Masami Imoto

Histochemical properties of vascular and sinusoidal endothelial cells in liver diseases

SummaryLiver biopsy specimens with or without liver diseases were examined immunohistochemically to determine the distribution of endothelial cell markers, factor VIII-related antigen (FVIII-RAg), Ulex europaeus agglutinin I (UEA-I) lectin and PAL-E. We also investigated the localization of laminin, a component of the basement membrane. In normal livers, FVIII-RAg, UEA-I and laminin were negative in sinusoidal endothelial cells, but positive in blood vascular endothelia of the portal area. The antigen detected by PAL-E was distributed in venous endothelial cells. PAL-E did not label endothelial cells of the artery. In the lobule, immunoreactivity with PAL-E was weakly detected only in some sinusoids of the periportal area. In chronic active hepatitis and liver cirrhosis, FVIII-RAg and UEA-I stained endothelial cells of neovasculatures in the enlarged portal areas of the fibrous septum surrounding pseudolobules. Some sinusoidal endothelial cells in cirrhotic livers were reactive to UEA-I and FVIII-RAg, whereas PAL-E-positive cells were found rarely in the pseudolobules. In carcinomatous sinusoidal endothelial cells, FVIII-RAg, UEA-I and PAL-E were strongly stained. Laminin underlay these carcinomatous sinusoids. These suggest capillarization of sinusoids in hepatocellular carcinoma. The histochemical approach using endothelial cell markers could be a practical tool in the diagnosis of hepatocellular carcinoma.

Immunohistochemical study on tissue inhibitors of metalloproteinases in normal and pathological human livers.

SummaryThe localization of tissue inhibitor of metalloproteinases (TIMP) in normal and pathological livers was examined by immunohistochemistry using monoclonal antibodies at the light microscopic level. In normal liver, immunoreactive TIMP was detected in smooth muscle cells and endothelial cells of blood vessels, fibroblasts, bile duct cells and Kupffer cells, indicating that TIMP is likely to be a general element of the liver. Immunoreactivity was observed in newly-formed blood vessels, proliferating bile ductules, and fibroblasts in the expanded portal area and fibrous septa of chronic active hepatitis and cirrhosis. TIMP was strongly stained in the capsule of hepatocellular carcinoma. The intensity of the immunoreaction in the capsule was generally greater than that in cirrhotic liver apart from the tumor mass. In three of five cases with hepatocellular carcinoma, endothelial walls in contact with tumor cells were positive.

Predominance of histamine H1 receptors on liver plasma membrane

We quantitatively determined the receptors for histamine H1, histamine H2, alpha- and beta-adrenergic, prostaglandin E2 and F2 alpha in liver plasma membranes. The number of the receptors was the greatest in histamine H1 receptors (4740 +/- 750 fmol/mg protein) and the second in alpha-adrenergic receptors (965 +/- 16). Although relatively small numbers of the receptors were observed for histamine H2 (116 +/- 11) and prostaglandin E2 (38.0 +/- 8.9), we could not determine the number and affinity of beta-adrenergic and prostaglandin F2 alpha receptors. In contrast to the number of receptors, there was not significant difference in the affinities of receptors for histamine H1, histamine H2, alpha-adrenergic hormone and prostaglandin E2. These results suggest that histamine and its receptor play some role in liver function.

Demonstration of Noradrenaline-Immunoreactive Nerve Fibres in the Liver

To demonstrate noradrenaline-immunoreactive nerve fibres in liver tissues, we used an antibody to noradrenaline in the immunostaining of liver tissues from rats, guinea-pigs and humans. The tissue specimens were fixed by perfusion or immersion with cacodylate buffer containing sodium metabisulphate and glutaraldehyde, and cryostat sections were prepared. An indirect peroxidase-labelled antibody method was used for staining noradrenaline. Noradrenaline-immunoreactive nerve fibres were localized around blood vessels in the portal area and around the central vein. There were differences between the species in the intralobular distribution of noradrenaline-immunoreactive fibres. Normal guinea-pig and human liver showed intralobular noradrenaline-immunoreactive fibres while rat liver did not. Noradrenaline-immunoreactive fibres were absent from regenerating nodules in a human cirrhotic liver. This method of demonstrating noradrenaline directly using perfusion- or immersion-fixation is appropriate for studying innervation in normal and damaged livers of various species including humans.

