Innate immune reactivity of the ileum-liver axis in nonalcoholic steatohepatitis.

BACKGROUND: Over-proliferation and bacterial translocation of Gram-negative bacilli within the intestinal flora, and increased portal venous levels of endotoxins, are involved in nonalcoholic steatohepatitis (NASH). AIM: To evaluate the innate immune response in the small intestine and liver using the rat NASH model. METHODS: We produced the NASH model by administering a choline-deficient amino acid-defined diet to F344 rats. We analyzed the serum and liver tissue to assess the effects of innate immune reactivity in this NASH model. RESULTS: Significant increases were detected in serum ALT levels and in the portal venous serum and whole-liver levels of TNF-α and IFN-γ in the NASH group. Strong Sirius red staining and TNF-α immune staining were seen in the NASH group, and real-time PCR revealed significantly increased expression of TNF-α and TLR4 mRNA in the NASH group. Higher TNF-α levels were detected in the Kupffer cells isolated culture supernatant in the NASH group than in the control group. Immune staining of the ileal tissue specimens resulted in greater staining of TNF-α, TLR4, and macrophage/dendritic cells, mainly in the submucosa, in the NASH group than in the control group. CONCLUSIONS: In the small intestine and liver of the rat NASH model, the possibility that enhancement of the innate immune response, mediated by the TLR4 signal, led to increased production of TNF-α was suggested. This interaction between the small intestine and liver may be involved in the onset and progression of NASH.

Therapeutic effects of cytokine modulator Y-40138 in the rat alcoholic liver disease model.

BACKGROUND AND AIM: Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-gamma (IFN-γ), induce liver injury in the rat alcoholic liver disease (ALD) model. Y-40138 is known to suppress the pro-inflammatory cytokines and augment the anti-inflammatory cytokines. We investigated whether or not Y-40138 may be effective as a novel immunotherapy in the rat ALD model. METHODS: Male Wistar rats were fed Lieber-DeCarli ethanol liquid diet. The effects of Y-40138 treatment in the ALD models were assessed by analyzing the serum and the liver tissues. RESULTS: The serum levels of alanine aminotransferase (ALT), TNF-α, and IFN-γ, and the liver levels of TNF-α and IFN-γ were significantly higher in the ethanol-fed group than in the pair-fed group. The immunohistochemistry of the liver TNF-α and 4-hydroxynonenal (4HNE), and the expressions of TNF-α and IFN-γ mRNA were increased, too. The gene expressions of interleukin-10 (IL-10) in the ethanol-fed group were suppressed as compared with the pair-fed group. The serum triglyceride (TG) and liver TG were increased, and Oil Red O and α-smooth muscle actin (α-SMA) staining showed greater expression by ethanol-fed feeding. After administration of Y-40138, enzyme linked immunosorbent assay and real-time polymerase chain reaction of the liver showed that the increased TNF-α and IFN-γ were suppressed, and that IL-10 was augmented. Moreover, ethanol-induced lipid accumulation in the liver was suppressed by administering Y-40138. CONCLUSIONS: Y-40138 decreased the inflammation, fibrosis, oxidative stress, and lipid synthesis, and augmented the anti-inflammatory cytokines of the liver. These results indicate that the multiple cytokine production modulator, Y-40138, is a promising novel therapy for ALD.

Salvage effect of E5564, Toll-like receptor 4 antagonist on d-galactosamine and lipopolysaccharide-induced acute liver failure in rats.

