All-trans retinoic acid suppresses liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

All-trans retinoic acid (ATRA) has been reported to exert major effects on the immune system, including monocytes/macrophages. The present study was designed to determine whether ATRA would modulate macrophage-associated liver injury induced by Propionibacterium acnes and lipopolysaccharide (LPS) in rats. All-trans retinoic acid administration alleviated the liver injury and reduced the incidence of death following hepatic failure. Serum alanine aminotransferase (ALT) levels 5 h after, and survival rates within 12 h after the administration of LPS were significantly lower in the ATRA-treated group (134 ± 119 IU/L and 72.7%) compared with the control group (713 ± 411 IU/L and 18.2%; P < 0.05). Histological findings supported these results. These effects may be due to suppression of tumour necrosis factor-α (TNF-α) and superoxide anions produced by activated macrophages. Serum levels of TNF-α 1 h after LPS administration were significantly lower in the ATRA-treated group (60.5 ± 7.0 ng/mL) as compared with the control group (105.2 ± 39.3 ng/mL; P < 0.05). Formazan deposition that was generated by the perfusion of the liver with nitroblue tetrazolium, also suggested suppression of the release of superoxide anions from hepatic macrophages. These results suggest that ATRA acts as an immunomodulator in liver injury by suppressing the activation of liver macrophages.     

Cytokine-induced neutrophil chemoattractant release from hepatocytes is modulated by Kupffer cells

To clarify the role of intercellular communication in the liver during accumulation of neutrophils, the release of cytokine-induced neutrophil chemoattractant (CINC) (interleukin-8 [IL-8] related protein in rodents) by hepatocytes was investigated in the presence of Kupffer cell-conditioned medium in vitro. Kupffer cells were prepared by perfusion of rat liver with collagenase followed by centrifugation on a metrizamide gradient and were cultured in the presence or absence of lipopolysacharide (LPS). The conditioned medium was collected after 24 hours, and rat hepatocytes were cultured in the presence or absence of Kupffer cell-conditioned medium. An amount of CINC in the culture supernatant was measured by western blotting analysis and enzyme-linked immunosorbent assay (ELISA), and expression of its messenger RNA (mRNA) was assessed by the polymerase chain reaction. LPS-stimulated Kupffer cell-conditioned medium enhanced an expression of CINC mRNA in hepatocytes and increased the production of CINC by hepatocytes. Enhanced production of CINC was not shown when the Kupffer cell-conditioned medium was pretreated with heat (56 degrees C, 30 minutes). The production of CINC by hepatocytes in the presence of the LPS-stimulated Kupffer cell-conditioned medium was reduced by an antibody against interleukin 1 beta (IL-1 beta), but not by antibodies against tumor necrosis factor alpha (TNF-alpha) or LPS. These results suggest that production of CINC by hepatocytes could be regulated by IL-1 beta released from Kupffer cells, leading to neutrophil accumulation during liver injury, because this protein is a strong chemoattractant for neutrophils. (Hepatology 1996 Feb;23(2):353-8)     

Lipo prostaglandin E1 reduces the production of CXC chemokines in endotoxin-induced rat liver injury

