Toshiyuki Kitazawa

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‐α), 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‐α 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.

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‐α) 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‐α. 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‐α mRNA and CD14 mRNA in the liver, spleen and lung of rats with GalN‐induced hepatitis.

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)‐α 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.

The production of tumor necrosis factor-α 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-a 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-a 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-a by Kupffer cells. With acute alcohol loading, splenic macrophages tended to produce more TNF-a. Alveolar macrophages produced more TNF-a than Kupffer cells, and although the production of TNF-a by alveolar macrophages tended to be suppressed by acute alcohol loading, the production of TNF-a 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-α in acute alcoholics with endotoxemia.

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.

Innate Immune Reactivity of the Ileum–Liver Axis in Nonalcoholic Steatohepatitis

BackgroundOver-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).AimTo evaluate the innate immune response in the small intestine and liver using the rat NASH model.MethodsWe 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.ResultsSignificant 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.ConclusionsIn 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.

Effect of prostaglandin e receptor subtype EP4 selective agonist on the secretion of tumor necrosis factor-α 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.