BACKGROUND AND AIMS: The transmembrane protein Toll-like receptor 4 (TLR4), which exists mainly in macrophages such as Kupffer cells of the liver, plays an important role in recognizing and mediating macrophage activation and pro-inflammatory cytokine release. Activation of the pro-inflammatory cytokine cascade, including tumor necrosis factor-alpha (TNF-alpha), has a pivotal role in the progression of severe liver injury. D-galactosamine (GalN) and lipopolysaccharide (LPS)-induced liver injury in rats is an experimental model of fulminant hepatic failure, where TNF-alpha plays a central role in the progression of liver injury. E5564, a synthetic analogue of the lipid A component of endotoxin, inhibits endotoxin-stimulated inflammation and is under study for patients with sepsis. In the present study, we sought to explore the salvage effect of TLR4 antagonist E5564 on GalN+LPS-induced acute liver failure (ALF) in rats. METHODS: ALF was induced in male Wistar rats by the intraperitoneal injection of GalN (500 mg/kg) and LPS (50 microg/kg). Immediately after GalN+LPS injection, rats were treated with intravenous injection of E5564 (3 mg/kg). The cumulative survival rates of GalN+LPS-induced ALF rats were compared between those with and without E5564 treatment. RESULTS: The intravenous injection of E5564 reduced the elevation of serum total bilirubin, aspartate aminotransferase, alanine aminotransferase and TNF-alpha levels in rats at 3 h after GalN+LPS injection, and improved the survival rate of GalN+LPS-induced ALF rats at 24 h (8% vs 43%). CONCLUSIONS: TLR4 antagonist E5564 reduced GalN+LPS-induced acute liver injury in rats and improved the overall survival rate of GalN+LPS-induced ALF rats. It may contribute to the treatment of ALF through blocking endotoxin-induced TNF-alpha overproduction of macrophages.

Therapeutic approach to regulate innate immune response by Toll-like receptor 4 antagonist E5564 in rats with D-galactosamine-induced acute severe liver injury.

BACKGROUND AND AIMS: Toll-like receptor 4 (TLR4) is a transmembrane protein, existing mainly in macrophages, such as Kupffer cells of the liver. It plays an important role in recognizing and mediating macrophage activation and pro-inflammatory cytokine release. Activation of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha is pivotal in the progression of liver injury. Gut-derived endotoxin has been considered to play an important role in the development and progression of a D-galactosamine (GalN)-induced acute severe liver injury (ALI) model. E5564, a synthetic analog of the lipid A component of endotoxin, inhibits endotoxin-stimulated inflammation and is under study for patients with sepsis. In this study, we seek to explore the effect of TLR4 antagonist E5564 on GalN-induced ALI in rats. METHODS: ALI was induced in male Wistar rats by the i.p. injection of 1 g/kg bodyweight of GalN and immediately after GalN injection they were treated with an i.v. injection of 3 mg/kg bodyweight of E5564. At 24 h after GalN injection with or without E5564, serum levels of total bilirubin (T.Bil), alanine aminotransferase (ALT) and TNF-alpha were analyzed. Expression levels of TNF-alpha, TLR4 and CD14 mRNA in the whole liver of rats was detected by reverse transcription polymerase chain reaction analysis. RESULTS: The i.v. injection of E5564 reduced the elevation of serum T.Bil, ALT and TNF-alpha levels in rats treated with GalN. The expression level of TNF-alpha mRNA in the whole liver, which was increased at 24 h after GalN injection, was also reduced by i.v. injection of E5564. CONCLUSION: TLR4 antagonist E5564 reduced GalN-induced ALI in rats. It may contribute to the treatment of acute liver failure through blocking endotoxin-induced TNF-alpha overproduction of macrophages.

Expression of Toll-like receptor 4 in various organs in rats with D-galactosamine-induced acute hepatic failure.

BACKGROUND AND AIMS: Activation of the pro-inflammatory cytokine cascade, including tumor necrosis factor alpha (TNF-alpha) is considered to play an important role in the pathophysiology and clinical outcome of severe liver injury. Kupffer cells, resident macrophages of the liver, have a transmembrane protein Toll-like receptor 4 (TLR4), which recognizes endotoxin (lipopolysaccharide; LPS) or LPS-CD14 complex and mediates macrophage activation and pro-inflammatory cytokine release. D-Galactosamine (GalN), a hepatocyte-specific inhibitor of RNA synthesis, is known to sensitize animals to the lethal effects of LPS and TNF-alpha. In the present study we seek to address TLR4-signaling in the development of GalN-induced acute hepatic failure (AHF) and explore the expression of TLR4 mRNA as compared to TNF-alpha mRNA and CD14 mRNA in the liver, spleen and lung of rats with GalN-induced hepatitis. METHODS: AHF was induced in male Wistar rats by the intraperitoneal injection of 1 g/kg bodyweight GalN. Expression levels of TNF-alpha, TLR4 and CD14 mRNA in the whole liver, spleen and lung of rats were detected by reverse transcription-polymerase chain reaction analysis. RESULTS: Expression level of TLR4 mRNA in the liver of rats with GalN-induced AHF was increased parallel with that of TNF-alpha and CD14 mRNA as compared to the control rats. However, expression levels of TNF-alpha, TLR4 and CD14 mRNA in the whole spleen and lung were not different between rats with AHF and control. CONCLUSIONS: There may be a difference of stimulatory effects of endotoxin on the innate immunity between the liver and other organs of rats with GalN-induced AHF.