The present study attempted to assess the effect of prostaglandin E1 (PGE1) incorporated into lipid microspheres (Lipo PGE1) on chemokine production in endotoxin-induced rat liver injury. Male Wistar rats weighing 200–250 g were injected with 2 mg lipopolysaccharide (LPS) per kg intravenously. Lipo PGE1 was administered simultaneously at various concentrations (0.002, 0.02, 0.2, 2 μg/kg) in the tail vein. Blood samples and liver specimens were taken from the rats at 1, 3, 8, 12 and 24 h after injection with LPS alone or with LPS and Lipo PGE1. Serum macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) levels were measured by the enzyme-linked immunosorbant assay using the corresponding antibodies. Liver specimens were fixed, and the number of neutrophils that had infiltrated each liver section was determined under a microscope. Serum alanine aminotransferase (ALT) levels were significantly lower in the rats injected with LPS and Lipo PGE1 compared with those in the rats injected with LPS alone, and this difference was expressed in a PGE1 dose-dependent manner. Serum MIP-2 levels were significantly lower at 3 h (141.4±95.5 pg/ml) and 8 h (44.9±44.7 pg/ml) after injection with LPS and Lipo-PGE1 (2 μg/kg) than at the same times after injection with LPS alone (342.9±35.9 and 358.3±23.4 pg/ml, respectively). Similarly, serum CINC levels were significantly lower at 8 h (482.7±156.0 ng/ml) after injection with LPS and Lipo-PGE1 (2 μg/kg) than at the same time after injection LPS alone (723.3±29.0 ng/ml). No significant differences were observed at any time between serum tumor necrosis factor-α (TNF-α) levels in rats injected with LPS alone and in rats injected with LPS and Lipo-PGE1 (2 μg/kg). The number of neutrophils that had infiltrated the liver was significantly lower at 8 h after injection with LPS and Lipo PGE1 than at the same time after injection with LPS alone. This difference was expressed in a Lipo PGE1 dose-dependent manner. In conclusion, Lipo PGE1 reduces liver injury and serum levels of MIP-2 and CINC, but not TNF-α, in rats injected with LPS and also reduces the number of neutrophils that infiltrate in the liver.     

Isolation and primary culture of rat Kupffer cells

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48–110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 μm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80–110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80–120 times 10⁶ Kupffer cells per liver.     

Rat liver slices as a tool to study LPS-induced inflammatory response in the liver

BACKGROUND/AIMS: Inflammation in the liver is a complex interaction between parenchymal and non-parenchymal cells, and therefore can not be studied in vitro in pure cultures of these cells. METHODS: We investigated whether Kupffer cells in the liver slice are still responsive to an inflammatory stimulus of lipopolysaccharide (LPS), and evoke an inflammatory response in the hepatocytes. RESULTS: TNFalpha, IL-1beta and IL-10 were significantly elevated in culture medium of LPS-stimulated rat liver slices. Nitric oxide (NO) production of LPS-treated slices gradually increased from 5 to 24 h (24 h: 81+/-5 microM vs. 14+/-2 microM in control P < 0.05), paralleled by inducible nitric oxide synthase (iNOS) in the hepatocytes, iNOS mRNA was induced after 3 h. NO production but not iNOS induction was significantly inhibited by NOS inhibitors S-methylisothiourea and N(G)-nitro-L-arginine methylester. Both pentoxifylline and dexamethasone inhibited TNFalpha and IL-1beta production, albeit to a different extent, iNOS induction and, as a result thereof, NO production. CONCLUSIONS: These results imply that non-parenchymal cells in liver slices are viable and can be activated by LPS. In addition, it is concluded that the upregulation of iNOS in hepatocytes by LPS is caused by cytokines produced by Kupffer cells because inhibition of TNFalpha and IL-1beta production attenuated iNOS induction.     

Immunohistochemical study of tumor necrosis factor-alpha in acute liver injury induced by Propionibacterium acnes and lipopolysaccharide in rats

