Lipid peroxidation as molecular mechanism of liver cell injury during reperfusion after ischemia

The pathophysiological importance of reactive oxygen species has been extensively documented in the pathogenesis of hepatic ischema-reperfusion injury. Kupffer cells and neutrophils were identified as the dominant sources of the postischemic oxidant stress. To test the hypothesis that a direct free radical-mediated injury mechanism (lipid peroxidation; LPO) may be involved in the pathogenesis, highly sensitive and specific parameters of LPO, i.e., hydroxy-eicosatetraenoic acids (HETES), and F₂-isoprostanes, were determined by gas chromatographic-mass spectrometric analysis in liver tissue and plasma during 45 min of hepatic ischemia and up to 24 h of reperfusion. A significant 60–250% increase of F₂-isoprostane levels in plasma was found at all times during reperfusion; the HETE content increased only significantly at 1 h of reperfusion and in severely necrotic liver tissue at 24 h with increases between 90–320%. On the other hand, in a model of LPO-induced liver injury (infusion of 0.8 μmol tert-butylhydroperoxide/min/g liver), the hepatic HETE content increased two to fourfold over baseline values at 45 min, i.e., before liver injury. A further increase to 12- to 30-fold of baseline was observed during moderate liver injury. Based on these quantitative comparisons of LPO and liver injury, it seems highly unlikely that LPO is the primary mechanism of parenchymal cell injury during reperfusion, although it cannot be excluded that LPO may be important as a damaging mechanism in a limited compartment of the liver, e.g., endothelial cells, close to the sources of reactive oxygen, e.g., Kupffer cells and neutrophils.     

Short term regulation of hepatocyte glutathione content by hepatic sinusoidal cells in co-culture

An inadequate balance between oxidant species and antioxidant mechanisms may constitute the primary mechanisms of a number of pathologies. The liver plays a central role in this balance: parenchymal hepatic cells contain and export especially high levels of the antioxidant glutathione and activated Kupffer cells release inflammation mediators and reactive oxygen species. There is growing evidence of a paracrine regulation of hepatic function by means of a fluent intercellular communication which must still be fully elucidated, especially in basal conditions. In vivo models provide often too complex results but, in vitro, tissue interactions are left aside; therefore it is important to find new experimental models to address cell communication studies. Here we propose the complementary use of three models to study liver glutathione system regulation in basal conditions: pure parenchymal cells primary cultures, addition of sinusoidal cell conditioned media to parenchymal cells and co-culture of sinusoidal cells using porous membranes. We have also developed a high specifity immunofluorescent method for the complete characterization of sinusoidal cell populations by flow cytometry and confocal microscopy. Our results show that Kupffer cells possess higher levels of reactive oxygen species than sinusoidal endothelial cells even in basal conditions. We also report that the glutathione content of hepatic parenchymal cells in basal conditions is regulated by a sinusoidal-parenchymal cells cross-talk and suggest the existence of a paracrine circuit in the management of liver oxidative stress.     

Tri-iodothyronine differentially induces Kupffer cell ED1/ED2 subpopulations

Thyroid calorigenesis is carried out by activation of cytochrome-c oxidase, as well as by induction of mitochondrial and nuclear genes that code for cell respiratory apparatus components and uncoupling proteins. These effects operate increments in basal metabolic rate and also lead to increased production of oxygen and nitrogen reactive species in liver parenchymal cells. The hepatic antioxidant system is also compromised, since superoxide dismutase and catalase activities, glutathione content and lipid soluble antioxidants are reduced. Liver macrophages contribute to the hepatic oxidative stress observed in T(3)-treated rats, and both Kupffer cell hyperplasia and hypertrophy are reported. Kupffer cells constitute the main fixed macrophage population in the body and are a heterogeneous group of cells, derived from a less numerous population of local precursors, which are morphologically fairly distinguishable from the mature lineage elements. ED1 and ED2 antigens have been particularly useful in the characterization of Kupffer cell subpopulations. In particular, antibodies against these antigens provided evidence that T(3)- induced Kupffer cell hyperplasia causes a shift on liver macrophage population phenotype, leaning towards younger cell types. Despite the fact that sinusoidal environment itself stimulates the proliferation of macrophage precursors and their differentiation into Kupffer cells, increased Kupffer cell turnover rates modify the sinusoidal environment and may imply further functional effects. Thus, Kupffer cell hyperplasia secondary to increased T(3) levels is potentially a pro-inflammatory event, which involves both, the expansion of Kupffer cell precursor population by means of circulating monocyte recruitment, and the differentiation of preexisting local Kupffer cell precursors into mature liver macrophages.     