Localization of synaptophysin immunoreactivity in the human liver.

The distribution of synaptophysin, specifically located in nerve terminals, was investigated immunohistochemically in normal and diseased human livers in 4 patients with normal liver, 6 with chronic active hepatitis, 12 with cirrhosis, and 8 with hepatocellular carcinoma. In normal liver and chronic hepatitis synaptophysin immunoreactivity was detected in the lobules and portal areas. In cirrhosis it was found in the fibrous septum but in no pseudolobules. Parenchymal innervation would thus appear to cease with the development of cirrhosis, and denervation from the parenchyma may lead to various functional abnormalities in liver cirrhosis. In hepatocellular carcinoma no synaptophysin immunoreactivity was found along carcinomatous sinusoids. Immunoreactive spots were present in the capsules of hepatocellular carcinoma to a much lesser extent than in the fibrous septum of cirrhosis. Neural functions may thus have little effect on the microcirculation of hepatocellular carcinoma.

High rate of mixed genotypes of hepatitis C virus in patients of an epidemic area in Japan

The subjects of this study were 151 patients (69 males and 82 females) who underwent examination and liver biopsy owing to liver dysfunction in an epidemic area with hepatitis C. Second generation hepatitis C virus antibody (HCV Ab) was positive in 116 (76.8%) of 151 cases. HCV‐RNA was detected in 120 (79.5%) by polymerase chain reaction (PCR). In 7 (4.6%) cases, HCV Ab could not be found, but HCV‐RNA was detected. Three (2.0%) cases were positive for HCV Ab but negative for HCV‐RNA. On the basis of variation in nucleotide sequence within a restricted region in the putative core gene of HCV, HCV genotypes were classed into four types of I, II, III and IV by PCR. The genotypes were identified in 120 cases. Ninety‐eight (81.7%) cases carried one of the four types. Type II was found in 76 (63.3%) cases and type III in 22 (18.3%). The other 22 (18.3%) carried simultaneously two different genotypes (mixed type): 21 (17.5%) cases with type II + III and one (0.8%) case with type II + IV. In comparison with the incidence of HCV mixed types in cases with hepatitits C in a non‐epidemic area, carriers of mixed types were found at a significantly higher rate in the epidemic area. Liver biopsy of 120 cases with identified HCV genotypes in the epidemic area showed 93 cases of chronic active hepatitis, nine of chronic lobular hepatitis, 10 of chronic persistent hepatitis and eight of liver cirrhosis. No significant correlation could be detected between liver histology and HCV genotypes. Of the 120 cases, 63 (52.5%), 54 (45.0%) and 12 (10.0%) cases had past histories of folk remedies accompanying bleeding, operation and transfusion, respectively. The repetition of these medications may have caused a high ratio of carriers of the mixed genotypes of HCV.

Detection of Noradrenaline-Immunoreactive Nerve Fibres in Rat Liver by Immunoelectron Microscopy

Noradrenergic innervation of rat liver was studied immunohistochemically using antibody to noradrenaline at the electron-microscopic level. Noradrenaline-immunoreactive nerve fibres were located in the portal tract and some were in close contact with the portal vein and hepatic artery. Noradrenaline-immunoreactive fibres were found to contain many vesicles that were reactive to anti-noradrenaline antibody. This preliminary study suggests that the method for detecting noradrenaline-immunoreactive fibres using the antibody is useful for studies at the electron-microscopic level.

