Mitsunari Yamamoto

IL-6 Is Required for the Development of Th1 Cell-Mediated Murine Colitis

Proinflammatory cytokines have been demonstrated to play a crucial role in the pathogenesis of Crohn’s disease. Among those cytokines, strong expression of IL-6 has been repeatedly demonstrated. To examine the role for IL-6 in the pathogenesis of Crohn’s disease, we introduced anti-IL-6R mAb to a murine model of colitis. Colitis was induced in C.B-17-scid mice transferred with CD45RBhigh CD4+ T cells from BALB/c mice. Anti-IL-6R mAb or rat IgG was administered weekly after T cell transfer. ICAM-1 and VCAM-1 expression were analyzed by immunohistochemistry. Colonic cytokine expression was determined by RT-PCR. Mice treated with mAb showed normal growth, whereas controls lost weight. The average colitis score was 0.64 for mAb-treated mice and 1.80 for controls. T cell expansion in treated mice was less remarkable than in the controls. Colonic ICAM-1 and VCAM-1 expression were markedly suppressed by mAb. IFN-γ, TNF-α, and IL-1β mRNA were reduced by the treatment. The results presented here show a crucial role for IL-6 in the pathogenesis of murine colitis and suggest a therapeutic potential of anti-IL-6R mAb for treatment of human Crohn’s disease.

Hepatoma-derived Growth Factor Stimulates Cell Growth after Translocation to the Nucleus by Nuclear Localization Signals

Hepatoma-derived growth factor (HDGF) is the original member of the HDGF family of proteins, which contains a well-conserved N-terminal amino acid sequence (homologous to the amino terminus of HDGF; hath) and nuclear localization signals (NLSs) in gene-specific regions other than the hath region. In addition to a bipartite NLS in a gene-specific region, an NLS-like sequence is also found in the hath region. In cells expressing green fluorescence protein (GFP)-HDGF, green fluorescence was observed in the nucleus, whereas it was detected in the cytoplasm of cells expressing GFP-HDGF with both NLSs mutated or deleted. GFP-hath protein (GFP-HATH) was distributed mainly in the nucleus, although some was present in the cytoplasm, whereas GFP-HDGF with a deleted hath region (HDGFnonHATH) was found only in the nucleus. Exogenously supplied GFP-HDGF was internalized and translocated to the nucleus. GFP-HATH was internalized, whereas GFP-HDGFnonHATH was not. Overexpression of HDGF stimulated DNA synthesis and cellular proliferation, although HDGF with both NLSs deleted did not. Overexpression of HDGFnonHATH caused a significant stimulation of DNA synthesis, whereas that of hath protein did not. HDGF containing the NLS sequence of p53 instead of the bipartite NLS did not stimulate DNA synthesis, and truncated forms without the C- or N-terminal side of NLS2 did not. These findings suggest that the gene-specific region, at least the bipartite NLS sequence and the N- and C-terminal neighboring portions, is essential for the mitogenic activity of HDGF after nuclear translocation.

Hepatoma‐derived growth factor is highly expressed in developing liver and promotes fetal hepatocyte proliferation

Hepatoma‐derived growth factor (HDGF) is a heparin‐binding protein, which has been purified from the conditioned media of HuH‐7 hepatoma cells. Recent studies have suggested the involvement of HDGF in development of the kidney and cardiovascular systems. In the present study, we investigated the possibility that HDGF was also involved in liver development. Northern blot and immunostaining revealed unique expression patterns of HDGF in liver development. HDGF expression was strongly detected in the fetal liver of the midgestation stage and was markedly decreased near birth. Its expression was mainly detected in stromal cells, including immature hepatocytes. Expression in hepatocytes decreased with differentiation. Administration of recombinant HDGF enhanced the growth of primary cultured fetal hepatocytes significantly, although the effect was small. The effect of exogenous HDGF on the proliferation of neonatal hepatocytes was also small and significant only at one point, despite the lower expression of endogenous HDGF, suggesting that the differences exist between fetal and neonatal hepatocytes. However, adenoviral introduction of HDGF antisense cDNA into the fetal hepatocytes significantly suppressed their proliferation, and the inhibitory effect of HDGF antisense virus was reversed by exogenous HDGF. In conclusion, HDGF helps regulate the hepatocyte proliferation in liver development. (HEPATOLOGY2002;36:1519–1527).

