibition also reversed hepatoprotection by ischemic preconditioning.CONCLUSION H2O2 preconditioning protects hepatocytes against warm ischemia/reperfusion injury via NADPH oxidase in Kupffer cells, and not directly. NADPH oxidase also mediates hepatoprotection by ischemic preconditioning.     

Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma.

A transgenic mouse strain that expresses the hepatitis B virus (HBV) large envelope protein in the liver was used to determine the extent of oxidative DNA damage that occurs during chronic HBV infection. This mouse strain develops a chronic necroinflammatory liver disease that mimics the inflammation, cellular hyperplasia, and increased risk for cancer that is evident in human chronic active hepatitis. When perfused in situ with nitroblue tetrazolium, an indicator for superoxide formation, the liver of transgenic mice displayed intense formazan deposition in Kupffer cells, indicating oxygen radical production, and S-phase hepatocytes were commonly seen adjacent to the stained Kupffer cells. Similar changes were not observed in nontransgenic control livers. To determine whether these events were associated with oxidative DNA damage, genomic DNA from the livers of transgenic mice and nontransgenic controls was isolated and examined for 8-oxo-2'-deoxyguanosine, an oxidatively modified adduct of deoxyguanosine. Results showed a significant, sustained accumulation in steady-state 8-oxo-2'-deoxyguanosine that started early in life exclusively in the transgenic mice and increased progressively with advancing disease. The most pronounced increase occurred in livers exhibiting microscopic nodular hyperplasia, adenomas, and hepatocellular carcinoma. Thus, HBV transgenic mice with chronic active hepatitis display greatly increased hepatic oxidative DNA damage. Moreover, the DNA damage occurs in the presence of heightened hepatocellular proliferation, increasing the probability of fixation of the attendant genetic and chromosomal abnormalities and the development of hepatocellular carcinoma.     

Kupffer cell-independent acute hepatocellular oxidative stress and decreased bile formation in post-cold-ischemic rat liver

The purpose of this study was to examine distribution and time history of oxidative stress during the hyperacute period of reperfusion in the liver grafts undergoing cold ischemia and to investigate roles of Kupffer cells as a potential oxidant source. Rat livers were harvested at 4 degrees C in University of Wisconsin solution and followed by reperfusion with Krebs-Henseleit buffer under monitoring bile excretion. To investigate oxidative changes, laser-confocal microfluorography was performed in reperfused livers preloaded with dichlorodihydrofluorescein diacetate succinimidyl ester, a fluorescence precursor sensing intracellular hydroperoxide generation. Livers undergoing the 16-hour cold storage displayed an impaired recovery of bile acid-dependent bile output concurrent with a marked increase in hydroperoxide generation in hepatocytes, which occurred as early as 5 minutes after the onset of reperfusion, whereas the status of lobular perfusion was well maintained. Pretreatment with liposome-encapsulated dichloromethylene diphosphonate, a Kupffer cell-depleting reagent, did neither alter the reperfusion-induced periportal oxidative changes nor improve the recovery of bile output in the graft. On the other hand, EPCK, a hepatotropic antioxidant composed of vitamin E phosphate ester bound to vitamin C, not only diminished the oxidative changes but also improved the reduction of bile acid-dependent bile output. Furthermore, the reagent was capable of inhibiting H(2)O(2)-induced oxidative stress in cultured hepatocytes. These results suggest that hepatocytes constitute a major site of the oxidative insult triggered through Kupffer cell-independent mechanisms and serve as an important cellular component to be protected by antioxidant therapeutics.     

Ischemic preconditioning protects hepatocytes via reactive oxygen species derived from Kupffer cells in rats