Mammalian cell injury induced by hypothermia- the emerging role for reactive oxygen species

Hypothermia is a well-known strategem to protect biological material against injurious or degradative processes and is widely used in experimental and especially in clinical applications. However, hypothermia has also proved to be strongly injurious to a variety of cell types. Hypothermic injury to mammalian cells has long been attributed predominantly to disturbances of cellular ion homeostasis, especially of sodium homeostasis. For many years, reactive oxygen species have hardly been considered in the pathogenesis of hypothermic injury to mammalian cells. In recent years, however, increasing evidence for a role of reactive oxygen species in hypothermic injury to these cells has accumulated. Today there seems to be little doubt that reactive oxygen species decisively contribute to hypothermic injury in diverse mammalian cells. In some cell types, such as liver and kidney cells, they even appear to play the central role in hypothermic injury, outruling by far a contribution of the cellular ion homeostasis. In these cells, the cellular chelatable, redox-active iron pool appears to be decisively involved in the pathogenesis of hypothermic injury and of cold-induced apoptosis that occurs upon rewarming of the cells after a (sublethal) period of cold incubation.     

Primary role of Kupffer cell–hepatocyte communication in the expression of oxidative stress in the post-ischaemic liver

It has been reported that hepatocyte metabolism and function can be modulated by the activated Kupffer cell through the release of different biomolecules like cytokines, eicosanoids, oxygen free radicals and enzymes. In relation to these paracrine factors involved in circuits of intercellular communication, the existence of a hepatic oxygen sensor located in the Kupffer cell has been postulated. According to this postulate the oxygen metabolism of the liver parenchymal cells could be under the control of the Kupffer cells. In order to study the role of the Kupffer cell in the reperfusion syndrome of the liver, a lobular ischaemia–reperfusion model was performed in rats with or without previous treatment with gadolinium chloride to block Kupffer cell function. Spontaneous chemiluminescence of the liver surface, oxygen uptake by tissue slices and tert-butyl hydroperoxide-initiated chemiluminescence determinations were performed to evaluate the oxygen metabolism and the oxy-radical generation by the liver. The lower basal photoemission, in parallel with a lower basal oxygen uptake registered in the hepatic lobes from the animals pretreated with gadolinium chloride clearly indicates that the gadolinium chloride-dependent functional inhibition of Kupffer cell leads to a downregulation of oxygen metabolism by the liver. Moreover, the intensity of oxidative stress exhibited by the postischaemic lobes appears to be closely linked with the Kupffer cell activity. On the basis of the data obtained we propose that a paracrine circuit between activated Kupffer cell and hepatocytes is an early key event in the induction of postischaemic oxidative stress in the liver. Furthermore the interference with the mitochondrial electron flow by some biomolecules released from the activated Kupffer cell, such as tumour necrosis factor, interleukins, eicosanoids, etc., would increase the rate of generation of reactive oxygen species by the inhibited mitochondrial respiratory chain. © 1998 John Wiley & Sons, Ltd.     

Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning

Hepatic ischemia/reperfusion injury has immediate and deleterious effects on the outcome of patients after liver surgery. The precise mechanisms leading to the damage have not been completely elucidated. However, there is substantial evidence that the generation of oxygen free radicals and disturbances of the hepatic microcirculation are involved in this clinical syndrome. Microcirculatory dysfunction of the liver seems to be mediated by sinusoidal endothelial cell damage and by the imbalance of vasoconstrictor and vasodilator molecules, such as endothelin (ET), reactive oxygen species (ROS), and nitric oxide (NO). This may lead to no-reflow phenomenon with release of proinflammatory cytokines, sinusoidal plugging of neutrophils, oxidative stress, and as an ultimate consequence, hypoxic cell injury and parenchymal failure. An inducible potent endogenous mechanism against ischemia/reperfusion injury has been termed ischemic preconditioning. It has been suggested that preconditioning could inhibit the effects of different mediators involved in the microcirculatory dysfunction, including endothelin, tumor necrosis factor-alpha, and oxygen free radicals. In this review, we address the mechanisms of liver microcirculatory dysfunction and how ischemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.     

