Miyoko Hirose

Role of Kupffer cells and gut-derived endotoxins in alcoholic liver injury1

The hepatotoxic effects of alcohol have been described in detail, but factors responsible for its hepatotoxicity have only partially been characterized. For example, it is known that chronic ethanol ingestion increases hepatotoxicity and produces fatty liver, hepatitis and cirrhosis. However, acute ethanol consumption reduces endotoxin hepatotoxicity. It now appears that Kupffer cells participate in several aspects of these phenomena. Previously, most studies on the effects of alcohol on liver function have focused chiefly on the hepatocyte. Recently, attention has been directed towards the effect of ethanol ingestion on Kupffer cell function, which is stimulated by gut‐derived endotoxins (lipopolysaccharides) via mechanisms dependent on increased gut permeability and the possible relationship between Kupffer cells and alcohol‐induced liver injury. Here we will review new evidence for the proposal that Kupffer cells and endotoxins play a pivotal role in hepatotoxicity following alcohol exposure, based on studies using the continuous intragastric enteral feeding model developed by Tsukamoto and French and an acute model developed by us.

Dietary glycine prevents chemical-induced experimental colitis in the rat

BACKGROUND & AIMS In this study, the effect of dietary glycine on experimental colitis induced by 2,4,6-trinitrobenzene sulphonic acid (TNBS) and dextran sulfate sodium (DSS) in the rat was evaluated. METHODS Male Wistar rats were fed a diet containing 5% glycine or casein as controls starting 3 days before experiments, and were given a single intracolonic injection of TNBS (50 mg/rat, dissolved in 50% ethanol). Similarly, some rats were given 3% DSS orally in drinking water for 5 days to induce colitis as a second model. The severity of colitis was evaluated pathologically, and tissue myeloperoxidase (MPO) activity was measured. Further, mRNA and protein levels for interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, cytokine-induced neutrophil chemoattractant (CINC), and macrophage inflammatory protein (MIP)-2 were detected by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS A diet containing glycine ameliorated diarrhea and body weight loss caused by TNBS, and improved both macroscopic and histologic scores of colitis significantly. TNBS-induced increases in MPO activities in the colonic tissue were blunted significantly in glycine-fed animals. Further, dietary glycine largely prevented increases in IL-1beta and TNF-alpha in the colon 2 days after TNBS, and TNBS induction of CINC and MIP-2 in the colonic tissue also was abrogated by glycine. Importantly, the protective effect of glycine was significant even when TNBS colitis was once established. Moreover, dietary glycine also was preventive in a second, DSS-induced colitis model. CONCLUSIONS Dietary glycine prevents chemical-induced colitis by inhibiting induction of inflammatory cytokines and chemokines. It is postulated that glycine may be useful for the treatment of inflammatory bowel diseases as an immunomodulating nutrient.

Role of actin and calmodulin in migration and proliferation of rabbit gastric mucosal cells in culture

Abstract The aim of this study was to develop a new cellular restoration model using primary cultured rabbit gastric mucosal cells, and to evaluate the role of cytoskeleton and calmodulin in this process. Confluent monolayer mucosal cell sheets consisting mainly of mucous cells were wounded, and a rotating silicon tip was used to make cell‐free areas of constant size. The process of restoration was monitored, and the cell‐free area was measured and analysed quantitatively.

How useful is the detection kit for antibody to Helicobacter pylori in urine (URINELISA) in clinical practice

OBJECTIVE:Increased knowledge of the significance of Helicobacter pylori (H. pylori) infection in gastric disorders has accelerated the trend of screening patients with dyspepsia for its infection. Serological examination of antibody for H. pylori has been widely performed. Recently, a urine-based enzyme-linked immunosorbent assay (URINELISA) kit for detection of antibody for H. pylori has been developed. Accordingly, we evaluated its diagnostic accuracy in clinical practice.METHODS:Subjects of this study were 132 patients who presented at our university hospital because of dyspeptic symptoms (81 men, 51 women; age, 41.5 ± 1.4 yr). 13C urea breath test, blood drawing for serological antibody for H. pylori infection by four different kits, and urine collection for the URINELISA test for detection of the antibody were performed. Diagnostic accuracy of the commercially available antibodies in serum and in urine were investigated using the results of the 13C urea breath test as the gold standard.RESULTS:Sensitivity, specificity, and accuracy of URINELISA were 86.3% (95% confidence intervals [CI], 76–93%), 91.5% (95% CI, 81–97%), and 88.6% (95% CI, 82–93%), respectively, which were comparable to those of imported serological kits.CONCLUSIONS:The URINELISA kit for detecting anti-H. pylori antibody in urine provides diagnostic accuracy comparable to that of imported kits for detecting antibodies in serum and is considered to be clinically useful for the diagnosis of H. pylori infection.