Innate immune reactivity of the liver in rats fed a choline-deficient L-amino-acid-defined diet.

AIM: To investigate the innate immune reactivity of tumor necrosis factor-alpha (TNF-alpha), Toll-like receptor 4 (TLR4), and CD14 in the liver of non-alcoholic steatohepatitis (NASH) model rats. METHODS: Male F344 rats were fed a choline-deficient L-amino-acid-defined (CDAA) diet. The rats were killed after 4 or 8 wk of the diet, and their livers were removed for immunohistochemical investigation and RNA extraction. The liver specimens were immunostained for TNF-alpha, TLR4, and CD14. The gene expressions of TNF-alpha, TLR4, and CD14 were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Kupffer cells were isolated from the liver by Percoll gradient centrifugation, and were then cultured to measure TNF-alpha production. RESULTS: The serum and liver levels of TNF-alpha in the CDAA-fed rats increased significantly as compared with the control group, as did the immunohistochemical values and gene expressions of TNF-alpha, TLR4, and CD14 with the progression of steatohepatitis. TNF-alpha production from the isolated Kupffer cells of the CDAA-fed rats was elevated by lipopolysaccharide stimulation. CONCLUSION: The expressions of TNF-alpha, TLR4, and CD14 increased in the NASH model, suggesting that TLR4 and CD14-mediated endotoxin liver damage may also occur in NASH.

Decreased phagocytic activity of Kupffer cells in a rat nonalcoholic steatohepatitis model.

AIM: To investigate Kupffer cell dynamics and phagocytic activity, using a rat nonalcoholic steatohepatitis (NASH) model. METHODS: Male F344 rats were fed either a control diet or a choline-deficient L-amino acid-defined (CDAA) diet, followed by contrast enhanced ultrasonography (CEUS) using Levovist. The uptake of latex beads by the Kupffer cells was determined by fluorescent microscopy. The status of the Kupffer cells was compared between the two groups, using the immunohistochemical staining technique. RESULTS: After 4 or more wk of the CDAA diet, CEUS examination revealed a decrease in the signal intensity, 20 min after intravenous Levovist. Fluorescent microscopic examination showed that the uptake of latex beads by the Kupffer cells was reduced at week 1 and 2 in the study group, compared with the controls, with no further reduction after 3 wk. Immunohistochemical staining revealed no significant difference in the Kupffer cell counts between the control group and the CDAA group. CONCLUSION: CEUS examination using Levovist demonstrated reduced contrast effect and phagocytic activity in the liver parenchymal phase, although the Kupffer cell numbers were unchanged, indicating reduced phagocytic function of the Kupffer cells in the rat NASH model. We believe that CEUS examination using Levovist is a useful screening modality, which can detect NASH in fatty liver patients.

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-alpha (TNF-alpha), interferon-gamma (IFN-gamma), 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-alpha). We then extracted Kupffer cells from the collagenase-perfused livers using the Percoll gradient centrifugation method, and measured the TNF-alpha levels in the supernatant (in vitro TNF-alpha 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-alpha and IFN-gamma 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-alpha immunohistochemical staining showed fewer stained cells in the NASH + Y-40138 group than in the NASH group. The TNF-alpha 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.

The production of tumor necrosis factor-alpha by macrophages in rats with acute alcohol loading.