The effect of intravenous injection of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) on the distribution of tumor necrosis factor-α (TNF-α) in different organs have not previously been investigated. Immunohistochemistry and histological examination were employed in evaluating the distribution of TNF-α in the liver, spleen, lungs and bone marrow in rats injected intravenously with P. acnes followed by LPS 7 days later. Granulomas containing ED1-positive macrophages were observed in the liver 7 days after P. acnes injection. Subsequent LPS injection resulted in proliferation of ED1-positive macrophages in the sinusoids and coagulation necrosis of hepatocytes after 6 h. TNF-α was detected in ED2-positive macrophages (Kupffer cells) 1 day after P. acnes injection and in macrophages constituting the granulomas 7 days later, but prior to LPS injection. TNF-α was also detected in ED1-positive macrophages in the spleen, predominantly in the marginal zone. When granulomas were formed 7 days after P. acnes injection, TNF-α was observed in macrophages of the granulomas. TNF-α was also detected in macrophages of the granulomas found in the lung 1 day after P. acnes injection. No macrophages expressing TNF-α were found in the granulomas of bone marrow. The highest expression was in the liver at any time interval and in macrophages constituting granulomas. Our results suggest that the high expression of TNF-α in the liver results in selective hepatic necrosis. The expression of TNF-α in macrophages of the liver after P. acnes injection and the subsequent development of hepatic necrosis after LPS injection suggest that P. acnes acts as an inducer of TNF-α production in macrophages while LPS acts as a trigger for the release of TNF-α from macrophages.     

Expression of tumor necrosis factor-α gene during allograft rejection following rat liver transplantation

Abstract: Background/Aims: Tumor necrosis factor-α (TNF-α) is believed to play a role in hepatic allograft rejection. However, the specific cellular population responsible for TNF-α production during hepatic allograft rejection is not known. Circulating monocyte-macrophage cells are the primary systemic sources of TNF-α. In the liver, Kupffer cells are the main producers of TNF-α. In this study, we determined which cells are involved in TNF-α production during allograft rejection after orthotopic liver transplantation. Methods: In situ hybridization was used to identify cells with TNF-α mRNA in the liver. Immunohistochemical staining with ED2 and ED3 was used to differentiate between cellular types (Kupffer cells versus infiltrating monocytes). To detect DNA fragmentation in liver cells, TdT-mediated biotin-dUTP nick-end labeling (TUNEL) was done. Studies were performed in the rat liver transplant model using rejecting (ACI to LEW) and non-rejecting (ACI to ACI) donor/recipient combinations. Results: In the control group, cells with TNF-α mRNA were rarely observed. In the rejection group, TNF-α mRNA was observed in mononuclear cells that were mainly within the vessels of the portal region and occasionally in the sinusoids. The cells with the signals for TNF-α mRNA were ED2-negative and ED3-positive. DNA fragementation was observed in hepatocytes as well as infiltrating mononuclear cells. Conclusions: The main producer of TNF-α may be infiltrating mononuclear cells such as monocyte-macrophage cells rather than Kupffer cells during allograft rejection after liver transplantation. Circulating monocyte-macrophages may play a role in the control of allograft rejection.     

Mechanism of neutrophil accumulation in sinusoids after extrahepatic biliary obstruction

BACKGROUND/AIMS: Polymorphonuclear neutrophil (PMN) infiltration represents a potential source of liver injury, but the precise mechanisms of PMN infiltration in cholestatic liver are not fully understood. METHODOLOGY: This study investigated hepatic expression of cytokine-induced neutrophil chemoattractant (CINC) 14 days after bile duct ligation, as well as the number of infiltrated PMNs in livers. Portal venous endotoxin levels were also evaluated. Furthermore, in vitro CINC production by isolated liver cells from obstructive jaundice (OJ) liver or sham-treated liver was evaluated after stimulation with tumor necrosis factor-alpha (TNFalpha), interleukin-1beta (IL-1beta) or LPS. RESULTS: The number of infiltrated PMNs in sinusoids significantly increased in OJ liver, as compared to sham-treated liver. CINC mRNA expression was also increased in OJ liver. Immunohistochemical study revealed that the majority of the CINC-positive cells were hepatocytes. In vitro study proved that CINC production by isolated hepatocytes was markedly enhanced by IL-1beta stimulation in OJ liver. Furthermore, IL-1beta production by LPS-stimulated Kupffer cells isolated from OJ liver was significantly increased, compared to those from sham-treated liver. Portal venous endotoxin was detectable only in OJ rats. CONCLUSIONS: Excessive production of IL-1beta by activated Kupffer cells, as a result of portal endotoxemia, may play an important role for increased CINC release from hepatocytes in cholestatic liver, leading to PMN infiltration.     