The interaction in vivo of transferrin and asialotransferrin with liver cells.

Rat transferrin or asialotransferrin doubly radiolabelled with 59Fe and 125I was injected into rats. A determination of extrahepatic and hepatic uptake indicated that asialotransferrin delivers a higher fraction of the injected 59Fe to the liver than does transferrin. In order to determine in vivo the intrahepatic recognition sites for transferrin and asialotransferrin, the liver was subfractionated into parenchymal, endothelial and Kupffer cells by a low-temperature cell isolation procedure. High-affinity recognition of transferrin (competed for by an excess of unlabelled transferrin) is exerted by parenchymal cells as well as endothelial and Kupffer cells with a 10-fold higher association (expressed per mg of cell protein) to the latter cell types. In all three cell types iron delivery occurs, as concluded from the increase in cellular 59Fe/125I ratio at prolonged circulation times of transferrin. It can be calculated that parenchymal cells are responsible for 50-60% of the interaction of transferrin with the liver, 20-30% is associated with endothelial cells and about 20% with Kupffer cells. For asialotransferrin a higher fraction of the injected dose becomes associated with parenchymal cells as well as with endothelial and Kupffer cells. Competition experiments in vivo with various sugars indicated that the increased interaction of asialotransferrin with parenchymal cells is specifically inhibited by N-acetylgalactosamine whereas mannan specifically inhibits the increased interaction of asialotransferrin with endothelial and Kupffer cells. Recognition of asialotransferrin by galactose receptors from parenchymal cells or mannose receptors from endothelial and Kupffer cells is coupled to active 59Fe delivery to the cells. It is concluded that, as well as parenchymal cells, liver endothelial and Kupffer cells are also quantitatively important intrahepatic sites for transferrin and asialotransferrin metabolism, an interaction exerted by multiple recognition sites on the various cell types.     

Free radicals, chemokines, and cell injury in HIV-1 and SIV infections and alcoholic hepatitis

Oxidative stress has been observed in HIV-1 infection and alcoholic liver disease. The formation of reactive oxygen species (ROS) contributes to cell injury through apoptosis and/or necrosis and secretion of proinflammatory cytokines and chemokines. The major sources of ROS and chemokines are the Kupffer cells. During chronic ethanol consumption they are primed and activated for enhanced formation of pro-inflammatory factors, probably as a result of ethanol-induced translocation of gut-derived endotoxin into the circulation. Pro-inflammatory factors produced in the liver stimulate neutrophilic and/or lymphocytic infiltration to this organ. The presence of inflammatory cells in the liver may compromise the hepatocytes to injury by releasing cytotoxic factors, i.e., ROS, cytolytic proteases. Kupffer cells also interact with the glycoprotein 120 of SIV and HIV-1, which can induce oxidative stress and chemokine release. CD4+ lymphocytes that are infected with the virus generate intracellular ROS, which in turn leads to apoptosis and cell death. Downregulation of CD4+ cells contributes to immunodeficiency, while enhanced sequestration of inflammatory cells in the liver during chronic ethanol use with or without HIV-1/SIV may result in hepatocyte injury and exacerbation of alcoholic liver disease.     

Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelial liver cells

Conditioned media of isolated Kupffer and endothelial liver cells were added to incubations of parenchymal liver cells, in order to test whether secretory products of Kupffer and endothelial liver cells could influence parenchymal liver cell metabolism. With Kupffer cell medium an average stimulation of glucose production by parenchymal liver cells of 140% was obtained, while endothelial liver cell medium stimulated with an average of 127%. The separation of the secretory products of Kupffer and endothelial liver cells in a low and a high molecular weight fraction indicated that the active factor(s) had a low molecular weight. Media, obtained from aspirin-pretreated Kupffer and endothelial liver cells, had no effect on the glucose production by parenchymal liver cells. Because aspirin blocks prostaglandin synthesis, it was tested if prostaglandins could be responsible for the effect of media on parenchymal liver cells. It was found that prostaglandin (PG) E1, E2, and D2 all stimulated the glucose production by parenchymal liver cells, PGD2 being the most potent. Kupffer and endothelial liver cell media as well as prostaglandins E1, E2, and D2 stimulated the activity of phosphorylase, the regulatory enzyme in glycogenolysis. The data indicate that prostaglandins, present in media from Kupffer and endothelial liver cells, may stimulate glycogenolysis in parenchymal liver cells. This implies that products of Kupffer and endothelial liver cells may play a role in the regulation of glucose homeostasis by the liver.     

Interaction in vivo and in vitro of apolipoprotein E-free high-density lipoprotein with parenchymal, endothelial and Kupffer cells from rat liver.

The interaction of apolipoprotein (apo) E-free high-density lipoprotein (HDL) with parenchymal, endothelial and Kupffer cells from liver was characterized. At 10 min after injection of radiolabelled HDL into rats, 1.0 +/- 0.1% of the radioactivity was associated with the liver. Subfractionation of the liver into parenchymal, endothelial and Kupffer cells, by a low-temperature cell-isolation procedure, indicated that 77.8 +/- 2.4% of the total liver-associated radioactivity was recovered with parenchymal cells, 10.8 +/- 0.8% with endothelial cells and 11.3 +/- 1.7% with Kupffer cells. It can be concluded that inside the liver a substantial part of HDL becomes associated with endothelial and Kupffer cells in addition to parenchymal cells. With freshly isolated parenchymal, endothelial and Kupffer cells the binding properties for apo E-free HDL were determined. For parenchymal, endothelial and Kupffer cells, evidence was obtained for a saturable, specific, high-affinity binding site with Kd and Bmax. values respectively in the ranges 10-20 micrograms of HDL/ml and 25-50 ng of HDL/mg of cell protein. In all three cell types nitrosylated HDL and low-density lipoproteins did not compete for the binding of native HDL, indicating that lipids and apo B are not involved in specific apo E-free HDL binding. Very-low-density lipoproteins (VLDL), however, did compete for HDL binding. The competition of VLDL with apo E-free HDL could not be explained by label exchange or by transfer of radioactive lipids or apolipoproteins between HDL and VLDL, and it is therefore suggested that competition is exerted by the presence of apo Cs in VLDL. The results presented here provide evidence for a high-affinity recognition site for HDL on parenchymal, liver endothelial and Kupffer cells, with identical recognition properties on the three cell types. HDL is expected to deliver cholesterol from peripheral cells, including endothelial and Kupffer cells, to the liver hepatocytes, where cholesterol can be converted into bile acids and thereby irreversibly removed from the circulation. The observed identical recognition properties of the HDL high-affinity site on liver parenchymal, endothelial and Kupffer cells suggest that one receptor may mediate both cholesterol efflux and cholesterol influx, and that the regulation of this bidirectional cholesterol (ester) flux lies beyond the initial binding of HDL to the receptor.     

Identification of the inhibitor of the plasminogen activator as the major protein secreted by endothelial rat liver cells

Freshly isolated Kupffer and endothelial liver cells exhibit a rate of 'de novo' protein synthesis which is twice as high per mg cell protein as that of parenchymal liver cells and contribute significantly (7.5% and 5.9%, respectively) to total liver protein secretion. In parenchymal cells the main secretory protein is a 68 kDa protein (containing 19% fo the secreted radioactivity, presumably albumin). In Kupffer cells a 49 kDa protein contains 8% of the secreted radioactivity, while in endothelial liver cells a 55 kDa protein is the most prominent secretory protein (containing 11% of the secreted radioactivity). By aid of a specific antibody the 55 kDa protein was identified as the inhibitor of the plasminogen activator and in the liver this protein was only secreted by the endothelial cells.     

