Sukeo Yamamoto's research while affiliated with Osaka General Medical Center and other places

ABSTRACT— When mononuclear phagocytes, including Kupffer cells, are activated by various agents, they synthesize and release cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF). In this study, we examined the effect of in vitro Kupffer cell activation by recombinant murine interferon (IFN) on IL-1 and TNF secretion. IFN enhanced TNF production in the presence or absence of lipopolysaccharide (LPS), but suppressed IL-1 production by Kupffer cells. Because IFN also stimulated prostaglandin E2 (PGE2) production, the effect of indomethacin, which is an inhibitor of cyclooxygenase and which inhibits PGE2 biosynthesis, on IL-1 and TNF production by Kupffer cells was examined. As a result, indomethacin enhanced TNF production by Kupffer cells, but had no effect on IL-1 synthesis. These results suggested that IFN modulates the production of IL-1 and TNF by Kupffer cells through different mechanisms.

Radionuclide angiography was used to generate curves of first-pass radioactivity vs. time for the right hepatic lobe and both kidneys following the rapid intravenous injection of 10 mCi of 99mTc-phytate. By analysis of the right hepatic curve, the arterial and portal components of the liver circulation were calculated in 21 healthy subjects and 183 patients, 39 with chronic hepatitis and 144 with cirrhosis of the liver. In the healthy subjects, the portal component (PC) of liver blood supply ranged from 64% to 79% (mean 71.5%). In patients with hepatitis the mean PC was 61.3%, and in patients with cirrhosis it was 37.9%. The PC was lower in cirrhotic patients with oesophageal varices than in those without (P < 0.001). The PC was assessed in 23 of the patients with cirrhosis before and after sclerotherapy for oesophageal varices. A paired t-test showed significant rises in this component immediately after the final treatment (P < 0.001). Two months after this treatment, the mean PC had dropped significantly, approaching the pre-treatment value (P < 0.001). One year after treatment, the PC had not dropped further. These findings suggest that this non-invasive method may be useful for the assessment of portal circulatory changes in cirrhosis, and of results of sclerotherapy for oesophageal varices.

The peptide leukotriene C4 D4, and E4 (LTC4, LTD4, and LTE4) are derived from arachidonic acid via a lipoxigenase pathway and are potent mediators of inflammation and allergy. It is also reported that Kupffer cells may have the capacity to influnece imflammatory events in chronic liver diseases. In this study, to clarify the relation of leukotrienes to liver injury, we have examined the release of LTC4, LTD4 and LTE4 from rat Kupffer cells in vitro. Kupffer cell suspensions (5×10⁵cells/ml) in modified Tyrode's solution incubated with the calcium ionophore A23187 (1μg/ml). The incubation proceeded for 0, 5, 10, 15, 30, 60min at 37°C and was stopped with 4-fold of ice-cold ethanol. Leukotrienes were assayed using combined technique of reverse-phase high-performance liquid chromatography and radioimmunoassay. When activated in vitro with the calcium ionophore A23187, Kupffer cells generated LTC4, LTD4, and LTE4. Thus, our studies indicate that Kupffer cells also could generate LTC4 LTD4, and LTE4 as well as polymorphonuclear leukocytes or monocytes. In addition, it is suggested that Kupffer cells maybe able to modify inflammatory and immunologic events in liver tissue by the release of LTC4 LTD4, and LTE4.

Influence of salicylazosulphapyridine, a medicine for inflammatory bowel disease, on peripheral blood lymphocytes in healthy subjects was examined by chromium release assay. The following results were obtained. 1. Inhibition by salicylazosulphapyridine on the PHA-induced cytotoxic action of human lymphocytes was obserbed by using mouse L cells as target cell. 2. The degree of inhibition for cytotoxicity was inversely proportionate to the viability of the lymphocytes. 3. Lymphocytes pretreated with salicylazosulphapyridine synthesized about similar amounts of DNA by PHA stimulation in comparison with DNA synthesis by untreated lymphocytes. 4. The toxic action of salicylazosulphapyridine was examined by using human lymphocytes, human erythrocytes, human Chang liver cells and mouse L cells as targets. As a result, lymphocytes were more sensitive than other cells to the toxic action of salicylazosulphapyridine, indicating that salicylazosulphapyridine has selectively the toxic action for the lymphocytes. The above results suggest that salicylazosulphapyridine has some inhibitory action for immune system in vivo.

