Kupffer cells and alcoholic liver disease

Células de Kupffer y hepatopatía alcohólica

F. J. Cubero and N. Nieto

Department of Medicine. Division of Liver Diseases. Mount Sinai School of Medicine. New York, USA

Correspondence

ABSTRACT

Liver disease is a major cause of illness and death worldwide. A central component in the complex network leading to the development of alcoholic liver disease is the activation of Kupffer cells by endotoxin and other soluble mediators. Alcohol consumption induces a state of ‘leaky gut' increasing plasma and liver endotoxin levels. When Kupffer cells become activated, they interact with a complex of proteins located on the extracellular membrane signaling to produce a wide array of soluble factors, including cytokines, chemokines, growth factors, cyclooxygenase and lipoxygenase metabolites, and reactive oxygen species such as superoxide anion, hydrogen peroxide, and nitric oxide, all of which provide physiologically diverse and pivotal paracrine effects on all other liver cell types and, ultimately, liver injury. Kupffer cells are also central to the liver homeostatic response to injury as upon cellular degenerative changes, they immediately respond to the insult and release mediators to orchestrate inflammatory and reparative responses. Thus, the homeostatic responses are initiated by Kupffer cell-derived mediators at the cellular level and underlie the liver's defense and reparative mechanisms against injury. In order to understand better the role of Kupffer cells in the onset of liver injury, animal models in which Kupffer cells are inactivated, and cell culture settings (e.g. co-cultures) are being used with promising results that advance our understanding of alcoholic liver disease.

Key words: Kupffer cells. Alcoholic liver disease. Reactive oxygen species. Lipopolysaccharide. Hepatic stellate cells.

Abbreviations(In alphabetical order)

Alcoholic liver disease (ALD); arachidonic acid (AA); carcinoembryonic antigen (CEA); c-Jun kinase (JNK); Cyclooxygenase (COX); dilinoleoylphosphatidylcholine (DLPC); early-immediate gene-1 (Egr-1); endotoxin or lipopolysaccharide (LPS); ethylenediamine tetra acetic acid (EDTA); extracellular membrane (ECM); extracellular signal-regulated protein kinase 1/2 (ERK1/2); glucose-6-phosphate dehydrogenase (G6PDH); glutathione (GSH); glutathione disulfide (GSSG); glutathione-S-transferase (GST); hepatic stellate cells (HSC); hydroxyl radical (OH^•); leukotrienes (LT); lipoxygenase (LOX); low-density lipoprotein (LDL); LPS-binding protein (LBP); major histocompatibility complex (MHC); nitrate (NO₃-); nitric oxide (NO); nitric oxide synthase (NOS); nitrite (NO₂-); nuclear factor-kappa beta (NF-κB); P₄₅₀ cytochrome 2E1 (CYP2El); peroxynitrite (ONOO-); phospholipase A₂ (PLA₂); platelet activation factor (PAF); platelet-derived growth factor (PDGF); propylthiouracil (PTU); prostaglandin D₂ (PGD₂); prostaglandin E₂ (PGE₂); prostaglandin H₂ (PGH₂); reactive oxygen species (ROS); superoxide anion (O₂^•-); superoxide dismutase (SOD); transforming growth factor-beta (TGF-β); transmembrane toll-like receptor (TLR); tumor necrosis factor-alpha (TNF-α); very-low-density lipoprotein (VLDL).

General achitecture of the liver

Kupffer cells

Kupffer cells are liver-specific macrophages which were first identified in the liver by von Kupffer (1876) (3). Kupffer cells are amoeboid in shape and adhere to the surface of fenestrated sinusoidal endothelial cells. In the cytosol of Kupffer cells, there are a number of dense bodies and electron-lucent vacuoles of various sizes including lysosomes. Golgi apparatus, coated vesicles, pinocytic vesicles, ribosomes, centrioles, microfilaments, and microtubules are also present in the cytosol (4). The nucleus of Kupffer cells is ovoid or indented, and occasionally lobulated (5). Wormlike structures, fuzzy coat, microvilli, and pseudopodia at the cell surface are characteristic structural components of Kupffer cells involved in endocytic mechanisms (6). Kupffer cells incorporate large particles such as erythrocytes and bacteria by phagocytosis and take up small particles and molecules via pinocytic vesicles (7-12).
Peroxidase activity is observed in the rough endoplasmic reticulum, nuclear envelope, and annulate lamellae (13,14). The majority of Kupffer cells display an endogenous peroxidase pattern typical of resident tissue macrophages and show positive staining for macrophage markers such as ED1, ED2, and Ki-M2R in rats and F4/80 in mice (6,15). Other markers are the presence of inducibility of major histocompatibility complex (MHC) class II antigens (16), receptors for fructose and galactose-exposing glycoproteins (17,18), peroxidase activity, and the capacity to phagocytose fluorescent labeled latex particles (19). Kupffer cells display high glucose-6-phosphate dehydrogenase (G6PDH) activity, which plays an important role in the metabolic response to phagocytosis. A key enzyme present in Kupffer cells is NADPH oxidase which plays a decisive role in the development of alcohol-induced liver injury (20). The cytochrome P₄₅₀ enzymes, and concretely P₄₅₀ 2E1 (CYP2E1), are also expressed in Kupffer cells. In addition, several glutathione-S-transferase (GST) isoenzymes and glutathione peroxidase may enable the Kupffer cell to detoxify potentially hepatoxic substances (21-23).