BACKGROUND: It is suggested that endotoxin, proinflammatory cytokines, and lipopolysaccharide-binding protein (LBP) play an important role in the development of alcoholic liver disease. Our previous study showed that splenic macrophages were important for endotoxin uptake and excessive production of tumor necrosis factor (TNF) in rats given large amounts of alcohol. To study the pathophysiological roles of macrophages in alcoholic liver diseases, we examined the production of TNF-alpha by rat Kupffer cells, splenic macrophages, and alveolar macrophages with acute alcohol loading in the presence or absence of LBP. METHODS: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from male Wistar rats given 5 mg/g body weight of ethanol intraperitoneally after an hour. The production of TNF-alpha by these cells incubated with endotoxin 100 ng/ml in the presence or absence of LBP (1% rat serum) was determined. RESULTS: Acute alcohol loading did not affect the production of TNF-alpha by Kupffer cells. With acute alcohol loading, splenic macrophages tended to produce more TNF-alpha. Alveolar macrophages produced more TNF-alpha than Kupffer cells, and although the production of TNF-alpha by alveolar macrophages tended to be suppressed by acute alcohol loading, the production of TNF-alpha by alveolar macrophages still remained high in the presence of rat serum. CONCLUSIONS: Splenic macrophages and alveolar macrophages may be related to excessive production of TNF-alpha in acute alcoholics with endotoxemia.

Effect of prostaglandin E receptor subtype EP4 selective agonist on the secretion of tumor necrosis factor-alpha by macrophages in acute ethanol-loaded rats.

BACKGROUND: It is suggested that endotoxin and proinflammatory cytokines play an important role in the development and progression of alcoholic liver disease. Recently, a prostaglandin receptor subtype EP4 agonist with cytoprotective effect has been developed. We examined the efficacy of an EP4 agonist ONO-AE1-437 on tumor necrosis factor-alpha (TNF-alpha) secretion of Kupffer cells, splenic macrophages, and alveolar macrophages in acute ethanol-loaded rats. METHODS: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from control and acute ethanol-loaded rats (5 mg/g body weight of ethanol, intraperitoneally). After the preculture in the medium that containing 0, 0.1, 1, 10, or 100 nmol/liter of ONO-AE1-437, TNF-alpha secretion of these cells stimulated by 100 ng/ml of endotoxin was determined for 3 hr. RESULTS: The amount of TNF-alpha secreted from alveolar macrophages was largest in both the control and the acute ethanol-loaded rats. Acute ethanol load enhances TNF-alpha secretion of splenic macrophages. The addition of ONO-AE1-437 significantly inhibited TNF-alpha secretion of Kupffer cells and splenic macrophages in both the control and the acute ethanol-loaded rats. Alveolar macrophages were less affected. CONCLUSIONS: An EP4 agonist ONO-AE1-437 suppresses excess TNF-alpha secretion from macrophages and seems promising for future trial in patients with severe alcoholic hepatitis.

Effect of alcohol on the secretion of tumor necrosis factor-alpha by macrophages in the presence of rat serum.

BACKGROUND: It is suggested that endotoxin, proinflammatory cytokines, and lipopolysaccharide-binding protein (LBP) play an important role in the development of alcoholic liver disease. Our previous study showed that splenic macrophages were important for endotoxin uptake and excessive production of tumor necrosis factor (TNF) in rats given large amounts of alcohol. To determine the pathophysiological roles of macrophages in alcoholic liver disease, we examined the effect of ethanol on TNF-alpha secretion of rat Kupffer cells, alveolar macrophages, and peritoneal macrophages in the presence or absence of LBP. METHODS: Kupffer cells, alveolar macrophages, and peritoneal macrophages were isolated from male Sprague Dawley rats. After the preculture in the medium containing 0, 10, 50, and 100 mmol/liter of ethanol, TNF-alpha secretion by these cells incubated with 100 ng/ml of endotoxin in the presence or absence of LBP (1% rat serum) was determined. RESULTS: In the absence of LBP, an addition of ethanol to the medium suppressed TNF-alpha secretion of alveolar macrophages. Kupffer cells and peritoneal macrophages were less affected. Addition of LBP led to marked enhancement (7- to 24-fold) of TNF-alpha secretion of macrophages either with or without ethanol in the medium. Although ethanol tended to suppress TNF-alpha secretion of these cells, alveolar macrophages were less affected in the presence of LBP. CONCLUSIONS: Serum LBP enhances the secretion of TNF-alpha by macrophages. Alveolar macrophages may be important for excessive production of TNF-alpha in chronic alcoholics with endotoxemia.