Anti-IL-6 receptor monoclonal antibody inhibits leukocyte recruitment and promotes T-cell apoptosis in a murine model of Crohn's disease

Background. To assess the contribution of IL-6 signaling to the physiopathology of Crohn’s disease, we introduced anti-IL-6 receptor monoclonal antibody to a murine colitis model. Methods. Colitis was induced in C.B-17-scid mice to which were transferred CD45RBhigh CD4+ T cells from Balb/c mice. Anti-IL-6 receptor monoclonal antibody or rat IgG was given intraperitoneally after T-cell transfer, followed by weekly injection. Vascular adhesion molecules and inducible nitric oxide synthase were visualized by immunostaining. Cytokine expression was determined by RT-PCR, and apoptotic cells were determined by the TUNEL method. Results. Mice treated with anti-IL-6 receptor monoclonal antibody showed normal growth while controls lost weight. Colitis was improved histologically with reduced infiltration of LFA-1+ monocytes/ macrophages and VLA-4+ T cells. ICAM-1 and VCAM-1 expression in the colonic vascular endothelium was markedly suppressed by the treatment, whereas no significant difference was seen in MAdCAM-1. IFN-γ, TNF-α, and IL-lβ mRNAs were markedly reduced, but no difference was observed in the expression of IL-4, IL-10, and TGF-β. Inducible nitric oxide synthase was upregulated in the mucosa of colitic mice and downregulated in the treated mice. Apoptotic cells were very sparse despite massive CD4+ T-cell infiltration in colitic mice, whereas increased apoptosis was seen in the treated mice with an apparently reduced number of T cells. Conclusions. Anti-IL-6 receptor monoclonal antibody abrogated murine colitis. It effectively blocked the expression of adhesion molecules, thereby blocking leukocyte recruitment, and increased T-cell apoptosis. These results strongly suggest the therapeutic potential of anti-IL-6 receptor monoclonal antibody for human Crohn’s disease.

Granulocyte-colony-stimulating-factor-producing hepatocellular carcinoma

Abstract: Patients with hepatocellular carcinoma (HCC) rarely show marked granulocytosis. We report an interesting case of rapidly growing poorly differentiated HCC associated with marked granulocytosis. The blood leukocyte count decreased following several treatments for HCC, including transcatheter arterial embolization, and increased again along with tumor regrowth. The serum levels of granulocyte colony-stimulating factor (G-CSF) were markedly elevated, and immunohistochemical study showed G-CSF staining in the cytoplasm of certain HCC cells. These findings confirm that HCC in this case was a G-CSF-producing tumor.

Triple therapy of initial high-dose interferon with ribavirin and amantadine for patients with chronic hepatitis C

Aims:  We previously reported the potential effect of combination therapy of an initial high‐dose interferon (IFN) and amantadine on the eradication of HCV‐RNA in patients with chronic hepatitis C. The additive effects of amantadine on interferon and ribavirin combination therapy remain controversial. In this study we investigated the efficacy of initial high‐dose IFN with ribavirin and amantadine on the virological response in patients with chronic hepatitis C with a high viral load of genotype 1b.

Role of Inducible Nitric Oxide Synthase in Murine Colitis

To examine the role of inducible nitric oxide synthase (iNOS) in the development of colitis, we characterized the expression of iNOS in a murine colitis model and the effects of a selective inhibitor of iNOS production, aminoguanidine (AMG). Furthermore, we examined the correlation between the production of inflammatory cytokines and nitric oxide (NO) in human colonic epithelial cells. Colitis was induced in C.B-17-SCID mice transferred with CD45RBhigh CD4+ T cells from BALB/c mice. Expression of iNOS in the intestine was determined by reverse transcriptase poly-merase chain reaction and immunohitochemistry. Anti-interleukin-6 receptor (anti-IL-6R) monoclonal antibody (mAb) or AMG was administered after T cell transfer. Furthermore, NO activity and iNOS production in HT-29 cells were examined after stimulation with interferon-γ (IFNγ), tumor necrosis factor-α (TNFα), IL-Iβ, IL-6, and soluble (s) IL-6R. There was increased expression of iNOS in colonic epithelial cells in the murine colitis model, but expression of iNOS and development of colitis were suppressed by administration of anti-IL-6R mAb. Severe colitis was induced when iNOS was inhibited by administration of AMG. In HT-29 cells, production of iNOS was induced in the presence of IFNγ. Although no induction of iNOS production was observed in the presence of IL-6 alone, NO production mediated by INFγ was increased in the presence of INFγ plus IL-6 and soluble IL-6R. The production of NO induced by IFNγ was thus stimulated by the addition of IL-6 in human colonic epithelial cells. In the colitis model, NO induced by inflammatory cytokines at the onset of colitis may suppress the development of inflammation.