To elucidate the immunopathogenesis of intrahepatic cholestasis in alcoholic liver disease, the authors investigated the possible participation of cholestatic factor. The peripheral lymphocytes from patients with intrahepatic cholestasis in alcoholic liver disease were stimulated with ethanol and liver specific lipoprotein in vitro and the cluture supernatant was fractionated by gel filtration using a Sephadex G-75 column. When a definitive fraction (fraction 4) was injected into the mesenteric vein of rats, a marked reduction of bile flow was observed. Similar results were obtained when blood serum of patients was fractionated in an identical manner and the same fraction was injected to rats via mesenteric vein. Histologically, a dilated bile canaliculus with the diminution of microvilli and increased vesicles around the dilated canaliculi were observed by an electron microscopy after injection of the active fraction into rats. These results strongly suggest that not only the sensitized lymphocytes produce the cholestatic factor(s) which caused the intrahepatic cholestasis by specific stimulation, but also this factor involves significantly in the pathogenesis of intrahepatic cholestasis which observed in the patients with intrahepatic cholestasis in alcoholic liver disease.

Although the lymphocyte transformation and lymphokine production have been frequently used for the identification of the causative drug in patients with drug-induced allergic hepatitis, these immunological parameters are not always positive in all patients. This may be reflected on the small population of the sensitized lymphocytes. In order to augment these immunological reactions, we deviced a method which interleukine-2 was added to the lymphocyte culture simultaneously with the antigen (drug and carrier). In four cases which did not show any positive lymphocyte transfromation and lymphokine (cholestatic factor) production by stimulating them with the drug in the presence of the suitable carrier, the addition of interleukine-2 was shown to enhance the immunological responses; lymphocyte transformation with th causative drug was significantly induced. Moreover, long-term culture of the sensitized lymphocytes was shown to be successful by cultivating th activated lymphocytes in the presence of interleukine-2 and lymphocyte transformation and cholestatic factor production were detectable in the culture at least two or three months after the cultivation.

In many drug-induced allergic hepatitis, peripheral lymphocytes were transformed by the stimulation with a given drug in the presence of autologous serum. However, when rat liver microsome fraction or soluble liver specific antigen fraction was added to the culture instead of autologous serum, the drug-induced lymphocyte transformation was more efficiently shown than autologous serum, while rat liver mitochondria fraction was less effective. On the other hand, in the cases of drug-induced allergic eruption, the addition of liver subcellular fractions were for less effective to induce the lymphocyte transformation than autologous serum. These results may suggest that liver subcellular components involve in pathogenesis of the drug-induced allergic hepatitis.

When the heat-killed Propionibacterium acnes (P. acnes) and a small amount of lipopolysaccharide (LPS) were injected intravenously in mice at a week interval, massive necrosis was observed on their livers. The liver adherent cells from mice previously injected with P. acnes were activated by LPS, to produce a hepatocytotoxic factor. However, the peritoneal exudate cells from the same treated mice did not produce such a cytotoxic factor even after stimuration with the same dose of LPS. Secondly we analysed cellular immunological changes which had been taken place during the liver injury induction. The spleen or the peripheral blood mononuclear cells from P. acnes injected mice less proliferated to the mitogenic stimulation then those from normal mice. Furthermore the mononuclear cells from liver injuried mice could not respond to mitogen stimuration at all. These results suggest that induction of liver injury in our experimental system simultaneously leads to the immuno-suppression.

Antibody-dependent cell-mediated cytotoxicity (ADCC) toward sheep red blood cells by peripheral blood mononuclear cells was modulated by pretreatment of effector cells with sex hormons: When peripheral blood mononuclear cells were pretreatmented with a given dosis of estrogen, the lysis of erythrocytes via ADCC reaction was enhanced significantry and this enhancement was suppressed by testosterone. A similar effect of sex hormone on ADCC reaction was also demonstrated when silicatreated mononuclear cells were used as a effector. Furthermore, although a excess of estrogen did not alter effector activity, simultaneous addition of low dosis of testosterone was shown to increase in ADCC reaction. These results suggest a possibility that sex hormones may not only modulate immune responses but also give some effect on ADCC reaction.

Eighty patients with unresectable hepatoma were treated with transcatheter arterial embolization, and 17 died within 3 months postoperatively. Of the 7 cases who died within 1 month, 4 died of hepatic failure and 3 died of renal failure. All of the 4 patients who died of hepatic failure showed obstruction of the portal trunk on arterial portogram. The three patients who died of renal failure complained of severe pain after embolization which was followed by anuria. Out of the 10 cases who died between 1 and 3 months after embolization, 5 died of hepatic failure, 2 intraperitoneal bleeding, 1 gastrointestinal bleeding, 1 sepsis and 1 brain metastasis. All of the 5 patients who died of hepatic failure had a large tumor which grew rapidly and showed early regrowth after a temporary reduction in size. The death of the other 5 patients was thought to be unrelated to their embolization. From the above mentioned results, embolization can be performed safely with careful technique and postoperative management apart from those in which the portal trunk are obstructed.