BACKGROUND AND AIMS: Hepatic ischemic preconditioning decreases sinusoidal endothelial cell injury and Kupffer cell activation after cold ischemia/reperfusion, leading to improved survival of liver transplant recipients in rats. Ischemic preconditioning also protects livers against warm ischemia/reperfusion injury, in which hepatocyte injury is remarkable. We aimed to determine whether ischemic preconditioning directly protects hepatocytes and to elucidate its mechanisms. METHODS: Rats were injected with gadolinium chloride to deplete Kupffer cells or with N -acetyl- l -cysteine, superoxide dismutase, or catalase to scavenge reactive oxygen species. Livers were then preconditioned by 10 minutes of ischemia and 10 minutes of reperfusion. Subsequently, livers were subjected to 40 minutes of warm ischemia and 60 minutes of reperfusion in vivo or in a liver perfusion system. In other rats, livers were preconditioned by H(2)O(2) perfusion instead of ischemia. In the other experiments, livers were perfused with nitro blue tetrazolium to detect reactive oxygen species formation. RESULTS: Ischemic preconditioning decreased injury in hepatocytes, but not in sinusoidal endothelial cells. Kupffer cell depletion itself did not change hepatocyte injury after ischemia/reperfusion, indicating no contribution of Kupffer cells to ischemia/reperfusion injury. However, Kupffer cell depletion reversed hepatoprotection by ischemic preconditioning. Reactive oxygen species formation occurred in Kupffer cells after ischemic preconditioning. Scavenging of reactive oxygen species reversed the effect of ischemic preconditioning, and H(2)O(2) preconditioning mimicked ischemic preconditioning. CONCLUSIONS: Ischemic preconditioning directly protected hepatocytes after warm ischemia/reperfusion, which is not via suppression of changes in sinusoidal cells as in cold ischemia/reperfusion injury. This hepatocyte protection was mediated by reactive oxygen species produced by Kupffer cells.     

Interaction between isolated and purified liver cells and small unilamellar liposomes

ABSTRACT— Aims/Background: The mechanism of interaction and the role played by the vesicle lipid composition for the selective association between liposomes and liver cells were studied, at the ultrastructural level, by investigating both in situ and in vitro the interaction between hepatocytes, Kupffer and endothelial liver cells with egg-phosphatidylcholine (eggPC) or eggPC/stearylamine (9:1; mol:mol) reverse-phase evaporation (REV) liposomes. Methods: Liver cells from rats, isolated by enzymatic perfusion and purified by differential centrifugation, were incubated, in a rotating bath at 37°C, with liposomes (2.5 mM final liposomal lipid concentration). Cell aliquots were withdrawn and processed for electron microscope observation at fixed time intervals. Parallel experiments were carried out by in situ liver perfusion with liposome suspensions. Results and Conclusions: Our first conclusions are: 1) lipidic composition affects the rate of liposomes uptake and internalization by hepatocytes; 2) liposome uptake by hepatocytes or Kupffer cells is likely an endocytic process; 3) endothelial cells internalize lipid vesicles as well; 4) liposome uptake was due to a phagocytic activity for all isolated liver cells, while in the in situ observation endothelial cells seem to use another mechanism (fusion); and 5) the rate of internalization is related to the viability of the treated cells. Experimental data seem to indicate that differential behaviour in the internalization of lipid vesicles exists among parenchymal, Kupffer and endothelial liver cells. These differences suggest that clearance of liposomes by these cells involves two mechanisms (i.e., endocytosis or fusion) with different rates of uptake and internalization that facilitate the design of carriers that can deliver drugs preferentially to a specific liver cell type.     

Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short-term ethanol perfusion in the rat

BACKGROUND/AIMS: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O2*-) release into the hepatic sinusoids during short-term exposure to ethanol. METHODS: The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert-buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O2*- production, MCLA (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one), a Cypridina luciferin analogue, was simultaneously infused and MCLA-enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL3) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine-threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4-methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol-induced chemiluminescence were also evaluated. Sites where O2*- could be released were determined by histochemical detection of nitro blue tetrazolium reduction. RESULTS: Both ethanol and tert-buthanol rapidly caused O2*- release. GdCL3 suppressed the ethanol-induced O2*- release by 61%. Staurosporine and DPI, but neither IB nor 4-MP, also significantly inhibited the ethanol-induced O2*- release. In the histochemical examination, ethanol-stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl3-pretreated liver had the precipitate only on SECs. CONCLUSIONS: This study shows that ethanol itself stimulates both SECs and KCs to release O2*- via activation of NADPH oxidase probably involving protein kinase C (PKC).     

