Kazuo Kamii

Oxygen-derived free radical generating capacity of polymorphonuclear cells in patients with ulcerative colitis.

Oxygen-derived free radical generating capacity of polymorphonuclear cells in 27 patients with ulcerative colitis, 10 with acute bacterial diarrhea and 20 healthy volunteers, was measured by the luminol-dependent chemiluminescence method by stimulation of formyl-methionyl-leucyl-phenylalanine. Oxidative free radical generating capacity of polymorphonuclear cells in patients with active ulcerative colitis was markedly enhanced as compared with control (p less than 0.01), while this enhanced free radical production by the cells was not detected at remission stage. Serial analyses revealed that oxidative free radical production by the cells in patients with ulcerative colitis was markedly enhanced with clinical deterioration of the disease, but it returned to normal level with an improvement of clinical features. These results suggest that the increased oxidative free radical production by polymorphonuclear cells could be related to the pathogenesis or aggravation of ulcerative colitis.

Modulation of ischemia-reperfusion-induced hepatic injury by Kupffer cells.

To elucidate the role of Kupffer cells in ischemia-reperfusion-induced hepatic injury, hepatic injury induced by ischemia-reperfusion was analyzed after modulation of Kupffer cell function. Ischemia of the liver was performed by occlusion of both the portal vein and hepatic artery, which enter into the left lateral and median lobes of the liver. Blood flow in the ischemic lobe was reduced, in contrast to an increased blood flow in the nonischemic lobe during occlusion of the veins. Although hepatocyte damage was not demonstrated by ischemia for <60 min, hepatic injury was found after reperfusion of the liver, and activation of Kupffer cells was morphologically demonstrated by electron microscopies. Suppression of Kupffer cells, induced by previous administration of gadolinium chloride or latex particles, reduced the grade of hepatic injury induced by ischemia-reperfusion. On the other hand, stimulation of Kupffer cell phagocytosis, induced by administration of latex particles at the time of reperfusion, aggravated the ischemia-reperfusion-induced hepatotoxicity, which was then reduced by simultaneous administration of superoxide dismutase. Kupffer cells, isolated from the rats treated with the ischemia-reperfusion procedure, have been found to release increased amounts of oxygen radical intermediates. These results suggest that hepatic injury induced by ischemia-reperfusion is modulated by the function of Kupffer cells and that superoxide anion released from Kupffer cells cound play an important role in ischemia-reperfusion hepatic injury.

Role of macrophages in regeneration of liver

In an attempt to clarify the role of macrophages and their mediators during regeneration of the liver, the difference of liver regeneration among C3H/HeN (LPS-responsive strain) and C3H/HeJ (LPS-resistant strain) mice was investigated. After a 67% partial hepatectomy, an increase in the weight of regenerating liver was significantly delayed in the C3H/HeJ mice, as compared with C3H/HeN mice. The number of hepatocytes labeled with antibody against PCNA reached maximum levels 48 hr after partial hepatectomy, but the PCNA labeling index in C3H/HeJ mice was 20% less than that for C3H/HeN mice. In addition, TNF-α activities in serum were enhanced shortly after partial hepatectomy in C3H/HeN strain mice, but were not increased in C3H/HeJ strain mice. Serum IL-6 levels were markedly enhanced in both C3H/HeN and C3H/HeJ mice, but a bimodial peak (14 and 48 hr after partial hepatectomy) was demonstrated in C3H/HeN mice, in contrast to a single peak (at 24 hr) in C3H/HeJ mice. Suppression of Kupffer cells by previous administration of gadolinium chloride in C3H/HeN mice reduced the increase in both serum TNF-α and IL-6 concentrations, reduced PCNA labeling index of hepatocytes by 20%, and disturbed the regeneration of the liver. Previous administration of antibody against TNF-α reduced the PCNA labeling index of hepatocytes by 20% after partial hepatectomy in C3H/HeN strain mice. These results suggest that LPS-responsive macrophages in the liver and their mediators, especially TNF-α, could partly play a role in liver regeneration.

