Export of intracellular HBsAg in chronic hepatitis B virus infection is related to viral replication

Abstract

Serum and liver HBsAg bear an inverse relation to each other during the evolution of chronic hepatitis B virus infection and the quantity of HBsAg in tissue rises gradually with time. In this study, intracellular and extracellular levels of HBsAg were measured by radioimmunoassay in primary culture of hepatocytes from 30 patients with chronic hepatitis B virus infection to determine a possible relationship with hepatitis B virus replication. Serum levels of HBsAg correlated with markers of active viral replication (serum hepatitis B virus DNA, p less than 0.005, and tissue HBcAg, p less than 0.02) but inversely with tissue HBsAg (p less than 0.05). In similar fashion, in vitro export of HBsAg was also related to the presence of active viral replication markers (serum hepatitis B virus DNA, p less than 0.02, and tissue HBcAg, p less than 0.05) and negatively with tissue HBsAg (p less than 0.001). Export of HBeAg also correlated positively with markers of active viral replication (serum hepatitis B virus DNA, p less than 0.05 and tissue HBcAg, p less than 0.05). Further experiments indicated that intrahepatic pre-S1 and pre-S2 correlated closely with intrahepatic HBsAg, indicating that a failure to export HBsAg was unlikely to be attributable to deficient intracellular expression of pre-S1 or pre-S2. These data indicate that in vitro primary hepatocyte culture of hepatitis B virus-infected cells provides an accurate reflection of in vivo export of HBsAg and that this is closely related to the presence of active viral replication.

Effect of interferon-γ on hepatitis B viral antigen expression in primary hepatocyte culture

“…Cells were then counted and resuspended at a concentration of 200,000 cells in 3 ml of medium in Falcon tubes (Becton Dickinson & Co., Rutherford, NJ) and cultured in suspension in the absence or presence of 1 and 10 U/ml of recombinant human IFNy (kind gift of Dr. M. Page, Wellcome Research Laboratory, Beckenham, UK) in a humidified incubator at 37" C for 18 hr. Evaluation by phase-contrast microscopy demonstrated that the isolated hepatocytes prepared using this method retain structural integrity with an intact plasma membrane (14,151. Hepatocyte viability after 18-hr culture also exceeded 80% (range = 82% to 91%) with or without IFNy as assessed by trypan blue dye exclusion and plastic adherence.…”

“…Primary Hepatocyte Culture. The hepatocyte isolation procedure was that described by Trevisan et al (13) with minor modifications (14). The medium used was HEPES-buffered RPMI 1640 media (Gibco Europe, Uxbridge, UK) supplemented with antibiotics (penicillin 200 U/ml, streptomycin 100 kg/ml) and 10% heat-inactivated FBS (Gibco) (pH 7.4, complete medium).…”

“…Intracellular viral antigens were then released by sonicating the cell samples over ice at 10 amplitude microns for 30 sec (Soniprep 150, 23 kHz, MSE, Fisons, Leicestershire, UK). Viral antigens were not destroyed by this treatment (14). The supernatant and the cell sonications were stored at -20" C until tested in batches using commercially available RIAs for HBsAg and HBeAg (Abbott).…”

“…The HBsAg results were quantified by constructing a standard positive curve with a standard positive sample containing 20 n g / d HBsAg. The HBeAg results were expressed with reference to a positive standard supplied with the kit (14). The same HBsAg and HBeAg positive and negative controls were used throughout to minimize interassay variability.…”

“…Zmmunocytochemical Staining. The effect of IFNy on the intracellular distribution of HBV antigen was assessed by immunocytochemical staining (14). Cell smears were prepared using a cytospin (Shandon, Cheshire, UK) to obtain 20,000 cells per slide (which were previously coated with 0.1% poly-L-lysine [Sigma] to enhance cell adhesion), and slides were stored at -20" C before staining.…”