Function of kupffer cells

Kupffer cells have many specific functions that are essential for the preservation of homeostasis in the liver under several conditions. Endocytosis, pivotal for maintaining homeostasis, is not only essential for the removal of several (plasma) proteins and other material from the blood, but the receptor-mediated uptake of these substances is directly coupled with a metabolic response, leading to the production of cytokines and eicosanoids. Both cytokines and eicosanoids may act subsequently in an autocrine and/or paracrine fashion.
Clearance of galactose-terminated particles from the circulation is performed by a galactose-specific receptor in Kupffer cells (24). The Mannose/N-acetylglucosamine receptor is probably involved in the clearance of larger mannose-exposing particles, like potentially hazardous microorganisms such as bacteria, yeast, and parasites (25). Low-density lipoproteins (LDL) are almost exclusively taken up by Kupffer cells (26). Kupffer cells possess an additional receptor which binds oxidatively modified LDL, very low-density lipoprotein (VLDL), and lipoprotein A (27-29). ]]> Like endothelial cells, Kupffer cells bind IgG immune complexes, which may contribute significantly to the normal host defense (30,31). The uptake of IgA immune complexes is mediated by a specific receptor present in Kupffer cells, which recognizes the Fc part of IgA (32, 33). Under several pathological conditions, like primary biliary cirrhosis, obstructive jaundice, and ethanol consumption, the capacity of Kupffer cells to take up immunoglobulin immune complexes is reduced, which may lead to a reduction of the host defense capacity. Kupffer cells possess receptors for the complement components C1q and C3b (30,34). Complement factors adhere to immunoglobulins (35), DNA, bacteria, and platelets (36-38). Human Kupffer cells also express several complement receptors, and the presence of CR1, CR3, and CR4 receptors provides Kupffer cells with an optimal capacity to remove complexes coated with complement from the circulation (39).
The effects of platelet activation factor (PAF) on the liver are mediated by Kupffer cells that possess specific binding sites for PAF (40). Carcinoembryonic antigen (CEA) is a gut-derived glycoprotein that is cleared from the circulation by Kupffer cells (41). These binding sites may be important for the development of liver metastases (42). Several cellular components, like DNA and cell-derived enzymes, are released during cell death in several clinical situations. DNA can be removed from the circulation by binding to a receptor on the Kupffer cells without opsonization (43).

Kupffer cells isolation, purification and culture

One of the first methods to isolate Kupffer cells was based in the in vivo uptake of iron particles by Kupffer cells, washing the sinusoidal cells out of the liver, and purifying the Kupffer cells using magnets (44). Later, chelators such as citrate, tetraphenylboron, or ethylenediamine tetra acetic acid (EDTA) were used to prepare liver cell suspensions, and iron-loaded Kupffer cells were isolated using magnets (45-48). These methods provided only low numbers of viable sinusoidal cells and contamination with other cell populations remained an issue of concern. Alternative isolation procedures described by Berry and Friend (49) and Seglen and Gjessing (50), and modified by Arahuetes et al. (51), utilized collagenase for the perfusion and dispersion of the liver, and were used to isolate parenchymal and non-parenchymal cells simultaneously from the same liver. Pronase, which preferentially destroys parenchymal liver cells, can be used directly for digestion of the liver or can be used in addition to collagenase digestion.
When centrifugal elutriation was intruduced in the protocol, the yield was much higher as contamination of the endothelial and Kupffer cell fractions by blood lymphocytes and so-called blebs was minimized (52). Percoll density gradients, followed by selective adherence of both Kupffer and endothelial cells, have been described in order to obtain pure cell fractions from the crude non-parenchymal liver cell suspensions (53). Centrifugal elutriation after an initial gradient in Histodenz or Nycodenz to separate Kupffer cells and endothelial cells from sellate cells is generally the method of choice (54).
Finally, separation by velocity sedimentation, which is based on the same physical properties as elutriation, normally gives less satisfactory results unless high-resolution equipment is used and diminishes the viability of both fractions as longer times are required (55). These cultured cells are still capable of phagocytosis of colloidal carbon and latex particles and may preserve their functions, with regard to endocytosis, for at least two weeks in culture.

Alcoholic liver disease (ald) and kupffer cells

Endotoxin

Multiple mammalian receptors for LPS have been identified, including two glycoproteins: LBP and CD14. CD14 binds to the LPS-binding protein (LPS-LBP) complex but is not, by itself, capable of initiating a transmembrane activation signal (6). It has been postulated that LPS/CD14 complexes interact with a transmembrane toll-like receptor (TLR) responsible for signal transduction (59).

Both in vivo and in vitro data suggest that chronic ethanol sensitizes Kupffer cells to at least some LPS-mediated responses. For example, chronic ethanol consumption increases the susceptibility of rats to LPS-induced liver injury (60). Hijioka et al. showed that binge ethanol drinking rapidly inactivated voltage-dependent Ca²⁺ channels in Kupffer cells becoming activated to release other critical mediators (61). Thus, inactivation of these channels may be involved in mechanisms of rapid tolerance to ethanol (62).

Shibayama et al. showed that acute administration of ethanol enhanced endotoxin hepatotoxicity. In his work, Kupffer cells became sensitized to LPS in cells isolated 24 hours after ethanol administration as reflected by increased intracellular Ca²⁺, tumor necrosis factor-alpha (TNF-α) production, and large increases in CD14. These effects were all blocked by antibiotics, indicating that sensitization of Kupffer cells by ethanol is also mediated by gut-derived LPS (63).

Oxidative stress

Oxidative stress caused by generation of reactive oxygen species (ROS) during alcohol consumption is suggested as one of the major mechanisms of alcohol-induced liver injury (56,61-71). ROS can be generated by a variety of enzymes including, but not limited to CYP2E1, NADH/NADPH oxidase, xanthine oxidase, and arachidonic pathways, such as lipoxygenase (LOX) and cyclooxygenase (COX). Induction of ROS can be also triggered by damaged mitochondria as it happens in ALD (72-76).