Tumor Necrosis Factor-α Cell-Surface Receptors of Liver Parenchymal and Nonparenchymal Cells during Acute and Chronic Alcohol Administration to Rats

Tumor necrosis factor-α (TNF-α) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-α could be due, at least in part, to alterations in TNF-α cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchymal (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 /μg/100 g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (K_d) and capacity (B_max) of binding sites, using recombinant human-[¹²⁵l]TNF-α as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (K_d1, in the range of 150–200 PM), low-capacity (B_max1, in the range of 2–3 fmol/10⁶ cells) binding site and a low-affinity (K_d2, in the range of 2–9 nm), high-capacity (B_max2, in the range of 3–15 fmol/10⁶ cells) binding site. One binding site was detected on hepatocytes isolated from control rats (K_d1= 1.0 nm and a B_max= 95 fmol/10⁶ cells). Acute ETOH administration caused an increase in K_d1 on both Kupffer and sinusoidal endothelial cells; a decrease in K_d1 on hepatocytes; and an increase in K_d2, B_max1, and B_max2 on endothelial cells. A second binding site on hepatocytes (K_d2= 5.8 nm, S_max2= 186 fmol/10⁶ cells) was observed only in the ETOH-treated group after LPS administration. Chronic alcohol exposure markedly elevated K_d1 and S_max1 on hepatocytes. Overall, LPS-induced changes mimicked those induced by alcohol, except for a decrease in B_max1 on Kupffer cells of rats in the acute treatment group. Also, the liquid diet containing alcohol or dextrin abrogated the high-affinity, low-capacity binding sites on both Kupffer cells and sinusoidal endothelial cells, and low-affinity, high-capacity binding sites on the hepatocyte. After LPS administration, the high-affinity binding sites on Kupffer and sinusoidal endothelial cells were reexpressed. These data show that: (1) ETOH induces changes in both the capacity (B_max) and affinity (K_d) of TNF-α cell-surface receptors of hepatocytes, Kupffer cells, and sinusoidal endothelial cells; and (2) ETOH-induced changes are consistent with an increased sensitivity of these cells to the action of TNF-α.     

Tumor necrosis factor alpha has a crucial role in increased reactive oxygen species production in platelets of mice injected with lipopolysaccharide

Increased reactive oxygen species (ROS) production leads to tissue damage observed in sepsis and lipopolysaccharide (LPS)-exposed animals. LPS stimulates cytokines releasing, including tumor necrosis factor alpha (TNF-α), that is important to ROS production. Platelets, considered inflammatory cells, generate ROS when exposed to LPS in vivo, but not when they are incubated in vitro with this compound. Therefore, we investigated the role of TNF-α on the increased intraplatelet ROS levels after LPS treatment. Mice were injected with LPS (1 mg/kg) or TNF-α (10 ng/kg), and blood was collected to prepare the washed platelets. Animals were treated with infliximab (anti-TNF-α antibody), R-7050 (non-selective TNF-α receptor antagonist) or apocynin (NADPH oxidase inhibitor). At 48 h after LPS or TNF-α injection, the ROS levels in ADP (25 µM)-activated platelets were evaluated by flow cytometry. Our data showed that injection of mice with LPS increased by 4-fold the ROS production (p < 0.05), which was significantly reduced by the treatments with infliximab, R-7050 or apocynin. Injection of mice with TNF-α markedly elevated the ROS formation in platelets (p < 0.05) that was reduced by infliximab, R-7050 or apocynin treatments. In separate experiments, platelets from saline-injected mice were incubated with TNF-α (30 to 3000 pg/mL) in absence or presence of infliximab, R-7050, apocynin or GKT137831 (NOX1/NOX4 inhibitor) before ROS measurements. TNF-α in vitro markedly increased the ROS levels, an effect significantly reduced by all treatments. Therefore, platelets are involved in the oxidative stress induced by LPS through TNF-α action, and NADPH oxidase takes part in this effect.     