Endotoxin affects the expansion modes of liver macrophages after partial hepatectomy

We studied the effect of increased endotoxin levels on the expansion modes of liver macrophages after two‐thirds partial hepatectomy using anti‐endotoxin polymyxin B. The expansion consists of dual modes: local proliferation and immigration. Local proliferation of mature cells was evaluated by the S‐phase proportion measured by flow cytometric DNA analysis. This proportion (11.8 ± 1.3% before hepactectomy) decreased to 6.0 ± 1.1% at 12 h and reached a maximum of 21.9 ± 2.7% on the 5th day after hepatectomy. In polymyxin B treated rats, the proportion reached a maximum of 21.1 ± 1.6% at 48 h without any preceding decrease. Immigration of macrophage precursors was evaluated by the decreasing proportion of latex beads‐containing cells that were marked as resident liver macrophages by injection of latex beads before hetatectomy. This proportion (98.7 ± 0.2% before hepatectomy) was significantly decreased to 90.5 ± 1.6% at 48 h. In polymyxin B treated rats, however, the proportion showed no significant decrease within 3 days. These results indicate that endogenous endotoxin, which suppresses the local proliferation and promotes extrahepatic recruitment, regulates the number of liver macrophages in regenerating rat liver.

Prostaglandin production by isolated Kupffer cells

Kupffer cells have been supposed to participate in the pathogenesis of liver injury through the synthesis of eicosanoids, arachidonic acid metabolites, which show prominent biological activity. In the present study, we estimated production of prostaglandins (PGs), derivatives of arachidonic acid through the cyclooxygenase pathway, using cultured rat Kupffer cells. The nonparenchymal cells of male Wistar rats were isolated by perfusion and incubation of liver tissue with pronase. Kupffer cells were then purified from the nonparenchymal cells by selective adherence to plastic and were cultured in RPMI 1640 medium plus 20 % calf serum for 48 h prior to their use in experiments. Arachidonic acid (final concentration, i0 ~M) or calcium ionophore A23187 (20 ~M) was added to the medium. The medium was eluted with methyl formate using Sep-Pak C18 cartridge. The contents of PGs were analyzed using high performance liquid chromatography, as described previously (i). As a result, stimulated rat Kupffer cells produced mainly PGD2 among the cyclooxygenase products, i.e. PGD2, E2, F2~, 6-keto-PGFl~, and tromboxane B2. Exposure to arachidonic acid for 60 and 120 min released 93.5 • 15.3 and 143.0 + 26.2 ng/ixlO 6 cells of PGD2, respectively. Exposure to calcium ionophore A23187 for 60 and 120 min released 70.6 • 34.7 and 79.8 • 9.7 ng/IxlO 6 cells, respectively. The previous study demonstrated the possible involvement of the increase of PGD2 in liver tissue in the pathogenesis of D-galactosamine-induced liver injury (i). The present findings suggest that stimulated Kupffer cells participate in this pathogenesis through the synthesis of PGD2. Besides the peak of PGD2, two unidentified peaks (arrows) were detected in the chromatograms obtained from the samples of stimulated cells. These peaks were abolished by the administration of indomethacin, a potent cyclooxygenase inhibitor, along with arachidonic acid or calcium ionophore A23187. The above unidentified peaks might be attributed to PG analogues derived from cyclooxygenase reaction, e.g. PGD2 derivatives.

Clinical evaluation of serum 3β-hydroxy-5-cholenoic acid in hepatobiliary diseases

SummarySerum 3β-hydroxy-5-cholenoic acid (3β-OH-Δ5) was analyzed in 100 cases (90 patients with hepatobiliary diseases, 10 normal subjects) and its clinical significance investigated. The measurement of 3β-OH-Δ5 was performed by high performance liquid chromatography (HPLC) with immobilized 3β-hydroxysteroid dehydrogenase (3β-HSD) as the enzyme column. Esterified 3β-OH-Δ5 was measured after enzymatic hydrolysis with sulfatase andβ-glucuronidase. 3β-OH-Δ5 was hardly detected in normal cases. On the other hand, serum 3β-OH-Δ5 levels were remarkably high in cholestatic cases and also high in other cases with high bilirubin levels. The ratio of glycineto taurine-conjugates (G/T ratio) was effective in discriminating cholestasis from hepatocellular damage such as in cases of acute hepatitis or fulminant hepatitis. More than 90% of the 3β-OH-Δ5, which is toxic, was sulfated or glucuronidated, suggesting detoxification by esterified bile acids. Significant increases of taurine-conjugated 3β-OH-Δ5 were observed in cases with pruritus, and a relationship between taurine-conjugates and pruritus was presumed. Therefore, analysis of 3β-OH-Δ5 is considered to be effective in clarifying the pathogenesis of hepatobiliary diseases.