Efficacy of hypothermic perfusion using University of Wisconsin solution in extended hepatectomy with hepatic inflow occlusion in a canine model

This study was designed to elucidate the efficacy of University of Wisconsin (UW) solution for preventing liver injury, when used as a hypothermic perfusate infused into the systemic circulation during extended hepatectomy with hepatic inflow occlusion. Adult mongrel dogs (9.5–17.5 kg, n= 14) were subjected to 75% hepatectomy under 60 min hepatic inflow occlusion. The animals were divided into two groups. The UW group (n= 7) underwent hypothermic perfusion using 4°C UW solution (core temperature of the liver: 12.3±0.2°C). The control group designated as the Ringer's lactate (LR) group (n= 7) underwent hypothermic perfusion using 4°C LR solution. The perfusate was introduced into the systemic circulation via the hepatic vein. Blood from the hepatic vein was sampled, and alanine amino-transferase, purine nucleoside phosphorylase activities and the ammonia concentration were measured. The 7 day survival rate was higher in the UW group than in the LR group. The parameters of liver function were less significantly altered in the UW group than in the LR group. The plasma ammonia concentration was significantly (P<0.05) lower 6h after reperfusion in the UW group than in the LR group. A small volume of hypothermic perfusion of the liver using UW solution was safe if it returned to systemic circulation. Hypothermic perfusion of the liver using UW solution may be effective for preventing hepatic tissue injury during extended hepatectomy with hepatic vascular occlusion.     

Evidence for enhanced secretory function of hepatic macrophages after long-term ethanol feeding in rats

ABSTRACT— Rats were pair-fed nutritionally adequate liquid diets, containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4–6 weeks. Ruffle formation of hepatic macrophages in the periportal area observed with a transmission electron microscope (which reflects their extent in activation) was more remarkable in ethanol-fed rats than in control rats. The ability of hepatic macrophages to produce superoxide anions assessed in situ by formazan deposition after liver perfusion with nitro-blue tetrazolium and phorbol myristate acetate was enhanced after such ethanol feeding. A similar result was seen 24 h after withdrawal of ethanol feeding. These findings suggest that long-term ethanol consumption may activate hepatic macrophages in secretory function.     

Kupffer cells participate in early clearance of syngeneic hepatocytes transplanted in the rat liver

BACKGROUND & AIMS: Kupffer cells are activated shortly after deposition of hepatocytes in liver sinusoids, with clearance of a significant fraction of transplanted cells, especially when cells are entrapped in portal spaces. We determined whether perturbation of Kupffer cells would improve transplanted cell engraftment. METHODS: Dipeptidyl peptidase IV-deficient rats were used as recipients of syngeneic Fischer 344 rat hepatocytes. Kupffer cell function was analyzed by measuring phagocytic activity with carbon particle or (99m)Tc-sulfur colloid incorporation. Transplanted cell survival and integration in the liver parenchyma was determined by histochemical analysis of tissues. Transplanted cell proliferation was analyzed in rats conditioned with retrorsine and partial hepatectomy. RESULTS: Gadolinium chloride significantly impaired Kupffer cell function, especially in periportal areas, where transplanted cells were localized. Transplanted cell survival increased by approximately 2-fold in animals treated with gadolinium chloride 24 hours before cell transplantation. In gadolinium-treated rats, more transplanted cells were observed in portal vein radicles, as well as in liver sinusoids, albeit integration of cells in the liver parenchyma was slower in gadolinium-treated rats and cells separated from other hepatocytes in portal vein radicles that failed to exhibit bile canalicular reconstitution. Finally, hepatocyte transplantation in rats primed with retrorsine and partial hepatectomy showed accelerated kinetics of liver repopulation in animals pretreated with gadolinium chloride. CONCLUSIONS: Perturbation of Kupffer cell activity will benefit liver repopulation with cells and further analysis of clinically suitable approaches to exploit this mechanism will be appropriate.     