Arachidonic acid stimulates TNFα production in Kupffer cells via a reactive oxygen species-pERK1/2-Egr1-dependent mechanism

Kupffer cells are a key source of mediators of alcohol-induced liver damage such as reactive oxygen species, chemokines, growth factors, and eicosanoids. Since diets rich in polyunsaturated fatty acids are a requirement for the development of alcoholic liver disease, we hypothesized that polyunsaturated fatty acids could synergize with ethanol to promote Kupffer cell activation and TNFα production, hence, contributing to liver injury. Primary Kupffer cells from control and from ethanol-fed rats incubated with arachidonic acid showed similar proliferation rates than nontreated cells; however, arachidonic acid induced phenotypic changes, lipid peroxidation, hydroperoxides, and superoxide radical generation. Similar effects occurred in human Kupffer cells. These events were greater in Kupffer cells from ethanol-fed rats, and antioxidants and inhibitors of arachidonic acid metabolism prevented them. Arachidonic acid treatment increased NADPH oxidase activity. Inhibitors of NADPH oxidase and of arachidonic acid metabolism partially prevented the increase in oxidant stress. Upon arachidonic acid stimulation, there was a rapid and sustained increase in TNFα, which was greater in Kupffer cells from ethanol-fed rats than in Kupffer cells from control rats. Arachidonic acid induced ERK1/2 phosphorylation and nuclear translocation of early growth response-1 (Egr1), and ethanol synergized with arachidonic acid to promote this effect. PD98059, a mitogen extracellular kinase 1/2 inhibitor, and curcumin, an Egr1 inhibitor, blocked the arachidonic acid-mediated upregulation of TNFα in Kupffer cells. This study unveils the mechanism whereby arachidonic acid and ethanol increase TNFα production in Kupffer cells, thus contributing to alcoholic liver disease.     

Hypoxia, reactive oxygen, and cell injury

Hypoxia usually decreases the formation of reactive oxygen species by oxidases and by autoxidation of components of cellular electron transfer pathways and of quinoid compounds such as menadione. In the case of menadione reactive oxygen species are liberated to a significant extent only at non-physiologically high oxygen partial pressures (PO2). At physiological and hypoxic PO2 values electron shuttling of menadione in the mitochondrial respiratory chain predominates. In contrast, lipid peroxidation induced by halogenated alkanes, such as carbon tetrachloride, in liver leads to an increase in the formation of reactive oxygen and thus in cell injury under hypoxic conditions. Reactive oxygen species may also be generated during reoxygenation of a previously hypoxic tissue. Based on experiments with isolated hepatocytes a three-zone-model of liver injury due to hypoxia and reoxygenation is presented; 1) a zone where the cells die by hypoxia; 2) a zone where the cells are destroyed upon reoxygenation, presumably mediated by an increase in the cellular ATP content; and 3) a zone where cell injury occurs upon reoxygenation, mediated by reactive oxygen species possibly liberated by xanthine oxidase.     

Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice

Cholestasis-induced liver injury during bile duct obstruction causes an acute inflammatory response. To further characterize the mechanisms underlying the neutrophil-induced cell damage in the bile duct ligation (BDL) model, we performed experiments using wild-type (WT) and ICAM-1-deficient mice. After BDL for 3 days, increased ICAM-1 expression was observed along sinusoids, along portal veins, and on hepatocytes in livers of WT animals. Neutrophils accumulated in sinusoids [358 +/- 44 neutrophils/20 high-power fields (HPF)] and >50% extravasated into the parenchymal tissue. Plasma alanine transaminase (ALT) levels increased by 23-fold, and severe liver cell necrosis (47 +/- 11% of total cells) was observed. Chlorotyrosine-protein adducts (a marker for neutrophil-derived hypochlorous acid) and 4-hydroxynonenal adducts (a lipid peroxidation product) were detected in these livers. Neutrophils also accumulated in the portal venules and extravasated into the portal tracts. However, no evidence for chlorotyrosine or 4-hydroxynonenal protein adducts was detected in portal tracts. ICAM-1-deficient mice showed 67% reduction in plasma ALT levels and 83% reduction in necrosis after BDL compared with WT animals. The total number of neutrophils in the liver was reduced (126 +/- 25/20 HPF), and 85% of these leukocytes remained in sinusoids. Moreover, these livers showed minimal staining for chlorotyrosine and 4-hydroxynonenal adducts, indicating a substantially reduced oxidant stress and a diminished cytokine response. Thus neutrophils relevant for the aggravation of acute cholestatic liver injury in BDL mice accumulate in hepatic sinusoids, extravasate into the tissue dependent on ICAM-1, and cause cell damage involving reactive oxygen formation.     