Kupffer cells contain superoxide dismutase (SOD) that dismutates O₂^•- (superoxide anion) to yield H₂O₂. H₂O₂ and O₂^•- can interact via the Fenton reaction to produce more powerful and cytotoxic radicals, such as OH^• (hydroxyl radical) (77). However, under non-pathologic conditions intracellular accumulation of OH^• is also limited because H2O2 is metabolized by glutathione peroxidase and/or catalase to form H₂O and O₂. Thus, together with glutathione (GSH), SOD, glutathione peroxidase, and catalase are the major endogenous antioxidant enzyme systems responsible for limiting intracellular accumulation of O₂^•- and H₂O₂ during normal aerobic metabolism (78).

Nitric oxide synthase (NOS) has been shown to modulate levels of ROS in a variety of cells (79,80). There are three isoforms of NOS: two are constitutively expressed (endothelial and neuronal; eNOS and nNOS, respectively) and one is an inducible isoform (iNOS). Nitric oxide (NO) generated by NOS can interact with O₂^•-. This pathway for detoxification of O₂^•- does not result in the formation of H₂O₂, making it a more benign decomposition pathway than SOD. However, one of the potential products of this reaction that has received a great deal of attention is peroxynitrite (ONOO-), a potent oxidizing agent. ONOO- has been implicated as the causative agent in a variety of pathologies (81-84). Alternatively, ONOO- has been implicated as a protective agent (85-87). The latter can either spontaneously rearrange to form nitrate (NO₃-) or undergo cleavage to generate OH-like radicals and nitrite (NO₂-).

In ethanol binge drinking, Kupffer cells are primed for enhanced ROS release, due in part, to complement activation (71). Acute alcohol administration causes accumulation of reactive oxygen species, including O₂^•-, OH•, and H₂O₂ (88). Lipid peroxidation and mitochondrial dysfunction have been detected in both animal models and humans receiving acute doses of alcohol (72-76,89,90). Acute ethanol administration also enhanced iNOS mRNA in hepatocytes and Kupffer cells (68). There is also a decrease in GSH levels along with an increase in glutathione disulfide (GSSG) levels (68,91). However, the effect of chronic ethanol consumption on hepatic GSH levels is complex, with reports of no change, decreases, or even increases (64,67,68,70,72-76,89,90,92, 93).

Chronic ethanol consumption increases the cytosolic activity of iNOS and may increase O₂∑- production via activation or increasing the levels of CYP2E1, xanthine oxidase, NADPH oxidase, or damaged mitochondria, resulting in enhanced production of both NO and O₂∑-. Dietary supplementation with phosphatidylcholine has been shown to attenuate ethanol-induced fibrosis acting as a ‘sink' for free radicals (94).

Kupffer cells synthesize eicosanoids and are responsible for about 65% of the total amount of eicosanoids produced in the liver (95). Arachidonic acid (AA) can be released from the cell membrane by the action of phospholipase A₂ (PLA₂). This enzyme is activated by an increase in the intracellular Ca²⁺ concentration, whereas higher intracellular cAMP levels inhibit PLA₂ (96). COX₂ catalyzes the conversion of arachidonic acid into prostaglandin H₂ (PGH₂), which is subsequently converted to other prostaglandins and thromboxanes, the so called cyclooxigenase pathway (97). COX₂ is induced by endotoxin, cytokines, and oxidative stress (98). Conversion of AA by LOX and subsequent enzymes leads to the production of leukotrienes, the so called lipoxygenase pathway (59).

Leukotrienes and prostaglandins are collectively called eicosanoids. The main product of Kupffer cells is prostaglandin D₂ (PGD2), representing 55% of the total amount of eicosanoids produced by them (96). Kupffer cells lack the capacity to produce very potent leukotrienes (LT), like LTB₄ or LTC₄ (99). LPS is removed primarily by Kupffer cells that are activated, leading to rapid increases in COX₂ and intracellular Ca₂₊, the latter of which, in turn, activates PLA₂ (96). Increased prostaglandin E₂ (PGE₂) causes triglycerides' accumulation in hepatocytes, and therefore, a state of steatosis (97). This pathway is one of many physiological processes involved in mechanisms of fatty liver caused by ethanol (100,101). Alterations of the hepatic metabolic state during binge drinking has been also attributed to the release of mediators such as prostaglandins from activated Kupffer cells, and this effect can be attenuated by the anti-thyroid drug, propylthiouracil (PTU) (94).

Role of cytokines and nuclear factor-kappa B (NF-κB)

Production of inflammatory cytokines is a highly regulated process; regulation has been reported at the levels of transcription, translation, and secretion. Mechanistic studies have demonstrated that endotoxin binds to the LPS CD14/TLR₄ complex on Kupffer cells causing nuclear factor-kappa beta (NF-κB) activation, which in turn leads to TNF-α production and liver injury (102-104). Increasing experimental evidence supports that TNF-α signaling, generates an increase in mitochondrial ROS generation in the hepatocte through ubiquinone cycling via the electron transport chain (105-107).

Recent studies in rodents have confirmed a role of Kupffer cell-derived TNF-α in alcoholic liver injury (102) demonstrating increased immunostaining for TNF-α, IL-1, IL-6, and IL-8 on bile duct epithelial cells and Kupffer cells (108).

In unstimulated cells, NF-κB, a ubiquitous transcription factor, is sequestered in the cytoplasm with the Ikb family of inhibitors (59). On stimulation, Ikb is phosphorylated with subsequent release of NF-κB. NF-κB then translocates to the nucleus, in which it binds cis-acting elements in the promoter of target genes such as TNF-α and other proinflammatory cytokines (109).

Stimulation of macrophages with LPS activates tyrosine kinases, protein kinase C, NF-κB, as well as members of the mitogen-activated protein kinase family including extracellular signal-regulated protein kinase 1/2 (ERK1/2), p38, and c-Jun kinase (JNK) (110). Chronic ethanol feeding differentially regulates the expression of TNF-α and IL-1 in Kupffer cells. These differential responses are associated with impaired LPS activation of NF-kB counteracted by enhanced activation of ERK1/2 and early-immediate gene-1 (Egr-1) (111).

