Hironobu Kohda

Induction of Mn-superoxide dismutase by Tumor Necrosis Factor, Interleukin-1 and Interleukin-6 in human hepatoma cells

Effects of Tumor Necrosis Factor (TNF), Interleukin-1 (IL-1), Interleukin-6 (IL-6) and Interferon-gamma (IFN-gamma) on the expression of Mn-superoxide dismutase (Mn-SOD) protein were investigated in human hepatoma cells, Hu-H1, which revealed resistance to the cytotoxicity of TNF and IL-1. Both TNF and IL-1 enhanced the Mn-SOD production to the level of 30- to 40-fold. IL-6 also increased the enzyme protein to 2- to 3-fold of the basal level without any cell proliferative effect. A specific antibody against IL-6 almost completely inhibited the induction of Mn-SOD. IL-6, as well as TNF and IL-1, appears to play some role in the Mn-SOD protein expression in human hepatoma cells.

Flow cytometric and functional analysis of mononuclear cells infiltrating the liver in experimental autoimmune hepatitis

Experimental autoimmune hepatitis was produced by immunizing Wistar rats with syngeneic liver proteins. Mononuclear cells infiltrating the liver tissue were identified by immunohistochemical techniques using monoclonal antibodies specific for subpopulations of rat lymphocytes. The strong infiltration of CD8+ cytotoxic T lymphocytes (CTL) were found in the portal areas. Subpopulations of mononuclear cells infiltrating the liver, spleen cells and peripheral blood lymphocytes were identified by flow cytometry. Flow cytometric analysis revealed the presence of CD5‐ and CD8+ lymphocytes in the liver tissues. Mononuclear cells infiltrating the liver were isolated from Wistar rats having autoimmune hepatitis to determine whether those exhibit cytotoxicity against syngeneic hepatocytes; they exhibited cytotoxicity against isolated syngeneic hepatocytes, but failed to lyse K562 cells, syngeneic concanavalin A‐activated splenocytes and allogeneic hepatocytes. Depietion of CD8+ T cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating into the liver against syngeneic hepatocytes. These findings support the idea that liver cell injury in experimental autoimmune hepatitis may at least in part be mediated by CTL.

Inhibition of interleukin-1 beta release from cultured human peripheral blood mononuclear cells by prednisolone

Prednisolone, a water-soluble glucocorticoid hormone, suppressed the secretion of interleukin-1 beta from human peripheral blood mononuclear cells in culture. The prednisolone-induced suppression of the monokine release was doserelated and the half maximal response was observed at 0.1 nM.

The effect of IL-6 on the des-gamma-carboxy prothrombin synthesis in human hepatoma cells

SummaryEffects of several cytokines on des-gamma-carboxy prothrombin (PIVKA II) synthesis in human hepatoma cells were investigated to know the process of PIVKA II production during a liver allograft rejection. Human recombinant interleukin-6 (IL-6) significantly stimulated the PIVKA II synthesis without any influence on the cell proliferation. The effect was almost completely neutralized by the specific anti-IL-6 antibody. Neither tumor necrosis factor (TNF), interleukin-1 (IL-1) nor interferon-gamma (IFN-gamma) had such a stimulative effect. IL-6 appears to stimulate PIVKA II production, and would be a candidate of factors that enhance the production of PIVKA II during a liver allograft rejection.

Characteristics of the PIVKA-II found in hepatocellular carcinoma, investigation using monoclonal antibodies MU-3 and 19B7

Abstract We classified PIVKA-II into two subtypes using a new monoclonal antibody (19B7) to PIVKA-II and a previously available monoclonal antibody (MU-3) and demonstrated differences in composition of PIVKA-II subtypes between HCC and benign liver diseases. These two monoclonal antibodies recognize almost the same site of PIVKA-II molecule, and the three-dimensional conformation of this site due to Gla formation might be important in differences of recognition by the two antibodies.

Effects of stress on growth of transplanted hepatic tumours and immune responses

Growth of transplanted hepatic tumours (T‐9) was enhanced in immune rats under stress, compared with immune rats in an unstressed condition. Compared with unstressed immune rats, killer activity of mononuclear cells infiltrating the tumours against T‐9 cells was significantly reduced in stressed immune rats. In contrast, killer activity of splenocytes obtained from stressed immune rats against T‐9 cells was elevated compared with that from unstressed immune rats. In addition, natural killer cell activity of mononuclear cells infiltrating the tumours obtained from stressed immune rats was significantly reduced compared with that from unstressed immune rats. Cell populations infiltrating tumour tissues were identified by flow cytometric analysis. The percentage of CD8+ cells in mononuclear cells isolated from tumour tissues of stressed immune rats was reduced compared with that of unstressed immune rats. Furthermore, interleukin‐2 responsiveness of splenocytes was suppressed in stressed immune rats, whereas T cell function as reflected by phytohaemagglutinin‐ or Concanavalin A‐reactivity was unaffected by stress. Collectively, it is likely that stress suppressed the generation of cytotoxic cells from the spleen cells of immune rats.

Importance of cytotoxic T lymphocytes in the rejection of transplanted hepatocellular carcinoma.