Mechanisms of Liver Injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions

Polymorphonuclear leukocytes (neutrophils) are a vital part of the innate immune response to microbial infections and tissue trauma, e.g., ischemia-reperfusion injury, in many organs including the liver. However, an excessive inflammatory response can lead to a dramatic aggravation of the existing injury. To design interventions, which selectively target the detrimental effects of neutrophils, a detailed understanding of the pathophysiology is critical. Systemic or local exposure to proinflammatory mediators causes activation and priming of neutrophils for reactive oxygen formation and recruits them into the vascular beds of the liver without causing tissue injury. However, generation of a chemotactic signal from the parenchyma will trigger extravasation and an attack on target cells (e.g., hepatocytes). Adhesion to the target induces degranulation with release of proteases and formation of reactive oxygen species including hydrogen peroxide and hypochlorous acid, which can diffuse into hepatocytes and induce an intracellular oxidant stress and mitochondrial dysfunction. Various neutrophil-derived proteases are involved in transmigration and cell toxicity but can also promote the inflammatory response by processing of proinflammatory mediators. In addition, necrotic cells release mediators, e.g., high-mobility group box-1, which further promotes neutrophilic hepatitis and tissue damage. On the basis of these evolving insights into the mechanisms of neutrophil-mediated liver damage, the most selective strategies appear not to interfere with the cytotoxic potential of neutrophils, but rather strengthen the target cells' defense mechanisms including enhancement of the intracellular antioxidant defense systems, activation of cell survival pathways, or initiation of cell cycle activation and regeneration.     

NADPH oxidase-derived oxidant stress is critical for neutrophil cytotoxicity during endotoxemia

Neutrophils can cause liver injury during endotoxemia through generation of reactive oxygen species. However, the enzymatic source of the oxidant stress and the nature of the oxidants generated remain unclear. Therefore, we investigated the involvement of NADPH oxidase in the pathophysiology by using the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) in the galactosamine/endotoxin (700 mg/kg Gal:100 microg/kg ET) model of liver injury. In addition, we measured chlorotyrosine as indicator for hypochlorous acid formation by myeloperoxidase. Gal/ET treatment of male C3HeB/FeJ mice resulted in sinusoidal neutrophil accumulation and parenchymal cell apoptosis (14 +/- 3% of cells) at 6 h. At 7 h, 35% of neutrophils had transmigrated. The number of apoptotic cells increased to 25 +/- 2%, and the overall number of dead cells was 48 +/- 3%; many of them showed the characteristic morphology of necrosis. Hepatocytes, which colocalized with extravasated neutrophils, stained positive for chlorotyrosine and 4-hydroxynonenal (4-HNE) protein adducts. In contrast, animals pretreated with DPI (2.5 mg/kg) were protected against liver injury at 7 h (necrosis = 20 +/- 2%). These livers showed little chlorotyrosine or 4-HNE staining, but apoptosis and neutrophil accumulation and extravasation remained unaffected. However, DPI-treated animals showed serious liver injury at 9 h due to sustained apoptosis. The results indicate that NADPH oxidase is responsible for the neutrophil-derived oxidant stress, which includes formation of hypochlorous acid by myeloperoxidase. Thus NADPH oxidase could be a promising therapeutic target to prevent neutrophil-mediated liver injury. However, the long-term benefit of this approach needs to be investigated in models relevant for human liver disease.     