Dilinoleoylphosphatidylcholine (DLPC) decreases lipopolysaccharide-induced TNF-α generation by Kupffer cells of ethanol-fed rats by blocking p38, ERK1/2, and NF-κB activation (112). DLPC also decreases TNF-α induction by acetaldehyde, a toxic metabolite released by ethanol oxidation (57). Iron has long been implicated in the pathogenesis of chronic liver disease, including ALD. It is believed that iron accumulates in chronic liver inflammation and catalyzes hydroxyl radical-mediated oxidative injury, activating the NF-κB pathway (102).

Paracrine effects on hepatic stellate cells

In normal liver, the space of Disse contains a non electron-dense basement membrane-like matrix which is essential for maintaining the differentiated function of all resident liver cells. However, as the liver becomes fibrotic, the total content of collagens and non-collagenous components greatly increases and it is accompanied by a shift in the type of extracellular matrix in the subendothelial space from the normal low density basement membrane-like matrix to interstitial type matrix containing fibril-forming collagens.

Under normal conditions, HSC remain quiescent and produce small amounts of extracellular membrane (ECM), such as laminin and collagen type IV which are essential components of basement membrane (115). Upon exposure to soluble factors from damaged hepatocytes and from activated Kupffer cells, HSC will lose their lipid content (retinyl palmitate), and undergo morphological transition to myofibroblast-like cells (70,98, 116-121).

Activated HSC produce then large amounts of extracellular matrix components e.g. collagen I in an accelerated fashion, triggering a fibrogenic response (70,98, 116-121). During the cross-talk of both liver cell types, mediated by different cytokines, reactive oxygen species, and other soluble factors, hepatocellular damage is initiated, and subsequent establishment of liver fibrosis occurs.

In summary, a conceptual issue that has emerged in the field of ALD is the importance of cell-type specific research. It is well-known that Kupffer cells actively participate in the pathogenesis of ALD. The role of Kupffer cells in both the autocrine and paracrine mode of proinflammatory and cytotoxic actions has been supported by different experimental strategies. Among them, the development of co-culture models of Kupffer cells and HSCs for investigating the effects of various fibrogenic mediators represents a promising tool for the study of ALD aiming at finding treatment strategies for this disease.

References

1. Rojkind M. Extracellular matrix. In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz DA, editors. The liver: biology and pathobiology. New York: Raven Press; 1988. p. 707-16.        [ Links ]

2. Wisse E, Knook DL, editors. Kupffer cells and other liver sinusoidal cells. Amsterdam: Elsevier/North Holland; 1977.        [ Links ]

3. Kupffer CV. Ueber Sternzellen der Leber. Arch Miksrosk Anat 1876; 12: 353-8.        [ Links ]

4. Wisse E. Observations on the fine structure and peroxidase cytochemistry of normal rat liver Kupffer cells. J Ultrastruct Res 1974; 46: 393-426.        [ Links ]

5. Wisse E. Kupffer cell reactions in rat liver under various conditions as observed in the electron microscope. J Ultrastruct Res 1974; 46: 499-520.        [ Links ]

6. Naito M, Hasegawa G, Ebe Y, Yamamoto T. Differentiation and function of Kupffer cells. Med Electron Microsc 2004; 37: 16-28.        [ Links ]

7. Bouwens L, De Bleser P, Vanderkerken K, Geerts B, Wisse E. Liver cell heterogeneity: functions of non-parenchymal cells. Enzyme 1992; 46: 155-68.        [ Links ]

8. Wake K, Decker K, Kirn A, Knook DL, McCuskey RS, Bouwens L, et al. Cell biology and kinetics of Kupffer cells in the liver. Int Rev Cytol 1989; 118: 173-229.        [ Links ]

9. Laskin DL, Weinberger B, Laskin JD. Functional heterogeneity in liver and lung macrophages. J Leukoc Biol 2001; 70: 163-70.        [ Links ]

10. Arii S, Imamura M. Physiological role of sinusoidal endothelial cells and Kupffer cells and their implication in the pathogenesis of liver injury. J Hepatobiliary Pancreat Surg 2000; 7: 40-8.        [ Links ]

11. Decker K. The response of liver macrophages to inflammatory stimulation. Keio J Med 1998; 47: 1-9.        [ Links ]

12. Sleyster EC, Knook DL. Relation between localization and function of rat liver Kupffer cells. Lab Invest 1982; 47: 484-90.        [ Links ]

13. Braet F, Wisse E. Structural functional aspects of liver sinusoidal endothelial cell fenestrae: a review. Comp Hepatol 2002; 23: 1.        [ Links ]

14. Sleyster EC, Knook DL. Relation between localization and function of rat liver Kupffer cells. Lab Invest 1982; 47: 484-90.        [ Links ]

15. Blouin A, Bolender RP, Weibel ER. Distribution of organelles and membranes between hepatocytes and non-hepatocytes in the rat liver parenchyma. J Cell Biol 1977; 72: 441-55.        [ Links ]

16. Ramadori G, Dienes HP, Burger R, Meuer S, Rieder H. Expression of Ia-antigens on guinea pig Kupffer cells. Studies with monoclonal antibodies. J Hepatol 1986; 2: 208-17.        [ Links ]

17. Lehrman MA, Hill RL. The binding of fructose-containing glycoproteins by hepatic lectins. J Biol Chem 1986; 261: 7419-25.        [ Links ]

18. Kolb-Bachofen V, Schlepper-Schäfer J, Vogel W. Electron microscopic evidence for an asialoglycoprotein receptor on Kupffer cells: localization of lectin mediated endocytosis. Cell 1982; 29: 859-66.        [ Links ]

19. Widmann JJ, Cotran RS, Fahimi HH. Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat liver sinusoids by endogenous peroxidase activity. J Cell Biol 1972; 52: 159-70.        [ Links ]