Chlamydia pneumoniae replicates in Kupffer cells in mouse model of liver infection

INTRODUCTIONChlamydia pneumoniae(C.pneumoniae)is a common cause of respiratory infections in humans[1,2],and it is also associated with outcomes other than respiratory disease,including coronary heart disease and myocardial infarction[3,4].Systemic diseas…     

Pathogenesis of alcoholic liver disease: interactions between parenchymal and non-parenchymal cells

The development of alcoholic liver disease (ALD) is a complex process involving both the parenchymal and non-parenchymal cells in the liver. The impact of ethanol on hepatocytes can be characterized as a condition of organelle stress with multifactorial changes in hepatocellular function accumulating during ethanol exposure. These changes include oxidative stress, mitochondrial dysfunction, decreased methylation capacity, endoplasmic reticulum stress, impaired vesicular trafficking and altered proteasome function. Injury to hepatocytes is attributed, in part, to ethanol metabolism by the hepatocytes. Changes in the structural integrity of hepatic sinusoidal endothelial cells, as well as enhanced inflammation in the liver during ethanol exposure are also important contributors to injury. Activation of hepatic stellate cells initiates the deposition of extracellular matrix proteins characteristic of fibrosis. Kupffer cells, the resident macrophages in the liver, are particularly critical to the onset of ethanol-induced liver injury. Chronic ethanol exposure sensitizes Kupffer cells to activation by lipopolysaccharides via toll-like receptor 4. This sensitization enhances the production of inflammatory mediators, such as tumor necrosis factor-α and reactive oxygen species that contribute to hepatocyte dysfunction, necrosis and apoptosis of hepatocytes and the generation of extracellular matrix proteins leading to fibrosis. In this review we provide an overview of the complex interactions between parenchymal and non-parenchymal cells in the liver during the progression of ethanol-induced liver injury.     

Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short‐term ethanol perfusion in the rat

Abstract: Background/Aims: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O₂^·?) release into the hepatic sinusoids during short‐term exposure to ethanol. Methods: The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert‐buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O₂^·? production, MCLA (2‐methyl‐6‐[p‐methoxyphenyl]‐3,7‐dihydroimidazo[1,2‐a]pyrazin‐3‐one), a Cypridina luciferin analogue, was simultaneously infused and MCLA‐enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL₃) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine–threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4‐methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol‐induced chemiluminescence were also evaluated. Sites where O₂^·? could be released were determined by histochemical detection of nitro blue tetrazolium reduction. Results: Both ethanol and tert‐buthanol rapidly caused O₂^·? release. GdCL₃ suppressed the ethanol‐induced O₂^·? release by 61%. Staurosporine and DPI, but neither IB nor 4‐MP, also significantly inhibited the ethanol‐induced O₂^·? release. In the histochemical examination, ethanol‐stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl₃‐pretreated liver had the precipitate only on SECs. Conclusions: This study shows that ethanol itself stimulates both SECs and KCs to release O₂^·? via activation of NADPH oxidase probably involving protein kinase C (PKC).     

Deranged blood coagulation equilibrium as a factor of massive liver necrosis following endotoxin administration in partially hepatectomized rats

Activated Kupffer cells provoke massive liver necrosis after endotoxin stimulation through microcirculatory disturbance caused by sinusoidal fibrin deposition in rats undergoing 70% hepatectomy. In these rats, serum activities of purine nucleoside phosphorylase (PNP) and alanine transaminase (ALT) were increased at 1 and 5 hours, respectively, following endotoxin administration. When 70% resected liver was perfused with Dulbecco's modified Eagle medium (DMEM) containing heat-inactivated fetal calf serum, the increase in both enzyme activities was not affected by addition of endotoxin during perfusion, suggesting that activated Kupffer cells injured neither sinusoidal endothelial cells nor hepatocytes. The activity of tissue factor, an initiator of blood coagulation cascade, was much higher in Kupffer cells isolated from partially hepatectomized rats than in those from normal rats. In contrast, mRNA expressions of tissue factor pathway inhibitor (TFPI) as well as thrombomodulin were almost undetectable in normal and partially resected livers. When recombinant human TFPI was injected intravenously in 70% hepatectomized rats, TFPI was markedly stained on the surfaces of sinusoidal endothelial cells and microvilli of hepatocytes on immunohistochemistry. In these rats, endotoxin-induced liver injury was significantly attenuated compared with rats given no TFPI. Similar attenuation was also found in rats receiving recombinant human thrombomodulin. These results suggest that fibrin deposition developing in 70% hepatectomized rats after endotoxin administration may be caused by deranged blood coagulation in the hepatic sinusoids through increasing tissue factor activity in Kupffer cells and minimal TFPI and thrombomodulin in endothelial cells. The destruction of sinusoidal endothelial cells as well as hepatocytes may occur as a result of microcirculatory disturbance caused by such sinusoidal fibrin deposition.     