Hepatocyte Nitric Oxide Production Is Induced by Kupffer Cells

To investigate the cellular communication in theliver, nitric oxide (NO) production by sinusoidal cellsand hepatocytes by stimulation with cytokines andKupffer cell-conditioned medium was quantitatively analyzed. NO production by the cells wasmeasured by the Griess reaction, and nitric oxidesynthase (iNOS) transcription level by a competitiveRT-PCR assay using mutant iNOS mRNA as a standard. NOproduction and iNOS mRNA transcriptional levels in Kupffercells were markedly increased by stimulation withlipopolysaccharide (LPS) and interferon-γ(IFN-γ), and moderately by interleukin-1β(IL-1β). NO production by hepatocytes was notsignificantly enhanced by LPS, but was markedly enhancedby IL-1β or the combination of tumor-necrosisfactor-α (TNF-α) and IFN-γ. HepatocyteNO production and iNOS mRNA levels were markedly enhanced bythe LPS-activated Kupffer cell conditioned medium, butthese effects were reduced by heat treatment or anti-TNFantibody. Although NG-monomethyl-L-arginine acetate and dexamethasone reduced NO productionby the cells, the iNOS mRNA level was reduced bydexamethasone only. Gel-shift assay showed NF-κBactivation in hepatocytes during this activation. These data reinforce the importance of cellularcommunication between sinusoidal cells andhepatocytes.

Chemotactic factors released from hepatocytes exposed to acetaminophen

To clarify the mechanism of neutrophil infiltration in the liver of acetaminophen-induced hepatic injury, chemotactic factor released from hepatocytes exposed to acetaminophen has been investigated. Hepatocytes exposed to acetaminophen release nondialyzable chemotactic factor, although actaminophen in itself inhibits chemotaxis of neutrophils. Chemotactic activity of the nondialyzable chemotactic factor was reduced after treatment with heat (56°C, 30 min) or trypsin. Chemotactic activity was demonstrated at the molecular weights of around 25 and 55 kDa. Chemotactic activity of the conditioned medium was not significantly reduced in the presence of antibody against rat KC/gro protein (interleukin-8-related cytokine in rodent). Chemotactic activity of a 25-kDa factor was reduced by the antibody against KC/gro protein, but that of a 55-kDa factor was not reduced. Immunoblot analysis revealed that the peptide reacted with antibody against rat KC/gro protein was demonstrated at a molecular weight of around 20–25 kDa, but not at around 55kDa, when the conditioned medium of acetaminophen-treated hepatocytes was electrophoresed. These results suggest that hepatocytes exposed to acetaminophen release two types of chemotactic factors for neutrophils and that a major part of the chemotactic factor could be different from a member of interleukin-8 family.

Effect of anti-allergic agents on chemotaxis of neutrophils by stimulation of chemotactic factor released from hepatocytes exposed to ethanol

In an attempt to clarify a mechanism of neutrophil infiltration in the liver of alcoholics and possible therapeutic effect of antiallergic agents on accumulation of these cells in the liver, we investigated chemotaxis of neutrophils by stimulation of a chemotactic factor released from rat hepatocytes exposed to ethanol. When hepatocytes were incubated with more than 30 mM ethanol for 24 hr, chemotactic activity for both rat and human neutrophils was demonstrated in the conditioned medium. An enhanced chemotactic activity of the conditioned medium was reduced in the presence of antibody against KC/gro protein, one of the interleukin-8-related cytokines in rodents. Antiallergic agents such as azelastine or ketotifen at a concentration of >0.01 µM markedly reduced chemotaxis of neutrophils. Prednisolone at a concentration of >10 µM also reduced chemotaxis of neutrophils. These results suggest that neutrophil accumulation in the liver of human alcoholics could be induced by a chemotactic factor produced by the ethanol-treated hepatocytes and that antiallergic agents could be effective against the extent of alcoholic hepatitis by reducing chemotaxis of neutrophils.

