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Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines.

A wide range of receptors are located at the blood sinusoidal aspect of the hepatocyte plasma membrane. Many circulating ligands that bind to receptors on the cell surfaces are interiorized along two pathways. Asialoglycoproteins are transferred from the plasma membrane to lysosomes and degraded, whereas immunoglobulin A and bile acids are transported across the hepatocyte interior and released into bile. Asialotransferrin type 3 (ref. 6) follows a further pathway termed diacytosis. After binding to the asialoglycoprotein receptor, asialotransferrin is endocytosed and then returned to blood with a proportion of its carbohydrate side chains resialylated. We now describe in liver the properties of intracellular asialotransferrin-enclosing vesicles (diacytosomes) and show that they differ from Golgi, lysosome and plasma membrane fractions. Furthermore, we show that the asialoglycoprotein binding sites are located on the cytoplasmic (outer) surface of diacytosomes.

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