Interactions between isolated hepatocytes and Kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein

Like the rat peritoneal macrophage, the isolated Kupffer cell is capable of processing and releasing iron acquired by phagocytosis of immunosensitized homologous red blood cells. When erythrophagocytosis is restrained to levels which do not affect cell viability, about one red cell per macrophage, close to 50% of iron acquired from red cells is released within 24 hr in the form of ferritin. Immunoradiometric assay of the extracellular medium indicates that 160 ng ferritin are released by 10(6) Kupffer cells after 24-hr incubation at 37 degrees C. Iron release is temperature-dependent, the rate at 37 degrees C being nearly 5-fold greater than at 4 degrees C. As estimated by sucrose-gradient ultracentrifugation, ferritin released by the erythrophagocytosing Kupffer cell averages 2,400 iron atoms per molecule. When reincubated with isolated hepatocytes, this released ferritin is rapidly taken up by the cells. Via this process, hepatocytes may accumulate more than 160,000 iron atoms per cell per min. Such accumulation is not impeded by the presence of iron-loaded transferrin in the culture medium, but is markedly depressed by rat liver ferritin. In contrast to the conservation of transferrin during its interaction with hepatocytes, the protein shell of the ferritin molecule is rapidly degraded into trichloroacetic acid-soluble fragments. Ferritin-mediated transfer of iron from Kupffer cells to hepatocytes may help explain the resistance of the liver to iron deficiency as well as the liver's susceptibility to iron overload.     

Chlamydia pneumoniae replicates in Kupffer cells in mouse model of liver infection

AIM: To develop an animal model of liver infection with Chlamydia pneumoniae (C.pneumoniae) in intraperitoneally infected mice for studying the presence of chlamydiae in Kupffer cells and hepatocytes.METHODS: A total of 80 BALB/c mice were inoculated intraperitoneally with C. pneumoniae and sacrificed at various time points after infection. Chlamydiae were looked for in liver homogenates as well as in Kupffer cells and hepatocytes separated by liver perfusion with collagenase. C. pneumoniae was detected by both isolation in LLC-MK2 cells and fluorescence in situ hybridization (FISH). The releasing of TNFA-α by C. pneumoniae in vitro stimulated Kupffer cells was studied by enzymelinked immunosorbent assay.RESULTS: C. pneumoniae isolation from liver homogenates reached a plateau on d 7 after infection when 6 of 10 animals were positive, then decreased, and became negative by d 20. C. pneumoniae isolation from separated Kupffer cells reached a plateau on d 7 when 5 of 10 animals were positive, and became negative by d 20.The detection of C. pneumoniae in separated Kupffer cells by FISH, confirmed the results obtained by culture.Isolated hepatocytes were always negative. Stimulation of Kupffer cells by alive C. pneumoniae elicited high TNF-α levels.CONCLUSION: A productive infection by C. pneumoniae may take place in Kupffer cells and C. pneumoniae induces a local pro-inflammatory activity. C. pneumoniae is therefore, able to act as antigenic stimulus when localized in the liver. One could speculate that C. pneumoniae infection, involving cells of the innate immunity such as Kupffer cells, could also trigger pathological immune reactions involving the liver, as observed in human patients with primary biliary cirrhosis.     