Fischer rats became resistant to syngeneic hepatocellular carcinoma (FAA-HTC1) cells on repeated sensitization with mitomycin C-treated FAA-HTC1 cells. In contrast, FAA-HTC1 cells injected into the liver killed normal control Fischer rats within 2 months. Histopathological studies revealed massive accumulation of mononuclear cells in the tumor tissues of sensitized rats that rejected syngeneic FAA-HTC1 cells, whereas very few mononuclear cells were found in the tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were identified as mostly T cells, and two-thirds of these T cells were CD8-positive. Compared with the activity in control rats, the killer activity of mononuclear cells infiltrating tumors was significantly increased in the sensititized rats 7 days after tumor inoculation. Depletion of CD8(+) T cells significantly reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD16(+) cells reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from both control and sensitized rats. Furthermore, the CD16(+) cell-depleted fraction of mononuclear cells infiltrating tumors showed significant cytotoxicity against FAA-HTC1 cells, but failed to show cytotoxicity against other syngeneic tumor cells or allogeneic hepatoma cells.

Induction of interleukin 6 production by interleukin 1 and tumor necrosis factor in human hepatoma cells

Interleukin 6 (IL-6) is produced by not only the lymphoid cells but also various non lymphoid cells (1). IL-6 is also known as multifunctional cytokine such as B-cell stimulatory factor 2, interferon beta 2, 26-kDa protein and hepatocyte stimulating factor. In this study, we investigated the effects of interleukin 1 (IL-1) and tumor necrosis factor (TNF) on the induction of IL-6 production in human hepatoma cell line, huH-1 (2). HUH-1 cells ( lx l05 cells/well) were plated in 24-well plates and incubated for 24 to 72 hrs with or without the addition of IL-1 alpha and TNF alpha in the culture medium. After incubation, the supernatants were subjected to the determination of IL-6. IL-6 was measured with commercially available ELISA kits (Toray-Fuji Bionix Inc.) using the specific monoclonal antibody. The IL-6 contents were measured after the exposure of the cells to IL1 (100U/ml) or TNF (100ng/ml) various times. Maximal stimulation was seen in 72 or 48 hrs after exposure of the cells to IL-1 or TNF, respectively, whereas the IL-6 level was not detected with no addition of cytokine (Figure). The IL-6 level with an addition of IL-1 to concentrationof 1, 10, 100 and 500 U/ml was 0, 97, 412 and 435 pg/ml, respectively. These results indicate the possible ability of ILl and TNF to induce IL-6 production in human hepatoma cells, as it has been reported that IL-6 is produced by various human cell lines. The similarity in the actions of IL-1 and TNF suggests that they activate similar intracellular pathways involved in the induction of IL-6 in human hepatoma cells.

Analysis of mononuclear cells infiltrating tumor in solitary hepatoma model

Abstract Ten million syngeneic hepatocellular carcinoma (FAA-HTCI) cells injected into liver are sufficient to kill Fischer rats within 2 months. Fischer rats became resistant to FAA-HTCI cells by repeated sensitization with MMC-treated FAA-HTC1 cells. Histopathological studies revealed massive accumulation of mononuclear cells in tumor tissues of sensitized rats that were rejecting syngeneic FAA-HTCI cells, whereas very few mononuclear cells were found in tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by immunohistochemical techniques and flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were mostly identified as being T cells, and two-thirds of these T cells were CD8 positive. Compared to control rats, killer activity of mononuclear cells infiltrating tumor tissues was significantly increased in the sensitized rats. Depletion of CD8(+) T cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD 16(+) cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating tumors obtained from control rats.

Effects on interleukin 1 alpha and beta production of peripheral blood mononuclear cells from patients with hepatocellular carcinoma after transcatheter arterial embolization

To clarify the immunological effects of transcatheter arterial embolization (TAE) therapy for hepatocellular carcinoma (HCC), we investigated changes in the production of interleukin I (IL-I) alpha, as well as beta, determined by enzyme-linked immunosorbent assay (ELISA) before and after TAE therapy. Eleven patients with nonresectable HCC received intraarterial infusion chemotherapy and TAE using Gelfoam cubes. IL-I production was measured before and 7 days after TAE. Peripheral blood mononuclear cells obtained from patients were cultured in RPMI 1640 medium containing 10% fetal calf serum for 48 hr in the presence of lipopolysaccharide (I). Culture supernatants were recovered and assessed. IL-I alpha and beta were determined by ELISA (2,3). The amount of the sum of IL-I alpha and beta in the patients with HCC (1232 346 pg/ml;mean SD) was higher than that in healthy volunteers (968 159 pg/ml). Following one week of TAE, the sum of IL-I alpha and beta was significantly increased to 1804 903 pg/ml (p<0.05). Furthermore, the increase rate of IL-I beta from before to after TAE was significantly higher than that of IL-I alpha (p<0.05). The content of immunoreactive IL-I 2000 was increased after TAE, supporting the ~,L-f~ idea that TAE therapy may, at least ~,L-I~ ep<O.O5 in part, activate Kupffer cells and enhance immunological responses. These ~. immunomodulatory effects of TAE may influence therapeutic response. ~ooo