20. Kono H, Rusyn I, Yin M, Gabele E, Yamashina S, Dikalova A. NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease. J Clin Invest 2000; 106: 867-72.        [ Links ]

21. Steinberg P, Schlemper B, Molitor E, Platt KL, Seidel A, Oesch F. Rat liver endothelial and Kupffer cell mediated mutagenicity of polycyclic aromatic hydrocarbons and aflatoxin B1. Environ Health Perspect 1990; 88: 71-6.        [ Links ]

22. Koop DR, Chernosky A, Brass EP. Identification and induction of cytochrome P450 2E1 in rat Kupffer cells. J Pharmacol Exp Ther 1991; 258: 1072-6.        [ Links ]

23. Steinberg P, Schramm H, Schladt L, Robertson LW, Thomas H, Oesch F. The distribution, induction and isoenzyme profile of glutathione S transferase and glutathione peroxidase in isolated rat liver parenchymal, Kupffer and endothelial cells. Biochem J 1989; 264: 737-44.        [ Links ]

24. Van Berkel TJC, Kruijt JK, Spanjer HH, Nagelkerke JF, Harkes L, Kempen HJM. The efficacy of a water-soluble tris-galactoside-terminated cholesterol derivative on the fate of low density lipoproteins and liposomes. J Biol Chem 1983; 260: 2694-9.        [ Links ]

25. Ezekowitz RAB, Williams DJ, Koziel H, Armstrong MYK, Warner A, Richards FF, et al. Uptake of Pneumocystis carinii mediated by the macrophage mannose receptor. Nature 1991; 351: 155-8.        [ Links ]

26. Harkes L, van Berkel TJC. Quantitative role of parenchymal and non-parenchymal liver cells in the uptake of 14C-sucrose labeled low-density lipoprotein in vivo. Biochem J 1984; 224: 21-7.        [ Links ]

27. Van Berkel TJC, De Rijke YB, Kruijt JK. Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells. J Biol Chem 1991; 266: 2282-9.        [ Links ]

28. De Rijke YB, Hessels EM, van Berkel TJC. Recognition sites on rat liver cells for oxidatively modified á-very lowdensity lipoproteins. Arterioscler Thromb 1992; 12: 41-9.        [ Links ]

29. De Rijke YB, Jurgens G, Hessels EM, Hermann A, van Berkel TJC. In vivo fate and scavenger receptor recognition of oxidized lipoprotein[a] isoforms in rats. J Lipid Res 1992; 33: 1315-25.        [ Links ]

30. Loegering DJ. Kupffer cell complement receptor clearance function and host defense. Cir Shock 1986; 20: 321-33.        [ Links ]

31. Muro H, Shirasawa H, Maeda M, Nakamura S. Fc receptors of liver sinusoidal endothelium in normal rats and humans: a histological study with soluble immune complexes. Gastroenterology 1987; 83: 1078-85.        [ Links ]

32. Rifai A, Mannik M. Clearance of circulating IgA immune complexes is mediated by a specific receptor on Kupffer cells in mice. J Exp Med 1984; 160: 125-37.        [ Links ]

33. Sancho J, González E, Egido J. The importance of the Fc receptors for IgA in the recognition of IgA by mouse liver cells: its comparison with carbohydrate and secretory componentreceptors. Immunology 1986; 57: 31-42.        [ Links ]

34. Steer CJ, Richman LK, Hague NE, Richman JA. Identification of receptors for immunoglobulin and complement on mouse Kupffer cells in vitro. Gastroenterology 1978; 75: 988-90.        [ Links ]

35. Toth CA, Pohl D, Agnello V. Methods of detection of immune complexes by utilizing C1q or rheumatoid factors. In: Rose N, Friedman H, Fahey J, editors. Manual of clinical laboratory immunology. Washington DC: American Society for Microbiology; 1986. p. 204-10.        [ Links ]

36. Uwatakos S, Mannik M. The location of binding sites on C1q for DNA. J Immunol 1990; 144: 3484-8.        [ Links ]

37. Peerschke E, Ghebrehiwet B. Platelet C1q receptor interactions with collagen and C1q coated surfaces. J Immunol 1990; 145: 2984-8.        [ Links ]

38. Betz S, Isliker H. Antibody independent interaction between E. coli and human complement components. J Immunol 1981; 127: 1748-54.        [ Links ]

39. Hinglais N, Kazatchkine MD, Mandet C, Appay MD, Bariety J. Human liver Kupffer cells express CR1, CR3, and CR4 complement receptor antigens. An immunohistochemical study. Lab Invest 1989; 61: 509-14.        [ Links ]

40. Chao W, Liu H, DeBuysere M, Hanahan DJ, Olson MS. Identification of receptors for platelet activating factor in rat Kupffer cells. J Biol Chem 1989; 264: 13591-8.        [ Links ]

41. Thomas P, Zamcheck N. Role of the liver in clearance and excretion of circulating carcinoembryonic antigen (CEA). Dig Dis Sci 1983; 28: 216-24.        [ Links ]

42. Thomas P, Petrick AT, Toth CA, Fox ES, Elting JJ, Steele G, Jr. A peptide sequence on carcinoembryonic antigen binds to a 80kD protein on Kupffer cells. Biochem Biophys Res Commun 1992; 188: 671-7.        [ Links ]

43. Emlen W, Rifai A, Magilavy D, Mannik M. Hepatic binding of DNA is mediated by a receptor on nonparenchymal cells. Am J Pathol 1988; 133: 54-60.        [ Links ]

44. Rous P, Beard JW. Selection with the magnet and cultivation of reticuloendothelial cells (Kupffer cells). J Exp Med 1934; 59: 577-91.        [ Links ]