Post-ischemic intraportal adenosine administration protects against reperfusion injury of canine liver

Although adenosine has been postulated to inhibit ischemia‐reperfusion injury in various tissues, its in vivo cytoprotective mechanism is not fully known. The aim of this study was to determine the effect of intraportally infused adenosine on reperfusion injury in the canine liver. Two h ischemia and reperfusion of the liver were induced in beagle dogs by clamping the portal triad. Either adenosine or saline was infused in the portal vein after reperfusion for 60 min. Levels of serum aspartate aminotransferase and alanine aminotransferase and the survival of animals were examined. Hepatic levels of protein carbonyls and glutathione were also measured, as markers of oxidative stress. One h after reperfusion, the liver was perfused with nitroblue tetrazolium and the formation of formazan was observed to evaluate superoxide formation. Twenty‐four h after reperfusion, 100% of animals in the adenosine group and 33% of animals in the control group survived. Adenosine significantly decreased the reperfusion‐induced increase in serum levels of aspartate aminotransferase and alanine aminotransferase. Adenosine also suppressed the formation of protein carbonyls and the decrease in glutathione levels. Histologically, neutrophil infiltration, superoxide formation, and apoptosis were decreased by adenosine. These results suggest that intraportally infused adenosine attenuates reperfusion injury of the liver, presumably by suppressing the activation of neutrophils and oxidative stress.     

Morphological and functional changes after portal vein occlusion in rats

We investigated morphological and functional changes after portal vein occlusion in rats. Portal branches for the median and left lateral lobes were ligated, after which the lobes were examined micromorphologically. After embolization of the same branches, regenerative capacity was evaluated in normal livers and in livers with CCl₄-induced cirrhosis. Indocyanine green elimination, antithrombin III activity, and Kupffer cell density were also investigated. In another set of rats, the embolized lobes were resected 0, 2, 4, or 7 days after portal vein embolization (PVE), and endotoxin was injected intravenously 48h after each hepatectomy. In the ligated lobes, apoptotic hepatocytes were detected mainly around a widespread necrotic area on day 2, and among normal hepatocytes on day 7. In the nonembolized livers of control rats, increases were noted in liver weight, ornithine decarboxylase (ODC) activity, DNA synthesis, and mitosis of hepatocytes. In the cirrhotic livers, ODC activity was stimulated in a fashion similar to that seen in control liver, but DNA synthesis and weight change was delayed, although not significantly. On days 2, 4, 7, and 14 PVE, Kupffer cell density was about twice that seen in rats before PVE. Endotoxin-induced liver injury was slight if the rats had received PVE 4-7 days before the hepatectomy.     

Kupffer cells promote lead nitrate-induced hepatocyte apoptosis via oxidative stress

BACKGROUND: Apoptosis and its modulation are crucial factors for the maintenance of liver health, allowing hepatocytes to die without provoking a potential harmful inflammatory response through a tightly controlled and regulated process. Since Kupffer cells play a key role in the maintenance of liver function, the aim of this study was to verify whether Kupffer cells are involved in the induction of liver apoptosis after i.v. injection of Pb(NO3)2 likely by secretion mechanisms. RESULTS: The in vivo hepatic apoptosis, induced by Pb(NO3)2 was prevented by a pre-treatment with gadolinium chloride (GdCl3), a Kupffer cells toxicant, that suppresses Kupffer cell activity and reduces to a half the apoptotic rate. In addition, in vivo Pb(NO3)2 administration deprives hepatocytes of reduced glutathione, whereas the loss of this important oxidation-preventing agent is considerably mitigated or abolished by pre-treatment with GdCl3. However, incubation of isolated hepatocytes and Kupffer cells and HepG2 cells with Pb(NO3)2 for 24 hours induced necrotic but not apoptotic cells. Apoptosis of hepatocytes and HepG2 cells was observed only after the addition of conditioned medium obtained from Kupffer cells cultured for 24 hours with Pb(NO3)2, thus indicating the secretion of soluble mediators of apoptosis by Kupffer cells. Apoptosis in the HepG2 cells was observed upon 24-hours incubation of HepG2 cells with 1 mM buthionine sulfoximine, a glutathione depleting agent, thus showing that there is an oxidative apoptogenic pathway in HepG2 cells. CONCLUSION: Pb(NO3)2 has, at most, a direct necrotic (but not apoptogenic) effect on hepatocytes and HepG2 cells, giving a clue about the regulatory role of Kupffer cells in the induction of liver apoptosis after a single Pb(NO3)2 injection without pre-treatment with GdCl3, probably via secreting soluble factors that trigger oxidative stress in target cells.