Hiroko Tsutsui

Defective NK Cell Activity and Th1 Response in IL-18–Deficient Mice

IL-18 is a cytokine that is secreted from activated macrophages and induces IFNgamma production. To investigate the in vivo role of IL-18, we generated IL-18-deficient mice. In Propionibacterium acnes (P. acnes)-primed IL-18-deficient mice, LPS-induced IFNgamma production was markedly reduced, despite normal IL-12 induction. Natural killer cell activity was significantly impaired. Th1 cell response after injection of P. acnes or Mycobacterium bovis (bacillus Calmette-Guerin [BCG]) was significantly reduced. Similar results were observed in IL-12-deficient mice. Interestingly, Th1 response was induced after BCG infection in IL-12-deficient mice. We therefore generated mice lacking both IL-18 and IL-12. In these mice, NK activity and Th1 response were further impaired. This demonstrates the important role of both IL-18 and IL-12 in NK activity, as well as in in vivo Th1 response.

There is No Correlation Between Function and Lymphokine Production of HBs‐Antigen‐Specific Human CD4+‐Cloned T Cells

The question whether antigen‐specific human CD4+ T cells can be classified on the basis of appropriate and fixed lymphokine production patterns and their corresponding functions still remains lo be elucidated. We generated ten CD4+ T‐cell clones specific for HBsAg from HBsAg‐positive but HBsAb‐negative individuals. Seven of these clones exhibited helper activity for HBsAb response, while the three other clones did not. Both helper‐ and non‐helper‐type T‐cell clones produced interleukin 4 (IL‐4) after antigenic stimulation. By stimulation with phytohaem‐agglutinin (PHA) plus phorbol myristate acetate (PMA). three of the seven helper‐type clones produced interleukin 2 (lL‐2) in addition toiL‐4. However, the other four helper‐type clones did not produce IL‐2 by such stimulation, although they continued the production of IL‐4. All non‐help)er‐type T‐cell clones produced a large amount of IL‐2. and some of them completely became an IL‐2 producer after certain stimulation. These results suggested that both helper‐ and non‐helper‐type CD4+ T‐cell clones specific for HBsAg might have no strict pattern of lymphokine production as m the TH1 /TH2 dichotomy of murine CD4+ T cells. The data also revealed that lymphokine‐producing capacity of individual cloned T cells is changeable depending upon the sort of activation.

Induction of apoptosis in a human hepatocellular carcinoma cell line by a neutralizing antibody to transforming growth factor-α

A cell line derived from a Japanese man with hepatocellular carcinoma was established in culture and designated OCUH-16. The cell line has the morphological and chromosomal features of hepatocellular carcinoma cells and has a short doubling time (≈33 h). OCUH-16 cells were shown to express transforming growth factor-α, (TGF-α) in addition to albumin, DNA polymerase-α, c-JUN, and the retinoblastoma gene product. Electron microscopy revealed TGF-α immunoreactivity associated with the cell membrane, but TGF-α was not detected in medium conditioned by OCUH-16 cells by enzyme-linked immunosorbent assay. Reverse transcription and polymerase chain reaction analysis revealed the presence of TGF-α messenger RNA in these cells. Culture of OCUH-16 cells in the presence of a neutralizing antibody to TGF-α inhibited cell proliferation and induced many cells to undergo apoptosis (programmed cell death). These observations suggest that endogenous TGF-α is necessary for OCUH-16 cell growth.

An experimentally-induced acute hepatic failure model in various strains of mice

SummaryWhen BALB/cAJcl mice are intravenously injected with heat-killedPropionibacterium acnes (P. acnes) followed by an intravenous injection of lipopolysaccharide (LPS) 7 days later, massive necrosis is induced in the liver tissue and most of the mice die within 24 hours of LPS injection. Using this experimental model, acute hepatic failure was induced in various strains of mice and the difference in the response was studied. As a result, as in BALB/cAJcl mice, acut hepatic failure was also induced in BALB/ cAJcl-nu, AKR/J, C3H/HeNJcl, C57BL/6NJcl and DDy mice. However, as an exception, hepatic cell necrosis was hardly seen and the survival rate was remarkable high in C3H/HeJ mice, which genetically do not respond to LPS stimulation. These results indicate that for this experimental induction of acute hepatic failure, macrophages must be activated by the two-step stimulation ofP. acnes and LPS.

