In the duck model of hepatitis B virus infection, endotoxin of gram negative bacteria inhibits the replication of duck hepatitis B virus (DHBV) in vitro. We reported that endotoxin activates liver macrophges, which are present in primary hepatocyte cultures, to release polypeptide mediators, and among these the cytokines IFN alpha and IFN gamma. The mediators act at an early step of hepadnaviral replication, presumably at the level of translation of viral RNAs. Presently, it is not possible to define the different mediators involved as the duck is imunologically not well characterized. We therefore aimed to establish a murine in vitro test system. For this, replication-competent hepatitis B virus genomes were introduced into different monse hepatocyte cultures using adenoviral vectors.
Primary mouse hepatocytes were isolated by differential centrifugation after two step collagenase perfusion of the mouse liver. To enrich the hepatocyte culture with different amounts of smaller non-parenchymal liver cells (e.g. liver macrophages, sinusoidal endothelial cells), hepatocytes were sedimented at 35, 70, or 200×g. Liver sinusoidal endothelial cells were purified by centrifugal elutriation, and after endotoxin treatment (100 ng/ml) the conditioned cell culture medium was collected. Hepatocyte cultures were infected with Adeno-DHBV (day 1 post plating, moi: 50), and treated with different concentrations of endotoxin (0,1 - 200 ng/ml) or conditioned cell culture medium (1:1). Neutralizing antibodies to various cytokines were added to the endotoxin treated mouse liver cell cultures.
Like in primary duck hepatocytes, endotoxin inhibited DHBV replication in primary mouse hapatocytes in a dose-dependent fashion. This effect was enhanced when mouse hepatocyte cultures were enriched for liver non-parenchymal cells. In contrast the conditioned cell culture medium of endothelial cells did not inhibit DHBV replication pointing to liver macrophages as the major source of antiviral mediators. Neutralizing antibodies to TNF alpha abolished a part of the antiviral effect indicating an involvement of TNF alpha.
The infection of primary mouse hepatocyte cultures enriched for non-perenchymal liver cells with adenoviruses transferring hepatitis B virus genomes allows us now to test the influence of immune mediators on hepatitis B virus replication in vitro, to analyze second mediators and to further define intracellular pathways activated by cytokines.