Pathogenesis of liver injury : virus-induced autoantibody response against asialoglycoprotein receptor in woodchuck (Marmota Monax) viral hepatitis

Abstract

The purpose of this research was to recognize the induction and pathogenic potential of autoantibodies against a liver-specific asialoglycoprotein receptor (ASGPR) in the course of hepadnavirus infection. Three sections of research work by using a woodchuck model of hepatitis B as an experimental system are presented in this thesis. -- First, woodchuck hepatic ASGPR (wASGPR) was purified and its polypeptide subunit composition, ligand binding properties and antigenicity were characterized. The isolated receptor is a hetero-oligomeric complex constituted of two subunits with molecular masses of 40- and 47-kDa. Antisera were generated in guinea pigs by challenge with the affinity-purified, bioactive wASGPR and rabbit ASGPR (rASGPR) preparations and in woodchucks by immunization with purified rASGPR. With the help of these antisera, a strong antigenic cross-reactivity of the wASGPR polypeptides with the ASGPRs from other species was demonstrated. Further, using antisera raised against isolated rASGPR polypeptides, the distinct antigenic specificity of each of the wASGPR subunits was revealed. Surface expression of wASGPR on the woodchuck hepatocytes was demonstrated by immunofluorescent staining of the isolated cells. Detailed analyses of the purified woodchuck hepatic plasma membranes (HPMs) showed that both subunits of wASGPR were present in the HPM preparations, although the membrane-bound 47-kDa subunit was identifiable only after exposure of HPMs to the ligand of the ASGPR. -- Second, the induction, dynamic patterns and specificity of autoantibodies to hepatic wASGPR were studied in animals with experimental woodchuck hepatitis virus (WHV) infection. Results showed that WHV infection induced anti-ASGPR autoantibodies or led to the rise in their levels in almost all animals experimentally infected with WHV (12 of 13). This demonstrated that the induction of this hepatocyte-specific autoreactivity was a common consequence of WHV infection. The relationship between anti-ASGPR reactivity existing prior to WHV inoculation and the outcome of experimental WHV hepatitis was also investigated. The results revealed that the occurrence of chronic hepatitis was significantly greater in the group of animals with pre-existing anti-ASGPR autoantibodies (54.6%) than that among woodchucks without autoantibodies in preinoculation sera (15.6%, P < 0.05), suggesting that the outcome of WHV infection might be influenced by the status of anti-ASGPR autoreactivity. Western blot analyses showed that WHV-induced anti-ASGPR autoantibodies recognized both 40- and 47-kDa subunits of wASGPR and rASGPR, implying that both receptor polypeptides may serve as targets for the pathogenic immune reactions. Furthermore, no antigenic cross-reactivity between wASGPR and WHV antigens could be identified by using antibodies against ASGPR or WHV envelope, suggesting that mechanisms other than viral protein mimicry could be involved in the induction of anti-ASGPR autoantibody in the course of WHV infection. -- Third, in vitro investigations of the pathogenic effects of the WHV-induced anti-ASGPR autoantibodies showed that these antibodies had the ability to inhibit specific ligand binding to the hepatocyte surface ASGPR. Also, Ig fractions from the same anti-ASGPR reactive sera were hepatocytotoxic in the presence of active complement. These findings indicate that virus-induced autoantibodies against hepatic ASGPR have the potential to directly contribute to liver injury and to the distortion of the hepatocyte clearance of asialoglycoproteins in viral hepatitis. Further careful studies are required to determine the significance of anti-ASGPR autoimmunity in the pathogenesis of liver injury in viral hepatitis. The woodchuck model of virus-induced ASGPR-specific autoimmunity could play an important role in these investigations.