Kyoko Toda

Tumour necrosis factor α signalling through activation of Kupffer cells plays an essential role in liver fibrosis of non-alcoholic steatohepatitis in mice

Background: While tumour necrosis factor α (TNF-α) appears to be associated with the development of non-alcoholic steatohepatitis (NASH), its precise role in the pathogenesis of NASH is not well understood. Methods: Male mice deficient in both TNF receptors 1 (TNFR1) and 2 (TNFR2) (TNFRDKO mice) and wild-type mice were fed a methionine and choline deficient (MCD) diet or a control diet for eight weeks, maintaining isoenergetic intake. Results: MCD dietary feeding of TNFRDKO mice for eight weeks resulted in attenuated liver steatosis and fibrosis compared with control wild-type mice. In the liver, the number of activated hepatic Kupffer cells recruited was significantly decreased in TNFRDKO mice after MCD dietary feeding. In addition, hepatic induction of TNF-α, vascular cell adhesion molecule 1, and intracellular adhesion molecule 1 was significantly suppressed in TNFRDKO mice. While in control animals MCD dietary feeding dramatically increased mRNA expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) in both whole liver and hepatic stellate cells, concomitant with enhanced activation of hepatic stellate cells, both factors were significantly lower in TNFRDKO mice. In primary cultures, TNF-α administration enhanced TIMP-1 mRNA expression in activated hepatic stellate cells and suppressed apoptotic induction in activated hepatic stellate cells. Inhibition of TNF induced TIMP-1 upregulation by TIMP-1 specific siRNA reversed the apoptotic suppression seen in hepatic stellate cells. Conclusions: Enhancement of the TNF-α/TNFR mediated signalling pathway via activation of Kupffer cells in an autocrine or paracrine manner may be critically involved in the pathogenesis of liver fibrosis in this NASH animal model.

Anti-fibrogenic effect of an angiotensin converting enzyme inhibitor on chronic carbon tetrachloride-induced hepatic fibrosis in rats

The tissue renin-angiotensin system has recently been demonstrated to reduce fibrogenesis in various organs. However, little has been clarified regarding its role in hepatic fibrosis. The purpose of this study was to investigate the effect of angiotensin-converting enzyme inhibitors on liver fibrogenesis induced in rats by low-dose chronic carbon tetrachloride administration. We used lisinopril that is absorbed in its active form and not metabolized in the liver to avoid any influence by the administration of the chemical. Carbon tetrachloride was administered twice a week i.p. Twelve and 24 weeks after the start of treatment, expanded periportal fibrosis or portal-portal bridgings and severe fat deposition were observed in the rats treated with carbon tetrachloride alone, and these findings were significantly reduced with the simultaneous treatment with lisinopril. The hydroxyproline content of the liver was significantly lower in the lisinopril-treated group. Angiotensin II up-regulated mRNA of pro alpha (I) collagen and transforming growth factor-beta in isolated hepatic stellate cells. These results suggest that the local tissue renin-angiotensin system plays a role in rat hepatic fibrogenesis induced by chronic carbon tetrachloride administration and that hepatic fibrogenesis is significantly reduced by ACE inhibitors.

Enzyme linked immunosorbent assay (ELISA) and immunoprecipitation studies on anti-goblet cell antibody using a mucin producing cell line in patients with inflammatory bowel disease.

Circulating anti-goblet cell antibody and its corresponding antigen in patients with inflammatory bowel disease were investigated. Anti-goblet cell antibody in the serum was examined by immunocytochemistry and enzyme linked immunosorbent assay (ELISA), using a colonic cancer cell line, HT29-18-N2, which differentiates into intestinal goblet cells. The frequencies of anti-goblet cell antibody detected by immunocytochemistry were 14 in 48 patients with ulcerative colitis (29%) and five in 15 patients with Crohns disease (33%). By ELISA, the frequencies of anti-goblet cell antibody were 38% in ulcerative colitis and 33% in Crohns disease. This antibody did not relate directly to anti-neutrophil cytoplasmic antibodies (ANCA), although the serum samples positive for anti-goblet cell antibody were commonly positive for ANCA in ulcerative colitis. Immunoprecipitation and SDS polyacrylamide gel electrophoresis (PAGE) study showed that the antibody in the ELISA positive serum samples recognised a > 200 kD goblet cell antigen, which remained unchanged after reduction, indicating that it consists of single chain polypeptides. These results suggest that there is a subgroup of inflammatory bowel disease that has circulating anti-goblet cell antibody reactive with a > 200 kD antigen. The antibody detected by newly established ELISA will be a disease marker for this group and the identification of the corresponding antigens may be important for the understanding of the underlying immune abnormalities.

