S. Morisawa

Effects of an anti-tumor polysaccharide, schizophyllan, on interferon-γ and interleukin 2 production by peripheral blood mononuclear cells

We examined the effect of schizophyllan, a neutral glucan isolated from the culture filtrate of Schizophyllum commune Fries, on the production of interferon-gamma (IFN-gamma) and interleukin 2 (IL 2) from the mitogen-stimulated human peripheral blood mononuclear cells (PBMC). When the levels of IFN-gamma and IL 2 in the culture medium of phytohemagglutin (PHA)- or concanavalin A (Con A)-stimulated PBMC were measured by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) respectively, a significant increase in the production of both cytokines by schizophyllan was demonstrated. These results suggest that the increased production of IFN-gamma and IL 2 may be responsible for the anti-tumor activity of this glucan.

Calcium-dependent prostaglandin biosynthesis by lipopolysaccharide-stimulated rat Kupffer cells

Isolated rat Kupffer cells produced and released prostaglandin (PG) E2, 6-keto-PGF1 alpha, and thromboxane B2 (TXB2) in response to lipopolysaccharide (LPS) stimulation. This elevation of PGE2, 6-keto-PGF1 alpha and TXB2 in the medium was not observed when cells were cultured in the absence of extracellular calcium or in the presence of an extracellular calcium chelator, EGTA. An intracellular calcium antagonist, TMB-8, also suppressed the production of PGE2, 6-keto-PGF1 alpha and TXB2 in a concentration-dependent manner. The intra-cellular calcium concentration of Kupffer cells elevated early after the addition of LPS determined by the use of fura-2 and a fluorescence microscopy. Moreover, calmodulin inhibitors, W-7 and W-13, apparently inhibited the production of PGF2, 6-keto-PGF1 alpha and TXB2. All these results suggest that LPS-induced PG production by stimulated rat Kupffer cells may be regulated by a calcium-calmodulin pathway.

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.

Changes in leukotrienes and prostaglandins in the liver tissue of rats in the experimental massive hepatic cell necrosis model

When heat-killed Propionibacterium acnes is intravenously injected into rats followed by an intravenous injection of a small amount of Gram-negative lipopolysaccharide (LPS) 7 days later, massive hepatic cell necrosis is induced and most of the rats die within 24 hours of LPS injection. Using this experimental model, we studied the changes in the levels of leukotrienes (LTs) and prostaglandins (PGs) in the liver tissue and bile of rats with experimentally-induced massive hepatic cell necrosis. Both the levels of LTs and PGs in the liver tissue and LTs in the bile increased before the microscopic appearance of hepatic cell necrosis. These results suggest that arachidonic acid metabolites may play an important role in the induction of liver cell injury.

Release of leukotriene B4 from rat Kupffer cells

In order to examine the production of leukotriene B4 (LTB4) from Kupffer cells, Kupffer cells isolated from the normal rat liver were incubated with calcium ionophore A23187, opsonized zymosan, or platelet activating factor (PAF), and the amount of LTB4 in the culture supernatant was determined by the combined technique of reverse-phase high-performance liquid chromatography and radioimmunoassay. As a result, when activated in vitro with calcium ionophore A23187, Kupffer cells generated LTB4. When Kupffer cells were stimulated with calcium ionophore after 10-min preincubation with AA861, a selective 5-lipoxygenase inhibitor, the release of LTB4 from Kupffer cells was markedly suppressed. PAF, which is a phospholipid mediator having a wide spectrum of biological activities, significantly enhanced the release of LTB4 from Kupffer cells stimulated with calcium ionophore or opsonized zymosan. Even when the Kupffer cell were not stimulated with calcium ionophore or opsonized zymosan, LTB4 production was significantly increased by PAF. Thus, our studies indicate that Kupffer cells could generate LTB4 as well as polymorphonuclear leukocytes and macrophages. In addition, it is suggested that Kupffer cells may be able to modify inflammatory and immunological events in the liver tissue by the release of LTB4.

