Kazuhiko Okada

Effect of interleukin-8 on production of tumor-associated substances and autocrine growth of human liver and pancreatic cancer cells.

Abstract We have previously reported that human liver cancer cell lines produce interleukin-8 (IL-8) at high levels. Those tumor cells appeared to express two kinds of IL-8 receptor on their surface. In order to analyze the role of IL-8 on the biological characteristics of those tumor cells, we suppressed IL-8 production from human liver (HuH-7 and HuCC-T1) and pancreatic cancer cell lines (HuP-T4) by treatment with IL-8 antisense oligonucleotides. Suppression of IL-8 production resulted not only in inhibition of cell growth, but also in an increase in the concentrations of some tumor-associated substances such as carbohydrate antigen 19-9 (CA19-9) in the medium. These data indicate that IL-8 produced by human liver and pancreatic tumors may act as an autocrine growth factor and may control the production of some tumor-associated substances. Furthermore, surface expression of sialyl-Lewisa, which is a ligand for ELAM-1 on human umbilical vein endothelial cells (HUVEC), HuCC-T1 and HuP-T4 cells was decreased and the attachment of these tumor cells to HUVEC was inhibited by treatment with IL-8 antisense oligonucleotide. Since the soluble form of CA19-9 (sialyl-Lewisa) was shown to inhibit the tumor cell binding to HUVEC, the decrease in release of CA19-9 into the medium and increase in the expression of sialyl-Lewisa on the cell surface may suggest that IL-8 production from the tumor cells enhances metastatic potential by augmenting the binding activity of the tumor cells to HUVEC. These data demonstrate that a cytokine produced by tumor cells may function as an autocrine growth factor and affect tumor cell dissemination.

Interleukin‐6 functions as an autocrine growth factor in a cholangiocarcinoma cell line

Abstract The tumour cells of a human cholangiocarcinoma cell line, HuCC‐T1, were found to express mRNA of interleukin‐6 (IL‐6) and to secrete a large amount of biologically active IL‐6 in the culture medium at the concentration of 22.6 ng/mL. Interleukin‐6 was demonstrated in the cytoplasm of the cells by immunohistochemical staining. Furthermore, these cells showed the presence of receptors for IL‐6 on the surface, and DNA synthesis of the cells was stimulated by the exogenous addition of recombinant human IL‐6 into the culture medium. The cell growth was significantly inhibited in the presence of anti‐human IL‐6 antibody in the culture medium. These findings indicate that IL‐6 is one of the autocrine growth factors of this cell line in vitro.

Analysis of T-cell receptor Vβ repertoire in liver-infiltrating lymphocytes in chronic hepatitis C

Abstract Background/Aims: To examine the T-cell repertoire which is involved in the immunopathogenesis of chronic hepatitis, we analyzed the T-cell receptor Vβ gene usage in liver-infiltrating lymphocytes by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical technique. Methods: Complementary DNA was synthesized from RNA which was extracted from 26 liver biopsy specimens and from peripheral blood lymphocytes from eight subjects, and amplified by RT-PCR. Radioactivity of each amplified product using 32 P-labeled primers was measured and the percentage of each Vβ expression was calculated. Results: The mean frequency of Vβ5.1 (11.1%) in liver-infiltrating lymphocytes of chronic hepatitis C was highest among those of all Vβ regions, and was significantly higher than that in both peripheral blood lymphocytes of chronic hepatitis C and liver-infiltrating lymphocytes of chronic hepatitis B. In the immunohistochemical analysis, Vβ5.1-positive cells were mostly observed in portal areas where inflammatory reactions occurred. The sequence of the complementarity determining region (CDR)3 on T-cell receptor expressing Vβ5.1 were examined in six patients with chronic hepatitis C. The sequences were similar to each other and all had one common amino acid (valine) irrespective of different HLA haplotype. Conclusions: These data suggest that Vβ5.1-positive cells are preferentially accumulated in the liver of chronic hepatitis C and are involved in the immunopathogenesis of the disease. Sequence analysis showed that Vβ5.1-positive cells recognize a common conventional antigen and valine recognized at the same position of the CDR3 may be a key residue in determining an antigen/major histocompatibility complex contact point.