Ursodeoxycholic acid therapy for chronic type C hepatitis: A multicenter, dose-finding trial

Abstract Ursodeoxycholic acid (UDCA) was administered orally for 12 weeks to 40 patients with chronic type C hepatitis. Patients were randomly assigned to receive UDCA 150 mg/day (n = 20) or 450 mg/day (n = 20). In the 450-mg group, alanine aminotransferase (ALT) decreased significantly ( P P P P

Phagocytic Properties of Hepatic Endothelial Cells and Splenic Macrophages Compensating for a Decreased Phagocytic Function of Kupffer Cells in the Chronically Ethanol-Fed Rats

The balance of phagocytic function among Kupffer cells, hepatic endothelial cells and splenic macrophages in the chronically ethanol-fed rats has been investigated. Clearance of latex particles in the blood was measured to estimate the function of the reticuloendothelial system. Phagocytosis of latex particles by Kupffer cells, hepatic endothelial cells or splenic macrophages in vivo was measured by counting the number of ingested particles in a cell after isolation of hepatic nonparenchymal cells or spleen cells following injection of different amounts of latex particles. Latex particle clearance was suppressed in the ethanol-fed rats, demonstrating a decreased phagocytic capacity of the reticuloendothelial system. Markedly decreased phagocytic function was found in 40% of Kupffer cells of the chronically ethanol-fed rats. In contrast, the number of latex particles in hepatic endothelial cells and in splenic macrophages was increased after injection of a triggering dose of latex particles. From these results it may be concluded that an increased phagocytosis of hepatic endothelial cells and splenic macrophages could compensate for the decreased phagocytic function of Kupffer cells.

Generation of chemotactic factor by hepatocytes isolated from chronically ethanol-fed rats

In an attempt to clarify a mechanism of polymorphonuclear cell and/or macrophage infiltration in alcoholic liver disease, we investigated a novel chemotactic and activating factor generated by rat hepatocytes isolated from the chronically ethanol-fed rats. Hepatocytes and hepatic macrophages were isolated from rat liver by perfusion and digestion with collagenase and subsequently by differential centrifugation on a metrizamide gradient. Rat polymorphonuclear cells were prepared from blood by the dextran sedimentation and Hypaque-Ficoll technique. Chemotactic activity was measured as migration of polymorphonuclear cells or hepatic macrophages using a chemotactic chamber. When hepatocytes isolated from the ethanol-fed rats were culturedin vitro, chemotactic activity for rat polymorphonuclear cells and hepatic macrophages was demonstrated in the culture supernatant. Inhibitors of transcription and protein synthesis reduced generation of chemotactic factor from these hepatocytes. Chemotactic activity of the conditioned medium was reduced after trypsin (0.25%, 37° C, 30 min) or heat (56° C, 30 min) treatment. The chemotactic activity was eluted at molecular weights of 20–25 kDa and 40–45 kDa following Sephadex G-150 chromatography. Superoxide anion production by polymorphonuclear cells and hepatic macrophages under the stimulation of phorbolmyristate acetate was enhanced in the presence of this chemotactic factor. This chemotactic factor may contribute to the pathogenesis of alcoholic liver disease.

Endotoxin induced cellular communication in the liver: Murine models for clarification of the role of LPS‐responsive macrophages in the pathogenesis of liver diseases

In several experimental models, lipopolysaccharide (LPS) plays an important role in the pathogenesis of liver diseases. Murine models of C3H/HeN and C3H/HeJ mice have been used to elucidate the role of LPS and its responsive‐macrophages in vivo, as C3H/HeN strain mice are known to be LPS‐responsive, while C3H/HeJ strain mice are LPS‐resistant. Furthermore, release of several kinds of biologically active mediators such as interleukin‐1, tumour necrosis factor‐α, colony stimulating factor and reactive oxygen radical is not enhanced in C3H/HeJ mice even after stimulation with LPS. Thus, these murine models could be suitable for clarification of endotoxin induced cellular communication in the liver.