Ammonia, hydrogen peroxide, and monochloramine retard gastric epithelial restoration in rabbit cultured cell model.

Ammonia (NH3), hydrogen peroxide(H2O2) and monochloramine(NH2Cl) produced by Helicobacter pyloriinfection might be responsible for mucosal injury. Theaim of this study was to evaluate the role ofNH3, H2O2, andNH2Cl in the restoration of rabbit primarycultured gastric mucosal cells with a wound repairmodel. Artificial wounds were made in confluentmonolayer gastric epithelial cell sheets by mechanicaldenudation, and changes in the size of the cell-freearea were analyzed quantitatively. Cell proliferation was assessed by bromodeoxyuridine staining. Incontrols, the wound healed within 48 hr. However,mucosal cell repair was inhibited by treatment withNH3, H2O2, andNH2Cl in a dose-dependent manner. These resultsindicate that NH3,H2O2, and NH2Clretarded the wound healing, which included epithelialcell migration and proliferation of gastric mucosa. Therefore, it is suggestedthat NH3, H2O2, andNH2Cl delays the healing process of pepticulcers.

Immunocytochemical localization of myosin in normal and phalloidin-treated rat hepatocytes

To clarify the intracellular distribution of myosin in normal rat hepatocytes and its alterations in phal-loidin-treated rat hepatocytes as a morphologic basis for the dysfunction of microfilaments, we performed indirect immunofluorescence using monospecific antibody raised against rat hepatocyte myosin. Cryostat rat liver sections analyzed by the use of this antibody showed a characteristic polygonal staining pattern, indicating that myosin is localized close to the plasma membrane including the region of bile canaliculi. The observed myosin staining pattern of normal liver coincides with the pattern of actin distribution as demonstrated by double-staining on the same liver section with antimyosin antibody and rhodamine-phalloidin. Upon administration of phalloidin to rats, the following changes in the myosin staining pattern were observed. (a) Peripheral fluorescence along the plasma membrane, especially around the bile canaliculi and sinusoids, was greatly enhanced. (b) Numbers of small fluorescent dots appeared in the cytoplasm of hepatocytes. These changes in the localization of myosin are shown to overlap with those of actin filament distribution. Accompanying these changes of localization, cellular myosin content appears to be increased, as the myosin marker-enzyme NH4+(-)ethylenediaminetetraacetic acid-adenosine triphosphatase activity in hepatocyte extracts was elevated threefold after 7 days of phalloidin treatment. This increase of myosin may be due to the previously observed stabilizing effect on microfilaments of phalloidin against cellular proteases. Thus, phalloidin, which primarily alters actin filament distribution, induces the changes in myosin localization and the increase in cellular myosin content without causing dissociation of myosin from actin in the hepatocyte.

Protective Effect of Thalidomide on Endotoxin-Induced Liver Injury

BACKGROUND Activation of Kupffer cells by lipopolysaccharide (LPS) plays a pivotal role in the onset of pathophysiological events that occur during endotoxemia, and intracellular calcium concentration ([Ca2+]i) is involved in LPS-stimulated cytokine production. Tumor necrosis factor (TNF)-alpha is produced exclusively by the monocyte-macrophage lineage, which is mostly made up of Kupffer cells, and thalidomide has been shown to reduce TNF-alpha production from macrophages. However, there is increasing evidence that TNF-alpha may play a role in the initiation or progression of multiple organ failure syndrome. Therefore, the purpose of this work was to determine whether thalidomide could prevent LPS-induced liver injury. METHODS Rats were given a single oral dose of thalidomide (5 mg/kg). To assess the sensitization of Kupffer cells, LPS (5 or 10 mg/kg) was administered intravenously, and mortality, liver histology, and transaminases were evaluated 24 hr later. Kupffer cells were isolated 2 hr after thalidomide treatment. After the addition of LPS, [Ca2+]i was measured by using a microspectrofluorometer with the fluorescent indicator fura-2, and TNF-alpha was measured by enzyme-linked immunosorbent assay. RESULTS LPS caused focal necrosis with neutrophil infiltration in the liver. Moreover, LPS dramatically increased transaminases. These pathologic parameters and increases of serum transaminases were diminished markedly by thalidomide. In isolated Kupffer cells, LPS-induced increases in [Ca2+]i and TNF-alpha production were suppressed by treatment with thalidomide. To further explore the mechanism by which thalidomide directly abrogated Kupffer cell sensitivity to LPS, we determined the effect of thalidomide (5 microM) in vitro on LPS-induced [Ca2+]i response and TNF-alpha production. With the addition of thalidomide (5 microM) in vitro to the culture media for 2 hr before LPS, these parameters were suppressed. CONCLUSIONS Thalidomide prevents LPS-induced liver injury via mechanisms dependent on the suppression of TNF-alpha production from Kupffer cells.