Effects of bile acids on liver cell injury by cultured supernatant of activated liver adherents cells

SummaryWhen heat-killedPropionibacterium acnes (P. acnes) is intravenously injected into mice followed by an intravenous injection of a small amount of lipopolysaccharide (LPS) 7 days later, most of the mice die of massive hepatic cell necrosis within 24 hours of LPS injection. In addition, when the liver adherent cells including Kupffer cells are separated from the mice 7 days after P.acnes injection and incubatedin vitro with LPS, remarkable activity of the cytotoxic factor is found in the culture supernatant. This cytotoxic factor is thought to cause liver injury. Using this experimental model, the effects of various bile acids on liver cell injury were studied. As a result, ursodeoxycholic acid and dehydrocholic acid suppressed liver cell injury induced by the cytotoxic factor. However, cholic acid, deoxycholic acid and chenodeoxycholic acid did not have any hepatocytoprotective effects.

Estradiol receptors in the cytosol of peripheral blood mononuclear cells in hepatitis B virus carriers treated with interferon-alpha.

SummaryEstradiol receptors in the cytosol of peripheral blood mononuclear cells and the effects of interferon-α (IFN-α) on estradiol receptors were studied in asymptomatic hepatitis B virus (HBV) carriers, patients with chronic hepatitis B and normal controls. The level of estradiol receptors in the cytosol of mononuclear cells was significantly lower in asymptomatic HBV carriers and patients with chronic hepatitis B, compared to normal controls. This low level of cytosol estradiol receptors in patients with chronic hepatitis B was increased by the administration of IFN-α. In addition, when peripheral blood mononuclear cells from patients with chronic hepatitis B were incubated with IFN-αvitro, the level of cytosol estradiol receptors also increased by increasing the concentration of IFN-α. We previously reported that the response of mononuclear cells to estrogen is impaired in HBV carriers, and our present results suggested that this may be due to the low level of estradiol receptors in the cytosol of mononuclear cells.

Enhancement of prostaglandin E2 production by liver macrophages (Kupffer cells) after stimulation with biological response modifiers

PGE2 production by liver macrophages (Kupffer cells) activated by biological response modifiers was examined. Kupffer cells obtained from a normal rat liver possessed cyclooxygenase activity and produced TXB2, PGD2, and PGE2 from (1-14C)arachidonic acid. The major product was PGD2. When Kupffer cells were incubated in the presence of lipo-polysaccharide (LPS), OK-432, or heat-killed Propionibacterium acnes for 24 h, the amount of arachidonate cyclooxygenase products increased and the major product changed from PGD2 to PGE2. When liver macrophages including Kupffer cells were prepared from rats after an injection of LPS, OK-432, or heat-killed P. acnes, it was noticed that the number of cells obtained and PGE2 production increased compared with those of normal rat. These results suggested that PGE2 production by rat liver was induced when they were treated with biological response modifiers.

Interferon γ stimulates prostaglandin E2 production by mouse Kupffer cells

When mononuclear phagocytes, including Kupffer cells, are activated by various agents, they synthesize and release arachidonic acid metabolites, prostaglandins (PGs) and leukotrienes (LTs). In this study, we examined the effect of in vitro Kupffer cell activation with recombinant murine IFNγ on PGE2 and LTB4 secretion. IFNγ enhanced PGE2 secretion, and this effect of IFNγ was stronger than that of IL-1 or TNF. Moreover, IFNγ promoted LTB4 release especially in the absence of PGs. On the other hand, dexamethasone and indomethacin inhibited and, EGTA and TMB-8, which reduce intracellular Ca++ Levels, blocked IFNγ induced PGE2 production, which suggested that the activation of phospholipase A2 and cyclooxygenase in Kupffer cells requires the elevation of intraceulullar Ca++ levels.

Tryptophan metabolism in D-galactosamine-induced liver injury

SummaryWe have reported that in rats with D-galactosamine-induced liver injury, the serum level of indoleacetic acid (IAA), a metabolite of tryptophan (TRP), increases before the increase in serum transaminase activity. To determine whether this IAA is derived from hepatocytes, isolated hepatocytes were treated with D-galactosamine and loaded with TRP, and the changes in TRP and IAA levels in the culture supernatant of the isolated hepatocytes were measured at various time intervals. As a result, IAA level in the culture supernatant of hepatocytes treated with D-galactosamine and loaded with TRP significantly increased in a time-dependent manner. This indicates that in D-galactosamine-induced liver injury, a metabolic pathway which produces IAA from TRP through tryptamine is present in hepatocytes.