Effect of Acute and Chronic Alcohol Administration to Rats on the Expression of Interleukin-6 Cell-Surface Receptors of Hepatic Parenchymal and Nonparenchymal Cells

Rats were treated with alcohol either acutely (continuous, 7-hr intravenous infusion; blood alcohol levels ～35 mM) or chronically (liquid diet, 12–14 weeks). Three hr before killing, the animals received Gram-negative bacterial lipopolysaccharide (LPS) or saline. Hepatocytes, Kupffer cells, and liver sinusoidal endothelial cells were isolated by liver collagenase perfusion and centrifugal elutriation, and used for measurements of recombinant human [¹²⁵I]interleukin-6 binding. Dissociation constant (K_d) and the amount of cell-surface receptors (B_max) were measured on whole cells, at 4°C. Two binding sites were detected on all three cell types: high-affinity (K_d1, from 20 to 125 PM) and low-affinity (K_d2, from 0.2 to 2 nM), with low B_max (B_max from 0.4 to 12 fmol/10⁶ cells) and high B_max (B_max2, from 10 to 210 fmol/10⁶ cells). Hepatocytes displayed an 8-fold higher binding capacity for high-affinity sites (B_max1) than the other two cell types. Acute ethanol treatment induced the following significant changes in the binding parameters: a decrease in K_d1 for hepatocytes and Kupffer cells, an increase in B_max2 for hepatocytes, and a decrease in B_max1 for Kupffer cells. Although the control (nonalcoholic) liquid diet per se completely suppressed the high-affinity binding sites, alcohol-containing diet induced only one change: a significant increase in K_d2 for hepatocytes. No changes in the binding parameters were seen after LPS administration to the chronically treated group. In the acute group, LPS mimicked alcohol action on hepatocyte binding parameters. Alcohol blunted LPS effects. No changes were observed in the cytokine binding to Kupffer cells after LPS injection. The results show that alcohol alters interleukin-6 cell-surface receptor properties and receptor amount on hepatocytes and Kupffer cells. By demonstrating the presence of interleukin-6 receptors on non-parenchymal liver cells, our data also suggest that these cells may be involved in an autocrine loop-like response, which could be a target for alcohol action on the liver.     

Immunotherapy for nonalcoholic steatohepatitis using the multiple cytokine production modulator Y-40138

AIM: To investigate the possible use of the multiple cytokine production modulator, Y-40138, as a novel immunotherapy in the rat nonalcoholic steatohepatitis(NASH) model.METHODS: We allocated 6-wk-old male F344 rats to choline-supplemented, L-amino acid-defined (CSAA) diet (control group), CSAA diet + Y-40138 (control +Y-40138 group), choline-deficient, L-amino acid-defined (CDAA) diet (NASH group), or CDAA diet + Y-40138 (NASH + Y-40138 group). In each group, we measured the plasma alanine aminotransferase (ALT) levels, and the plasma and liver levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-10 (IL-10). Tissue specimens of phosphate buffered saline-perfused liver were subjected to hematoxylin and eosin staining, Azan staining, Sirius red staining, and immunohistochemical staining (for Kupffer cells and TNF-α). We then extracted Kupffer cells from the collagenase-perfused livers using the Percoll gradient centrifugation method, and measured the TNF-α levels in the supernatant ( in vitro TNF-α production by Kupffer cells) using an enzyme-linked immunosorbent assay kit. RESULTS: In comparison to the NASH group, serum ALT elevation was mild, production of serum and liver TNF-α and IFN-γ was inhibited, and IL-10 production was increased in the NASH + Y-40138 group. Amelioration of liver histology was also noted in the NASH + Y-40138 group. Kupffer cell immunohistochemical staining revealed no differences between groups, whereas TNF-α immunohistochemical staining showed fewer stained cells in the NASH + Y-40138 group than in the NASH group. The TNF-α levels in the in-vitro Kupffer cell culture supernatant were lower in the NASH + Y-40138 group than in the NASH group.CONCLUSION: Administration of Y-40138 to NASH model rats reduced hepatic inflammation and suppressed fibrosis. These results indicate that the multiple cytokine production modulator, Y-40138, is promising as a novel treatment for NASH.     