Taurine preserves gap junctional intercellular communication in rat hepatocytes under oxidative stress.

Abstract: Gap junctional intercellular communication (GJIC) between hepatocytes is important for the maintenance of differentiated liver functions. Taurine is known to be cytoprotective, and is used clinically to improve liver functions. We evaluated the effect of taurine on GJIC in hepatocyte doublets under oxidative stress. Hepatocyte doublets were isolated from female Wistar rats, using a collagenase perfusion technique, and cultured in Leibovitz-15 medium containing fetal bovine serum (10%). H2O2 (2 mM) and/or taurine (0.1–1 mM) were added 2 h after inoculation, and the culture was incubated for 3 h. Fluorescent dye (Lucifer Yellow CH) coupling between adjacent cells was evaluated by microinjection. The distribution and quantity of connexin 32 (Cx32) in hepatocytes were detected using indirect immunofluorescence analysis and Western blotting. Steady state mRNA levels of Cx32 were detected by Northern blotting. The percentage of dye coupling 5 h after inoculation was 88 ± 6.3% in the control, however, this was decreased to almost half the control value by H2O2. Taurine prevented the decrease caused by H2O2 in a dose-dependent manner. Immunofluorescence analysis for Cx32 demonstrated numerous punctate fluorescent spots along the intercellular plasma membrane in controls, which were significantly decreased by H2O2. Taurine prevented the decrease of Cx32. Western blot analysis also showed the decrease of Cx32 protein levels by H2O2 treatment, which decrease was prevented by taurine. Interestingly, H2O2 and/or taurine treatments did not affect Cx32 mRNA levels. Our findings indicated that H2O2 treatment decreased GJIC between hepatocytes, most likely due to augmenting the degradation of Cx32 proteins, whereas taurine prevented this process. This effect of taurine is beneficial for the preservation of differentiated functions in the liver under oxidative stress.

Dimethylsulfoxide maintains intercellular communication by preserving the gap junctional protein connexin32 in primary cultured hepatocyte doublets from rats

Intercellular communication via gap junctions is one of the differentiated functions of cells. Dimethylsulfoxide (DMSO) is known to induce cell differentiation and maintain differentiated cellular functions in primary hepatocyte culture, but the mechanism of action of DMSO is unknown. Therefore, we investigated the effect of DMSO on cell‐cell communication via gap junctions of hepatocyte doublets, which are differentiated cells that lose differentiated functions with time in culture. In isolated rat hepatocyte doublets, we assessed the effects of 1, 2 and 3% DMSO in culture medium on morphological changes and dye‐coupling activity between pairs of cells by microinjection with fluorescent dye (Lucifer Yellow CH). The distribution of gap junction protein connexin32 (Cx32) was assessed by indirect immunofluorescence analysis and the Cx32 mRNA was detected by the reverse transcription‐polymerase chain reaction method. Dimethylsulfoxide delayed the morphological change of hepatocyte doublets from a spherical to a flattened shape. Dye‐coupling efficiency significantly decreased with time in culture in the control group, whereas in groups treated with 2 and 3% DMSO, dye‐coupling efficiency was retained after 6 and 9 h of inoculation (P < 0.05 and P < 0.01, respectively). Analysis by indirect immunofluorescence showed few fluorescent spots for Cx32 in the control group at 9 h of incubation, whereas many punctate fluorescent spots were seen in the 3% DMSO group at 9 h of incubation. The detection of Cx32 mRNA in the 3% DMSO group was also stronger than in controls. Dimethylsulfoxide significantly maintained intercellular communication via gap junctions in primary cultured rat hepatocytes through the preservation of functional Cx32 protein, thus maintaining cell differentiation.

Actin and myosin deposition around bile canaliculi: a predictor of clinical outcome in biliary atresia.

The role of pathological changes of the intrahepatic bile canaliculi in determining postportoenterostomy bile drainage in biliary atresia (BA) patients remains unestablished. It is known that canalicular contraction contributes an active force for bile flow in normal ductal systems. This motility is controlled by the bile canalicular membrane-associated filaments (BCMF), particularly actin and myosin. Wedge resected specimens of liver from 13 patients with BA were studied using histopathological markers of BCMF in order to clarify the morphological and functional changes of bile canaliculi. Histopathological data were also compared with clinical outcomes after portoenterostomy. Patients who showed sufficient bile flow after the operation showed an almost normal shape and level of BCMF accumulation around bile canaliculi, whereas there was markedly increased BCMF volume in patients who did not exhibit bile flow after surgery. The clinical staining patterns of BCMF in BA patients appears closely related to clinical outcome. These findings suggest that BCMF plays an important role in determining the rate of postoperative bile flow in BA patients.