Kupffer cell activation after no-flow ischemia versus hemorrhagic shock

Kupffer cell-derived oxidant stress is critical for reperfusion injury after no-flow ischemia. However, the importance of Kupffer cells as source of reactive oxygen formation is unclear in a hemorrhagic shock model. Therefore, we evaluated Kupffer cell activation after 60 or 120 min of hemorrhage and 90 min of resuscitation (HS/RS) in pentobarbital-anesthetized male Fischer rats. Plasma glutathione disulfide (GSSG) as indicator for a vascular oxidant stress showed no significant changes after HS/RS. Plasma ALT activities were only moderately increased (100-200 U/L). Kupffer cells isolated from postischemic livers did not generate more superoxide than cells from sham controls. In contrast, the 10-fold increase of plasma GSSG and the 9-fold higher spontaneous superoxide formation of Kupffer cells after 60 min of hepatic no-flow ischemia followed by 90 min of reperfusion demonstrated the activation of Kupffer cells in this experimental model. Plasma ALT activities (1930 +/- 240 U/L) indicated severe liver injury. These results demonstrate a fundamental difference in the degree of Kupffer cell activation between the two models of warm hepatic ischemia. Our findings suggest that different therapeutic strategies are necessary to ameliorate the initial injury after low flow ischemia (hemorrhage) compared to cold (transplantation) or warm (Pringle maneuver) no-flow ischemia.     

Complementary adhesion molecules promote neutrophil-Kupffer cell interaction and the elimination of bacteria taken up by the liver.

Most bacteria that enter the bloodstream are taken up by the liver. Previously, we reported that such organisms are initially bound extracellularly and subsequently killed by immigrating neutrophils, not Kupffer cells as widely presumed in the literature. Rather, the principal functions of Kupffer cells demonstrated herein are to clear bacteria from the peripheral blood and to promote accumulation of bactericidal neutrophils at the principal site of microbial deposition in the liver, i.e., the Kupffer cell surface. In a mouse model of listeriosis, uptake of bacteria by the liver at 10 min postinfection i.v. was reduced from approximately 60% of the inoculum in normal mice to approximately 15% in mice rendered Kupffer cell deficient. Immunocytochemical analysis of liver sections derived from normal animals at 2 h postinfection revealed the massive immigration of neutrophils and their colocalization with Kupffer cells. Photomicrographs of the purified nonparenchymal liver cell population derived from these infected mice demonstrated listeriae inside neutrophils and neutrophils within Kupffer cells. Complementary adhesion molecules promoted the interaction between these two cell populations. Pretreatment of mice with mAbs specific for CD11b/CD18 (type 3 complement receptor) or its counter-receptor, CD54, inhibited the accumulation of neutrophils in the liver and the elimination of listeriae. Complement was not a factor; complement depletion affected neither the clearance of listeriae by Kupffer cells nor the antimicrobial activity expressed by infiltrating neutrophils.     

Acute lung injury and cell death: how many ways can cells die?

Apoptosis has been considered as an underlying mechanism in acute lung injury/acute respiratory distress syndrome and multiorgan dysfunction syndrome. Recently, several alternative pathways for cell death (such as caspase-independent cell death, oncosis, and autophagy) have been discovered. Evidence of these pathways in the pathogenesis of acute lung injury has also come into light. In this article, we briefly introduce cell death pathways and then focus on studies related to lung injury. The different types of cell death that occur and the underlying mechanisms utilized depend on both experimental and clinical conditions. Lipopolysaccharide-induced acute lung injury is associated with apoptosis via Fas/Fas ligand mechanisms. Hyperoxia and ischemia-reperfusion injury generate reactive oxidative species, which induce complex cell death patterns composed of apoptosis, oncosis, and necrosis. Prolonged overexpression of inflammatory mediators results in increased production and activation of proteases, especially cathepsins. Activation and resistance to death of neutrophils also plays an important role in promoting parenchymal cell death. Knowledge of the coexisting multiple cell death pathways and awareness of the pharmacological inhibitors targeting different proteases critical to cell death may lead to the development of novel therapies for acute lung injury.