An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells

Abstract: Aims/Background: One function of Kupffer cells is the phagocytosis of nonviable hepatocytes. Our aims were to develop a model for phagocytosis of damaged hepatocytes by rat Kupffer cells in vitro, and to characterise prostaglandin E₂ (PGE₂), prostacyclin (PGI), and tumour necrosis factor-α (TNF) production in this model. Methods: Kupffer cells were incubated alone or with damaged hepatocytes for up to 18 h, then washed and cultured for up to 66 h. To compare mediator responses produced during inert particle phagocytosis, Kupffer cells were also incubated with latex beads. Results: Phagocytic uptake of hepatocyte debris was confirmed in at least 50% of Kupffer cells. A dissociation between TNF and PGI responses was found for both latex beads and damaged hepatocytes, such that a TNF secretory response was not triggered by either stimulus whereas PGI production was increased for both. Although phagocytosis of beads increased PGE₂ production, phagocytosis of hepatocytes did not. Conclusions: Phagocytosis of damaged hepatocytes by Kupffer cells results in the production of PGI but not PGE₂ or TNF.     

库普弗细胞条件培养基对原代培养肝细胞的细胞毒性

目的 探讨内毒素血症时库普弗细胞激活对肝细胞损伤的作用机制.方法 用链霉蛋白酶和胶原酶原位灌流,Percoll密度梯度离心分离、纯化SD大鼠肝细胞和库普弗细胞,制备内毒素(LPS)刺激的库普弗细胞的条件培养基(KCCM)并作用于肝细胞,检测肝细胞培养基丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)的含量,四甲基偶氮唑蓝(MTT)法检测KCCM对大鼠原代肝细胞活性的影响.结果 KCCM作用于肝细胞2 h后,肝细胞培养基中ALT、AST及LDH含量均明显升高(P＜0.05),在2～4 h内随时间延长ALT、AST及LDH含量均逐渐增加,呈明显的时间-效应关系;MTT结果显示,KCCM作用后12 h、24 h时间点对肝细胞具有明显损伤作用(P＜0.05).结论 内毒素诱导库普弗细胞释放的可溶性因子作用一定时间后可直接对肝细胞造成损伤.     

Interleukin-37 reduces liver inflammatory injury via effects on hepatocytes and non-parenchymal cells

Background and Aim: The purpose of the present study was to determine the effects of interleukin-37 (IL-37) on liver cells and on liver inflammation induced by hepatic ischemia/reperfusion (I/R). Methods: Mice were subjected to I/R. Some mice received recombinant IL-37 (IL-37) at the time of reperfusion. Serum levels of alanine aminotransferase, and liver myeloperoxidase content were assessed. Serum and liver tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2) and keratinocyte chemokine (KC) were also assessed. Hepatic reactive oxygen species (ROS) levels were assessed. For in vitro experiments, isolated hepatocytes and Kupffer cells were treated with IL-37 and inflammatory stimulants. Cytokine and chemokine production by these cells were assessed. Primary hepatocytes underwent induced cell injury and were treated with IL-37 concurrently. Hepatocyte cytotoxicity and Bcl-2 expression were determined. Isolated neutrophils were treated with TNF-α and IL-37 and neutrophil activation and respiratory burst were assessed. Results: IL-37 reduced hepatocyte injury and neutrophil accumulation in the liver after I/R. These effects were accompanied by reduced serum levels of TNF-α and MIP-2 and hepatic ROS levels. IL-37 significantly reduced MIP-2 and KC productions from lipopolysaccharide-stimulated hepatocytes and Kupffer cells. IL-37 significantly reduced cell death and increased Bcl-2 expression in hepatocytes. IL-37 significantly suppressed TNF-α-induced neutrophil activation. Conclusions: IL-37 is protective against hepatic I/R injury. These effects are related to the ability of IL-37 to reduce proinflammatory cytokine and chemokine production by hepatocytes and Kupffer cells as well as having a direct protective effect on hepatocytes. In addition, IL-37 contributes to reduce liver injury through suppression of neutrophil activity.     