In vitro anticolon antibody production by mucosal or peripheral blood lymphocytes from patients with ulcerative colitis.

Serum anticolon antibody and in vitro anti-colon antibody production by peripheral blood and mucosal lymphocytes was investigated in patients with ulcerative colitis. The frequency of serum anticolon antibody was 71% in 41 patients with ulcerative colitis, estimated by enzyme linked immunosorbent assay (ELISA) using isolated rat colon epithelial cells. This finding confirms our previous report on the frequency of serum anticolon antibody detected by flow cytometry analysis. The estimated frequencies of IgG anticolon antibody secreting cells were 1.5-12.5/10(6) cells in the colonic mucosa and 0.1-0.5/10(6) cells in peripheral blood, from patients with ulcerative colitis when Epstein-Barr virus (EBV) was used as a B cell polyclonal activator. Poisson analysis of limiting dilution culture showed that about one per 140 IgG cells in the colonic mucosa synthesised anticolon antibody. Two monoclonal IgG antibodies were obtained from EBV transformed anticolon antibody secreting cells by limiting dilution method. One reacted with goblet cells in the intestine, and the other reacted mainly with colonic epithelial cells. These results suggest that heterogeneous anticolon antibodies are present in patients with ulcerative colitis and that colonic mucosa may be the main source of anticolon antibody. Local autoimmune reaction might have an important role in causing the inflammation of colonic mucosa in this disease.

Rho/Rho kinase is a key enzyme system involved in the angiotensin II signaling pathway of liver fibrosis and steatosis.

Background and Aim:  The molecular mechanisms underlying the involvement of the renin‐angiotensin system in hepatic fibrosis are unclear. Recently, it was reported that a Rho kinase inhibitor prevented fibrosis of various tissues and that the Rho/Rho kinase pathway was involved in the renin‐angiotensin system of vascular smooth muscle cells. In this study, the involvement of the Rho/Rho kinase pathway on angiotensin II signaling in liver fibrogenesis and generation of steatosis was investigated.

Distribution and localization of caveolin-1 in sinusoidal cells in rat liver

 Caveolin, the principal structural protein in caveolae, is involved in signal transduction. The aim of the present study was to clarify the distribution and ultrastructural localization of caveolin-1 in hepatic sinusoidal endothelial cells (SECs) and hepatic stellate cell (HSCs) by confocal microscopy and the electron immunogold method. Liver tissue sections were prepared from male Wistar rats. SECs and HSCs were isolated from rat livers by collagenase infusion. For immunohistochemistry, liver sections were reacted with anticaveolin-1 antibody. The localization and distribution of caveolin-1 were identified by confocal immunofluorescence. The ultrastructural localization of caveolin-1 on SECs and HSCs was identified by electron microscopy using the immunogold method. Immunohistochemical studies using liver tissues localized caveolin-1 in sinusoidal lining cells, bile canaliculi, portal vein, and hepatic artery. By confocal microscopy, caveolin-1 was mainly demonstrated at the Golgi complex in SECs and HSCs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of sinusoidal endothelial fenestrae (SEF) and vesicles in SECs. Under an electron microscope, immunogold particles indicating the presence of caveolin-1 were demonstrated on the plasma membrane of caveolae and vesicles in HSCs. We concluded that caveolin-1 is localized from SEFs to the Golgi complex in SECs and from caveolae to the Golgi complex in HSCs.

Induction of hepatic stellate cell proliferation by LPS-stimulated peripheral blood mononuclear cells from patients with liver cirrhosis.

Abstract: We studied hepatic stellate cell proliferation in vitro. Peripheral blood mononuclear cells (PBMC) from patients with chronic active hepatitis C (CAH) and liver cirrhosis (LC) were cultured for 24 h in the presence or absence of Escherichia coli lipopolysaccharides (LPS). Hepatic stellate cell proliferation induced by the culture supernatants was measured, and interleukin-1 (IL-1) and IL-6 levels in the culture supernatants were quantified. Culture supernatants of LPS-stimulated PBMC from LC patients induced rat hepatic stellate cell proliferation by almost 2.8-fold (stimulation index, 2.83 ± 1.41) compared with when the cells were cultured without addition of PBMC culture supernatants. Production of IL-1β was significantly higher in the culture supernatants of both CAH and LC patients than in those of ten healthy controls (P < 0.01 and P < 0.05, respectively). But there was no significant correlation between IL-1 production and the induction of hepatic stellate cell proliferation by the culture supernatants. Although there were no significant differences in IL-6 production by LPS-stimulated PBMC among healthy controls and CAH and LC patients, we observed a significant correlation between IL-6 production and the induction of hepatic stellate cell proliferation in the culture supernatants of LC patients. Rat hepatic stellate cells themselves produced IL-6, and treatment with IL-6 antisense oligodeoxynucleotides suppressed the cell proliferation, suggesting that IL-6 is an autocrine growth factor for hepatic stellate cells. The addition of human recombinant IL-6 (hrIL-6) augmented rat hepatic stellate cell proliferation, indicating that excessive IL-6 may further facilitate cell proliferation. These findings suggest that a cytokine cascade including IL-6 may participate in hepatic stellate cell proliferation in LC patients when they are exposed to endotoxin.