Network of cytokine and arachidonic acid cascade in acute hepatic failure

SummaryWhen heat-killedPropionibacterium acnes (P. acnes) was intravenously injected into mice and 7 days later a small amount of lipopolysaccharide (LPS) endotoxin was administered, most of the mice died of massive liver necrosis. In this liver injury model, cytokines, immunomediators, and eicosanoids, inflammatory products, were produced by Kupffer cells and liver-infiltrated macrophages, which were thought to participate, directly or indirectly, in the induction of liver cell damage. Furthermore, these two networks seemed to regulate each other. Thus, this regulatory mechanism might play an important role in the induction of liver cell injury.

Modulation of cholestatic factor production by serum components.

SummaryWhen lymph node cells from sensitized guinea pigs were stimulated in vitro with a specific antigen and their culture supernatant was injected into the mesenteric vein of rats, a marked decrease in bile flow was demonstrated. The treatment of activated lymphocytes with a higher molecular weight fraction of normal human serum Fr-1 and Fr-2 was shown to decrease the reduction of bile flow. Conversely, a lower molecular weight fraction of serum (Fr-3) was found to augment the reduction of bile flow. These findings suggest that the serum components may regulate the production of a factor (or factors) causing the decrease in bile flow from the activated lymphocytes.

Studies on the mechanism of liver injury by macrophage-mediated cytotoxicity—partial purification of cytotoxic factor detected in the culture supernatant of activated macrophages

SummaryThe culture supernatant of activated lymphocytes was shown to contain macrophage activating factor (MAF), a kind of lymphokine, which activated the peritoneal macrophages prepared from guinea pigs. When the culture fluid of the MAF-activated macrophages was added to the isolated liver cells, a significant inhibition of their albumin biosynthesis was demonstrated. The active material was recovered in a definitive fraction by gel filtration using a Sephadex G-75 column and this was further fractionated into two fractions by DEAE-cellulose column chromatography, suggesting that at least two kinds of active substances existed. By isoelectric focusing electrophoresis it was shown that these two active principles were substances having different isoelectric points, by isoelectric focusing electrophoresis.These results suggest that some cytotoxic or cytostatic materials are produced from the lymphokine-activated macrophages and they may participate in the occurence of liver injury at least partially.

Lipid peroxide formation in isolated hepatocytes by cytotoxic factors produced from lymphokine-activated macrophages

SummaryWhen peripheral blood lymphocytes from patients with chronic active hepatitis were stimulated with liver specific lipoprotein (LSP), considerably higher frequencies of lymphocyte transformation and MIF production were induced. Peritoneal macrophages from guinea pigs were activated by lymphokine-containing lymphocyte culture supernatant and produced a cytotoxic (or cytostatic) factor acting on isolated hepatocytes in culture. The cytotoxic (or cytostatic) factor, which was fractionated by Sephadex G-75 column gel filtration followed by DEAE-cellulose column chromatography, had cytotoxic effect on isolated liver cells and produced a significant amount of lipid peroxide. These results suggested the possibility that the cytotoxic effects may be caused at least partially by the lipid peroxid formation.

Induction of liver cell injury by antibody-dependent monocytemediated cytotoxicity in vitro

SummaryThe isolated liver cells coated with the anti-liver cell membrane antibody were damaged by incubation with the peripheral blood mononuclear cells. This was demonstrated by measuring the reduction of protein synthesis in the target liver cells. Adherent cells from the peripheral blood mononuclear cells were shown to have a sufficient capacity acting on the isolated liver cells as an effector when they were separated from the peripheral blood of normal and patients with acute or chronic active hepatitis. However, those from patients with liver cirrhosis or hepatoma did not show such effector activity in antibody-dependent cell-mediated liver cell damage. These results suggest that possibly antibody-dependent macrophage-mediated cytotoxicity may play some role in the induction of liver cell injury because the anti-hepatocyte membrane antibody is frequently detected in patient’s sera, especially in those with chronic active hepatitis.