Detection and analysis of intracytoplasmic cytokines in peripheral blood mononuclear cells in patients with drug-induced liver injury

BACKGROUND/AIMS Idiosyncratic immune response to drugs causes two types of liver injury, cholestasis or hepatitis. However, the underlying immune mechanisms of drug-induced liver injury are presently unclear. METHODS We examined the cytokine production of peripheral blood mononuclear cells (PBMCs) from 17 patients with drug-induced liver injury and healthy controls during their incubation with and without the drug by flow cytometry. We also analyzed the cytokine production in PBMCs from eight patients after stimulation with the drug-pulsed HepG2 lysates to examine the possibility that the drug or its metabolites conjugated with a putative molecule derived from HepG2 cells might be more immunogenic. RESULTS Among several cytokines produced by the drug or the drug-pulsed HepG2 lysates, interferon-gamma production from CD8+ cells was associated with hepatocellular injury, and tumor necrosis factor-alpha production from CD14+ cells was with cholestasis. Especially, the latter was apparent when the drug-pulsed HepG2 lysates were used as stimulants, suggesting that a complex consist of the drug, or its metabolite, and a putative molecule derived from HepG2 cells might be more immunogenic than the drug itself. CONCLUSIONS The analysis of intracytoplasmic cytokine in PBMCs after stimulation with the drug or the drug-pulsed HepG2 lysates is useful to analyze the immune mechanism underlying drug-induced liver injury.

Analyses of proliferating cell nuclear antigen‐positive cells in hepatocellular carcinoma: Comparisons with clinical findings

Abstract Proliferating tumour cells in 92 patients with hepatocellular carcinoma (HCC) were identified by an immunohistochemical method using a monoclonal antibody against proliferating cell nuclear antigen (PCNA). The rate of PCNA‐positive cells in HCC tissues was positively correlated with histological grade and the tumour size and T factor of the tumour.

Modulation by cytokines on the surface expression and shedding of intercellular adhesion molecule-1 (ICAM-1) in human liver cancer cell lines

Abstract Surface expression and shedding of intercellular adhesion molecule-1 (ICAM-1) in the absence and presence of cytokines were measured in seven human liver cancer cell lines. All cancer cell lines were positive for ICAM-1 on the surface of the cells, and the percentages of positive cells ranged from 60 to 97% as analyzed by flow cytometry. Mean fluorescence was increased in the presence of IL-1β, TNF-α, IFN-α or IFN-γ, and the effect was augmented in a dose-dependent fashion. In the culture medium of all cancer cell lines, a soluble form of ICAM-1 was detected even in the absence of cytokines by enzyme-linked immunosorbent assay, and the concentration was increased by IL-1β, TNF-α, or IFN-γ. In two of seven cancer cell lines, the patterns of modulation by cytokines on the surface expression of ICAM-1 and the shedding of the molecules were clearly distinct, suggesting that the mechanisms for these two phenomena might be different. Soluble ICAM-1 was recently reported to suppress non-MHC-restricted cytotoxicity mediated by natural killer and lymphokine-activated killer cells. Our present data suggest that human liver cancer cells may escape from host immune surveillance by shedding of ICAM-1.