45. Anderson NG. The mass isolation of whole cells from rat liver. Science 1953; 117: 627-8.        [ Links ]

46. Jacob ST, Bhargava PM. A new method for preparation of liver cell suspensions. Exp Cell Res 1962; 27: 453-67.        [ Links ]

47. Rappaport K, Howze G. Dissociation of adult mouse liver by Na tetraphenyl boron: a potassium chelating agent. Proc Soc Exp Biol Med 1966; 121: 1010-21.        [ Links ]

48. St. George S, Friedman M, Byers SO. Mass separation of reticuloendothelial and parenchymal cells of rat´s liver. Science 1954; 120: 463-4.        [ Links ]

49. Berry MN, Friend DS. A high yield preparation of isolated rat liver parenchymal cells. J Cell Biol 1969; 43: 506-20.        [ Links ]

50. Seglen PO, Gjessing R. Effects of temperature and divalent cations on the substratum attachment of rat hepatocytes in vitro. J Cell Sci 1978; 34: 117-31.        [ Links ]

51. Arahuetes RM, Sierra E, Codesal J, García-Barrutia MS, Arza E, Cubero J, et al. Optimization of the technique to isolate fetal hepatocytes and assessment of their functionality by transplantation. Life Sciences 2001; 68: 763-72.        [ Links ]

52. Knook DL, Sleyster EC. Isolated parenchymal, Kupffer and endothelial rat liver cells characterized by their lysosomal enzyme content. Biochem Biophys Res Commun 1980; 96: 250-7.        [ Links ]

53. Smedsrod B, Pertoft H. Preparation of pure hepatocytes and reticuloendothelial cells in high yield from a single rat liver by means of percoll centrifugation and selective adherence. J Leuk Biol 1985; 38: 213-30.        [ Links ]

54. Brouwer A, De Leeuw AM, Praaning-van Dalen DP, Knook DL. Isolation and culture of sinusoidal liver cells: summary of a round table discussion. In: Knook DL, Wisse E, editors. Sinusoidal liver cells. Amsterdam: Elsevier/North Holland; 1982. p. 509-16.        [ Links ]

55. Morin O, Patry P, Lafleur L. Heterogeneity of endothelial cells of adult rat liver as resolved by sedimentation velocity and flow cytometry. J Cell Physiol 1984; 119: 327-34.        [ Links ]

56. Kesova IG, Ho YS, Thung S, Cederbaum AI. Alcohol-induced liver injury in mice lacking Cu, Zn-superoxide dismutase. Hepatology 2003; 38: 1136-45.        [ Links ]

57. Cao Q, Mak KM, Lieber CS. Dilinoleoylphosphatidylcholine decreases acetaldehyde-induced TNF-alpha generation in Kupffer cells of ethanol-fedrats. Biochem Biophys Res Commun. 2002; 299: 459-64.        [ Links ]

58. Nakamura Y, Yokoyama H, Higuchi S, Hara S, Kato S, Ishi H. Acetaldehyde accumulation suppresses Kupffer cell release of TNF-a and modifies acute hepatic inflammation in rats. J Gastroenterol 2004; 39:140-7.        [ Links ]

59. Nanji AA. Role of Kupffer cells in alcoholic hepatitis. Alcohol 2002; 27: 13-5.        [ Links ]

60. Fox ES, Cantrell CH, Leingang KA. Inhibition of the Kupffer cell inflammatory response by acute ethanol: NF-kappa B activation and subsequent cytokine production. Biochem Biophys Res Commun 1996; 5: 134-40.        [ Links ]

61. Hijioka T, Goto M, Lemasters JJ, Thurman RG. Effect of short-term ethanol treatment on voltage-dependent calcium channels in Kupffer cells. Hepatology 1993; 18: 400-5.        [ Links ]

62. Enomoto N, Ikejima K, Bradford B, Rivera C, Kono H, Brenner DA, et al. Alcohol causes both tolerance and sensitization of rat kupffer cells via mechanism dependent on endotoxin. Gastroenterology 1998; 115: 443-51.        [ Links ]

63. Shibayama Y, Asaka S, Nakata K. Endotoxin hepatotoxicity augmented by ethanol. Exp Mol Pathol 1991; 55: 196-202.        [ Links ]

64. Dey A, Cederbaum AI. Alcohol and oxidative liver injury. Hepatology 2006; 46: S63-74.        [ Links ]

65. Wu D, Cederbaum AI. Oxidative stress mediated toxicity exerted by ethanol-inducible CYP2E1.Toxicol Appl Pharmacol 2005; 207: 70-6.        [ Links ]

66. Wu D, Cederbaum AI. Alcohol, oxidative stress, and free radical damage. Alcohol Res Health 2003; 27: 277-84.        [ Links ]

67. Caro AA, Cederbaum AI. Oxidative stress, toxicology, and pharmacology of CYP2E1. Anu Rev Pharmacol Toxicol 2004; 44: 27-42.        [ Links ]

68. Carmiel-Haggai M, Cederbaum AI, Nieto N. Binge ethanol exposure increases liver injury in obese rats. Gastroenterology 2003; 125: 1818-33.        [ Links ]

69. Rojkind M, Domínguez-Rosales JA, Nieto N, Greenwel P. Role of hydrogen peroxide and oxidative stress in healing responses. Cell Mol Life Sci 2002; 59: 1872-91.        [ Links ]

70. Nieto N, Friedman SL, Cederbaum AI. Stimulation and proliferation of primary rat hepatic stellate cells by cytochrome P450 2E1-derived reactive oxygen species. Hepatology 2002; 35: 62-73.        [ Links ]

71. Wheeler MD, Kono H, Yin M, Nakagami M, Uesugi T, Arteel GE, et al. The role of Kupffer cell oxidant production in early ethanol-induced liver disease. Free Radic Biol Med 2001; 31: 1544-9.        [ Links ]