Changes of proliferative activity and phenotypes in spontaneous differentiation of a colon cancer cell line

We examined the alterations of proliferative activity and c‐myc expression of a colon cancer cell line (Caco‐2) during its spontaneous differentiation. Caco‐2 cells were cultured in various types of media and the degree of differentiation was monitored in terms of dome formation in cell monolayers and expression of alkaline phosphatase (ALP) activity. In Caco‐2 cells cultured with Eagles minimum essential medium (EMEM) containing 10% fetal calf serum (FCS), dome formation was demonstrated and ALP activity was markedly increased after the cells reached confluence. Five‐fold reduction of c‐myc mRNA and a marked decrease in S‐phase cells were observed in the differentiated cells. These changes were not induced in FCS‐free EMEM. The addition of insulin and transferrin to FCS‐free EMEM did not induce cell differentiation or reduction of c‐myc mRNA expression. When Caco‐2 cells were cultured with three different serum‐free media, the induction of dome formation and the increase of ALP activity were observed to varying degrees. Expression of c‐myc mRNA in the cells cultured with one serum‐free medium decreased to a level similar to that in fully differentiated cells cultured with EMEM containing 10% FCS. These results suggest that a spontaneous switch from a proliferative state with high c‐myc expression to differentiated phenotype occurs after cells reach confluence and depends on the culture conditions.

Inhibition of growth of human hepatoma cells by dual-function antisense IL-6 oligonucleotides

Hepatocellular lineage cell lines (two hepatoma and Chang liver cell lines) were found to produce interleukin-6 (IL-6). As the human hepatoma cell line, HCC-M expresses mRNA for both IL-6 and IL-6 receptor, we examined the possibility that IL-6 acted as an autocrine growth factor for HCC-M cells using two IL-6 antisense oligonucleotides (AS-1 and AS-2 oligomers) which were synthesized from different regions of an IL-6 cDNA clone. Both IL-6 antisense oligonucleotides inhibited the growth of HCC-M cells within 48 h (% inhibition by AS-1 and AS-2 oligomers was 53 and 21%, respectively). Although inhibition of HCC-M cell growth induced by AS-2 oligomer was restored by addition of exogenous recombinant IL-6 (rIL-6), the inhibition of growth induced by AS-1 oligomer was not fully restored by exogenous rIL-6, implicating the involvement of a nonantisense mechanism associated with four contiguous guanosine residues in this sequence. The inhibitory effect of AS-2 oligomer was attenuated after 72 h (% inhibition was 8%), whereas the AS-1 oligomer-induced inhibition of growth was sustained beyond 72 h (% inhibition was 38--39%). Therefore, these dual-function oligonucleotides that act via both an antisense and nonantisense mechanism may be of potential therapeutic value against hepatoma.

Immunoglobulin G subclass distribution of human anticolon antibodies in ulcerative colitis

Abstract Immunoglobulin G (IgG) subclasses of anticolon antibodies were studied in patients with ulcerative colitis (UC) using enzyme‐linked immunosorbent assay (ELISA). The concentrations of total serum IgG subclasses were also measured by ELISA. The values for total serum IgG subclasses in patients with UC were not significantly different from those in normal controls, while the ratio of IgG1 to IgG2 in the patients was significantly higher than that in normal controls. All four IgG subclasses of autoantibodies were demonstrated in the sera of the patients. IgG4 anticolon antibodies were detected most frequently (15 out of 18 patients, 83%). IgG2 was the next most prevalent (9 of 18 patients, 50%). The activity of anticolon antibodies in each subclass did not correlate with the concentration of the corresponding serum IgG subclass. Seven cell lines producing anticolon antibodies were obtained from the colonic mucosa of the patients by Epstein‐Barr virus (EBV) transformation. IgG subclasses of anticolon antibodies secreted by these cell lines were also varied. IgG4 subclass was secreted by three EBV transformed cell lines, all of which produced IgG4 anticolon antibodies. These results suggest that all four different IgG subclasses could respond to the colon antigens and that various antigens in colonic mucosa or lumen may contribute to the induction of those autoantibodies. In addition, the prominence of IgG4 anticolon antibodies may support the pathogenic role of this subclass in UC as in other autoimmune diseases.