CASE REPORT: Intrahepatic portal‐hepatic venous shunts associated with a huge pelvic leiomyoma

We present a case of portal‐systemic encephalopathy due to intrahepatic multiple portalhepatic venous shunts. A 71‐year‐old woman was admitted to our hospital because of recurrent episodes of disturbed consciousness. She showed no clinical signs of portal hypertension. Liver function was normal, except for an indocyanine green retention rate of 34% at 15 min and blood ammonia level of 282 μg/dL. Portal venography revealed dilatation of the portal vein and multiple portal‐hepatic venous shunts, and a liver biopsy specimen revealed almost normal liver. Further clinical examination revealed a huge pelvic tumour. At laparotomy, two dilated veins were seen to arise from the pelvic tumour with blood flow into the mesentery. The tumour was resected successfully and a histological diagnosis of leiomyoma was made. The blood ammonia concentration decreased to the normal range postoperatively. A follow‐up portal venogram demonstrated decreased portal vein dilatation and minor portalhepatic venous shunts, considered to be congenital in origin. It is concluded that liepatic encephalopathy was produced in this patient due to an excess portal blood flow from the huge pelvic leiomyoma via the mesentery, with portosystemic shunting through pre‐existent (probably congenital) intrahepatic anastomoses.

Variable Region Usage in T Lymphocytes Infiltrating Liver Tissues of Chronic Liver Diseases

To analyze the T cell repertoire of liver-infiltrating lymphocytes, 35 liver biopsy specimens taken for diagnostic purposes were stained by immunohistochemical technique using monoclonal antibodies (mAb) against CD3, CD8, CD45RO, and CD45RA and seven different variable (V) regions of the T cell receptor (TCR). Major populations of the infiltrating lymphocytes were CD3- and CD45RO- positive cells. The mean percentage (±SD) of Vβ5.1-positive cells was significantly (P < 0.05) higher in chronic hepatitis C (18.2 ± 13.3%, n = 15) than that in either chronic hepatitis B (5.7 ± 6.0%, n = 12) or autoimmune hepatitis (1.3 ± 1.2%, n = 3). In cases with primary biliary cirrhosis, only one case showed a high percentage (66%) of Vβ5.1-positive cells with mean ± SD of 19.3 ± 26.2% (n = 5). The percentages of Vβ5.1-positive cells correlated inversely with the Knodell histological activity index scores in cases with chronic hepatitis C. These data suggest that Vβ5.1-positive T cells may play a regulatory role in the immuno-pathogenesis of chronic hepatitis C.

Serum interleukin-8 levels in patients with hepatocellular carcinoma

Abstract Serum levels of interleukin-8 (IL-8) in 73 patients with hepatocellular carcinoma (HCC), 24 with liver cirrhosis (LC) and 18 with chronic hepatitis (CH), and in 20 healthy controls were measured by an enzyme-linked immunosorbent assay. The mean (± SD) level of serum IL-8 in patients with HCC was 48.4 ± 60.8 pg/ml, which were significantly (P

Induction of allogeneic tumour‐ and lymphokine‐activated lymphocytes against hepatocellular carcinoma

For clinical application of adoptive immunotherapy against hepatocellular carcinoma (HCC), it is not easy to prepare tumour specific effector cells such as cytotoxic T lymphocytes (CTL). To induce potent and broad‐spectrum effectors, allogeneic cultured hepatoma cell lines (JHH‐4 and HuH‐6) were used as stimulators of peripheral blood lymphocytes (PBL) instead of autologous HCC cells. Allogeneic tumour‐ and lymphokine‐activated killer cells (ATLAK) were generated by a mixed culture of lymphocytes and allogeneic cultured tumour cells with recombinant interleukin‐2 (rIL‐2). The tumour‐killing activity of ATLAK induced by HuH‐6 was confirmed against HuH‐6 and other different HCC cell lines (JHH‐2, HuH‐7 and PLC). These activated lymphocytes were significantly more potent than lymphokine‐activated killer cells (LAK) in [51Cr]‐releasing assay. The JHH‐4 stimulated ATLAK was reactive not only with JHH‐4 but also with JHH‐2. The lysis of allogeneic targets could be partially inhibited by anti‐CD8 and anti‐CD3 but not by anti‐CD4. Anti‐tumour cytotoxicity in these cultures might be mediated by CD3+CD56‐ and CD3+CD56+ effectors. These results imply that adoptive immunotherapy for HCC with ATLAK may be more feasible than that with LAK.