72. Kaplowitz N. Liver biology and pathobiology. Hepatology 2006; 43: S235-8.        [ Links ]

73. Han D, Matsumaru K, Rettori D, Kaplowitz N. Usnic acid-induced necrosis of cultured mouse hepatocytes: inhibition of mitochondrial function and oxidative stress. Biochem Pharmacol 2004; 67: 439-51.        [ Links ]

74. Kaplowitz N. Biochemical and cellular mechanisms of toxic liver injury. Semin Liver Dis 2002; 22: 137-44.        [ Links ]

75. Fernández-Checa JC, Kaplowitz N. Hepatic mitochondrial glutathione: transport and roleindiseaseandtoxicity. Toxicol Appl Pharmacol 2005; 204: 263-73.        [ Links ]

76. Fernandez-Checa JC. Alcohol-induced liver disease: when fat and oxidative stress meet. Ann Hepatol 2003; 2: 69-75.        [ Links ]

77. Granger DN, Grisham MB, Kvietys PR. Mechanisms of microvascular injury. In: Johnson LR, Alpers DH, Christensen J, Jacobson ED, Walsh JH, editors. Physiology of the gastrointestinal tract. New York: Raven Press; 1994. p. 1693-722.        [ Links ]

78. Cepinskas G, Rui T, Kvietys PR. Interaction between reactive oxygen metabolites and nitric oxide in oxidant tolerance. Free Radical Biology & Medicine 2002; 4: 433-40.        [ Links ]

79. Grisham MB, Jourd'heuil D, Wink DA. Nitric oxide. I. Chemistry of nitric oxide and its metabolites: implications in inflammation. Am J Physiol 1999; 276: G315-G321.        [ Links ]

80. Muriel P. Regulation of nitric oxide synthesis in the liver. J Appl Toxicol 2000; 20: 189-95.        [ Links ]

81. Kaur H, Halliwell B. Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett 1004; 350: 9-12.        [ Links ]

82. Szabo C, Salzman AL, Ischiropoulos H. Endotoxin triggers the expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in the rat aorta in vivo. FEBS Lett 1995; 363: 235-8.        [ Links ]

83. Rachmilewitz D, Stamler JS, Karmeli F, Mullins ME, Singel DJ, Loscalzo J, et al. Peroxynitrite-induced rat colitis -a new model of colonic inflammation. Gastroenterology 1993; 105: 1681-8.        [ Links ]

84. Oury TD, Piantadosi CA, Crapo JD. Cold-induced brain edema in mice. Involvement of extracellular superoxide dismutase and nitric oxide. J Biol Chem 1993; 268: 15394-8.        [ Links ]

85. Foresti R, Sarathchandra P, Clark JE, Green CJ, Motterlini R. Peroxynitrite induces haemoxygenase-1 in vascular endothelial cells: a link to apoptosis. Biochem J 1999; 339: 729-36.        [ Links ]

86. Lefer DJ, Scalia R, Campbell B, Nossuli T, Hayward R, Salamon M, et al. Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats. J Clin Invest 1997; 99: 684-91.        [ Links ]

87. Naseem KM, Bruckdorfer KR. Hydrogen peroxide at low concentrations strongly enhances the inhibitory effect of nitric oxide on platelets. Biochem J 1995; 310: 149-53.        [ Links ]

88. Bautista AP. Acute ethanol binge followed by withdrawal regulates production of reactive oxygen species and cytokine-induced neutrophil chemoattractant and liver injury during reperfusion after hepatic ischemia. Antioxid Redox Signal 2002; 4: 721-31.        [ Links ]

89. Coll O, Colell A, García-Ruiz C, Kaplowitz N, Fernández-Checa JC. Sensitivity of the 2-oxoglutarate carrier to alcohol intake contributes to mitochondrial glutathione depletion. Hepatology 2003; 38: 692-702.        [ Links ]

90. Fernández-Checa JC. Redox regulation and signaling lipids in mitochondrial apoptosis. Biochem Biophys Res Commun 2003; 9: 304: 471-9.        [ Links ]

91. Masini A, Ceccarelli D, Gallesi D, Giovannini F, Trenti T. Lipid hydroperoxide induced mitochondrial dysfunction following acute ethanol intoxication in rats. The critical role for mitochondrial reduced glutathione. Biochem Pharmacol 1994; 47: 217-24.        [ Links ]

92. Shaw KP, Lieber CS. Depressed hepatic glutathione and increased diene conjugates in alcoholic liver disease. Evidence of lipid peroxidation. Dig Dis Sci 1983, 28: 585-9.        [ Links ]

93. Kawase T, Kato S, Lieber CS. Lipid peroxidation and antioxidant defense systems in rat liver after chronic ethanol feeding. Hepatology 1989; 10: 815-21.        [ Links ]

94. Stewart SF, Day CP. The management of alcoholic liver disease. J Hepatol 2003; 38: 2-13.        [ Links ]

95. Kuiper J, Zijlstra FJ, Kamps JAAM, van Berkel TJC. Identification of prostaglandin D2 as the major eicosanoid from liver endothelial and Kupffer cells. Biochim Biophys Acta 1988; 959: 143-52.        [ Links ]

96. Caro AA, Cederbaum AI. Role of cytochrome P450 in phospholipase A2- and arachidonic acid-mediated cytotoxicity. Free Rad Biol Med 2006; 40: 364-75.        [ Links ]

97. Neyrinck AM, Margagliotti S, Gomez C, Delzenne NM. Kupffer cell-derived prostaglandin E2 is involved in regulation of lipid synthesis in ratlivertissue. Cell Biochem Funct 2004; 22: 327-32.        [ Links ]

98. Nieto N, Greenwel P, Friedman SL, Zhang F, Dannenberg AJ, Cederbaum AI. Ethanol and arachidonic acid increase alpha 2(I) collagen expression in rat hepatic stellate cells overexpressing cytochrome P450 2E1. Role of H2O2 and cyclooxigenase-2. J Biol Chem 2000; 275: 20136-45.        [ Links ]