The mechanisms of hepatic sinusoidal endothelial cell regeneration: A possible communication system associated with vascular endothelial growth factor in liver cells

Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver.     

Induction of anaphylatoxin C5a receptors in rat hepatocytes by lipopolysaccharide in vivo: mediation by interleukin-6 from Kupffer cells

BACKGROUND & AIMS: In normal rat liver, anaphylatoxin C5a induces glucose output from hepatocytes indirectly via prostanoids released from Kupffer cells. Correspondingly, it was found that hepatocytes, in contrast to Kupffer cells, did not express C5a receptors. Lipopolysaccharide (LPS) has been reported to enhance C5a receptor expression in murine livers. This might be the result of de novo expression in hepatocytes. METHODS: C5a receptor expression was investigated in hepatocytes after in vivo treatment of rats with LPS and in vitro stimulation of isolated cells with LPS and proinflammatory cytokines on messenger RNA (mRNA) and protein level, and functionally in isolated hepatocytes and perfused liver. RESULTS: In vivo treatment of rats with LPS induced C5a receptor mRNA and protein in hepatocytes with a maximum after 8-10 hours. At this time-point, C5a directly activated glycogen phosphorylase in isolated hepatocytes and enhanced glucose output in perfused livers without the involvement of prostanoids. LPS failed to induce C5a receptors in cultured hepatocytes in vitro, whereas interleukin (IL) 6 and IL-1beta, which are known to be released from Kupffer cells on stimulation with LPS, did so. In cocultures of hepatocytes with Kupffer cells, LPS induced C5a receptors in hepatocytes in an IL-6-dependent manner. CONCLUSIONS: Thus, IL-6 from Kupffer cells appears to be the main mediator of LPS-induced de novo expression of C5a receptors in hepatocytes.     

Taurine is an osmolyte in rat liver macrophages (Kupffer cells)

Background/Aims: The availability of betaine as an osmolyte was recently shown to interfere strongly with important cell functions of liver macrophages (Kupffer cells), such as eicosanoid and tumor necrosis factor-α production or phagocytosis. We therefore investigated whether taurine is also used as an osmolyte by Kupffer cells and whether it is involved in the control of Kupffer cell functions.Methods/Results: Hyperosmotic (hypoosmotic) exposure of cultured rat liver macrophages (Kupffer cells) for 6–12 h led to an increase (decrease) in the mRNA levels of the taurine transporter (TAUT) and an increase (decrease) in taurine transport into the cells. The hyperosmolarity-induced increase in TAUT-mRNA levels was diminished by 37±10% upon addition of taurine, but not upon addition of betaine. When Kupffer cells were preloaded with taurine, hypoosmotic exposure led to a rapid efflux of taurine from the cells, which was significantly delayed in the presence of the anion exhanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). Taurine efflux was also stimulated during phagocytosis of Latex particles; however, Latex was without effect on the hyperosmolarity-induced increase of TAUT mRNA levels. Lipopolysaccharide (LPS) led to an induction of cyclooxygenase-2, which was markedly enhanced during hyperosmotic conditions. Taurine diminished the induction of cyclooxygenase-2 and inhibited the LPS/hyperosmolarity-induced stimulation of prostaglandin E₂ formation.Conclusions: The data suggest that, in addition to betaine, taurine also acts as an osmolyte in Kupffer cells, and that taurine availability may be an important modulater of Kupffer cell functions such as eicosanoid synthesis.