99. Ouwendijk RJ, Zijlstra FJ, van den Broek AM, Brouwer A, Wilson JH, Vincent JE. Comparison of the production of eicosanoids by human and rat peritoneal macrophages and rat Kupffer cells. Prostaglandins 1988; 35: 437-46.        [ Links ]

100. Lieber CS. Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis. Alcohol 2004; 34: 9-19.        [ Links ]

101. Carmiel-Haggai M, Cederbaum AI, Nieto N. A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats. FASEB J 2005; 19: 136-8.        [ Links ]

102. Tsukamoto H. Redox regulation of cytokine expression in Kupffer cells. Antioxid Redox Signal 2002; 4: 741-8.        [ Links ]

103. Uesugi T, Froh M, Arteel GE, Bradford BU, Gabele E, Wheeler MD, et al. Delivery of IkappaB superrepressor gene with adenovirus reduces early alcohol-induced liver injury in rats. Hepatology 2001; 34: 1149-57.        [ Links ]

104. Wheeler MD, Yamashina S, Froh M, Rusyn I, Thurman RG. Adenoviral gene delivery can inactivate Kupffer cells: role of oxidants in NF-kappaB activation and cytokine production. J Leukoc Biol 2001; 69: 622-30.        [ Links ]

105. Zhou Z, Wang L, Song Z, Lambert JC, McClain CJ, Kang YJ. A critical involvement of oxidative stress in acute alcohol-induced hepatic TNF-alpha production. Am J Pathol 2003; 163: 1137-46.        [ Links ]

106. Lee FY, Li Y, Zhu H, Yang S, Lin HZ, Trush M, et al. Tumor necrosis factor increases mitochondrial oxidant production and induces expression of uncoupling protein-2 in the regenerating mice liver. Hepatology 1999; 29: 677-87.        [ Links ]

107. Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Fiers W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF-a. EMBO J 1993; 12: 3095-104.        [ Links ]

108. McClain CJ, Barve S, Barve S, Deaciuc I, Hill DB. Tumor necrosis factor and alcoholic liver disease. Alcohol Clin Exp Res 1998; 22: 248S-52S.        [ Links ]

109. Jokelainen K, Thomas P, Lindros K, Nanji AA. Acetaldehyde inhibits NF-kappaB activation through IkappaBalpha preservation in rat Kupffer cells. Biochem Biophys Res Commun 1998; 253: 834-6.        [ Links ]

110. Fox ES, Leingang KA. Inhibition of LPS-mediated activation in rat Kupffer cells by N-acetylcysteine occurs subsequent to NF-kappaB translocation and requires protein synthesis. J Leukoc Biol 1998; 63: 509-14.        [ Links ]

111. Akita K, Okuno M, Enya M, Imai S, Moriwaki H, Kawada N, et al. Impaired liver regeneration in mice by lipopolysaccharide via TNF-alpha/kallikrein-mediated activation of latent TGF-beta. Gastroenterology 2002; 123: 352-64.        [ Links ]

112. Cao Q, Mak KM, Lieber CS. DLPC and SAMe combined prevent leptin-stimulated TIMP-1 production in LX-2 human hepatic stellate cells by inhibiting HO-mediated signaltransduction. Liver Int 2006; 26: 221-31.        [ Links ]

113. Wu J, Kuncio GS, Zern MA. Human liver growth in fibrosis and cirrhosis. In: Strain AJ, Diehl AM, editors. Liver growth and repair. London: Chapman and Hall; 1998. p. 558-76.        [ Links ]

114. Gressner AM. Cytokines and cellular crosstalk involved in the activation of fat-storing cells. J Hepatol 1995; 22: S28-36.        [ Links ]

115. Wu J, Zern MA. Hepatic stellate cells: a target for the treatment of liver fibrosis. J Gastroenterol 2000; 35: 665-72.        [ Links ]

116. Nieto N, Cederbaum AI. S-adenosylmethionine blocks collagen I production by preventing transforming growth factor-beta induction of the COL1A2 promoter. J Biol Chem 2005; 280: 30963-74.        [ Links ]

117. Gong P, Cederbaum AI, Nieto N. Increased expression of cytochrome P450 2E1 induces heme-oxygenase-1 through ERK MAPK pathway. J Biol Chem 2003; 278: 29693-700.        [ Links ]

118. Nieto N, Cederbaum AI. Increased Sp1-dependent transactivation of the LAM gamma 1 promoter in hepatic stellate cells co-cultured with HepG2 cells overexpressing cytochrome P4502E1. J Biol Chem 2003; 278: 15360-72.        [ Links ]

119. Nieto N, Mari M, Cederbaum AI. Cytochrome P450 2E1 responsiveness in the promoter of glutamate-cysteine ligase catalytic subunit. Hepatology 2003; 37: 96-106.        [ Links ]

120. Nieto N, Domínguez-Rosales JA, Fontana L, Salazar A, Armendariz-Borunda J, Greenwel P, et al. Rat hepatic stellate cells contribute to the acute-phase response with increased expression of alpha1 (I) and alpha1(IV) collagens, tissue inhibitor of metalloproteinase-1, and matrix-metalloproteinase-2 messengerRNAs. Hepatology 2001; 3: 597-607.        [ Links ]

121. Nieto N, Friedman SL, Greenwel P, Cederbaum AI. CYP2E1-mediated oxidative stress induces collagen type I expression in rat hepatic stellate cells. Hepatology 1999; 30: 987-96.        [ Links ]

Correspondence:
Francisco Javier Cubero.
Department of Medicine.
Box 1123, 1425 Madison Avenue, Room 11-76.
10029 New York, USA.
Fax: 1-212-849-2574
e-mail: natalia.nieto@mssm.edu

Recibido: 19-05-06
Aceptado: 23-05-06