chuck serum albumin (Rb-WSA), and with control nonimmune guinea pig serum (NGPS) or normal ..... Marshall, JS, Williams S, Jones P, Hepner GW. Serum ...
Complement-Mediated Cytotoxicity and Inhibition of Ligand Binding to Hepatocytes by Woodchuck Hepatitis Virus–Induced Autoantibodies to Asialoglycoprotein Receptor JINGYU DIAO,1 NORMA D. CHURCHILL,1
Hepadnavirus invasion in woodchucks has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediate clearance of desialylated serum proteins. We evaluated the possible pathogenetic importance of anti-ASGPR triggered by woodchuck hepatitis virus (WHV), using anti-ASGPR–reactive serum immunoglobulins (Igs) from five animals with different stages of WHV hepatitis or self-limited WHV infection and isolated woodchuck hepatocytes or HepG2 cells as targets. The results revealed that WHV-induced anti-ASGPR can specifically inhibit asialoglycoprotein recognition by both homologous and heterologous liver cells, as tested in an asialofetuin (ASFN)-binding radioassay. However, the extent of the interference significantly varied (from 85% inhibition to none) for anti-ASGPR with similar titer from different animals, indicating a high degree of heterogeneity in the ASGPR epitope specificity and in the potential biological effects of these autoantibodies. The WHVtriggered anti-ASGPR also induced complement-mediated hepatocytolysis in a microculture tetrazolium (MTT) assay, which ranged from 8.9% 6 0.3% to 33.6% 6 3.6% (mean 6 SD) for different animals and target cell numbers. This cytopathic effect was strictly ASGPR-specific, complementdependent, and was not related to the anti-ASGPR ability to inhibit ligand-hepatocyte binding. Our findings indicate that among pathways by which anti-ASGPR autoimmunity could cause liver damage, hepadnavirus-induced antiASGPR might impair hepatocytes by both disrupting clearance of desialylated proteins and activation of the complement-mediated cytolysis. These cytopathic effects might contribute to the pathogenesis, aggravate severity, and
Abbreviations: anti-ASGPR, antibody to hepatic asialoglycoprotein receptor; WcASGPR, woodchuck ASGPR; WHV, woodchuck hepatitis virus; Igs, immunoglobulins; RbASGPR, rabbit ASGPR; ASFN, asialofetuin; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle medium; MTT, microculture tetrazolium. From 1Molecular Virology and Hepatology Research, Division of Basic Medical Sciences and 2Division of Pathology, Faculty of Medicine, Health Sciences Centre, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada. Received August 12, 1997; accepted February 9, 1998. Supported by a research grant MT-11262 from the Medical Research Council of Canada, Ottawa, Canada (to T.I.M). Ms. Diao was a recipient of a Memorial University Graduate Studentship. Address reprint requests to: Tomasz I. Michalak, M.D., Ph.D., Molecular Virology and Hepatology Research, Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John’s, NFLD, Canada A1B 3V6. Fax: (709) 737-7010. Copyright r 1998 by the American Association for the Study of Liver Diseases. 0270-9139/98/2706-0023$3.00/0
AND
TOMASZ I. MICHALAK1,2
prolong recovery from liver injury in viral hepatitis. (HEPA1998;27:1623-1631.)
TOLOGY
The prevalence of autoantibodies to hepatic asialoglycoprotein receptor (anti-ASGPR) at high frequencies in defined liver disorders, and the fact that they are directed against a molecule essentially unique to hepatocytes, raise an assumption about their relevance to the pathogenesis of liver damage.1 The targeted receptor is a hetero-oligomer of glycoproteins integrated into the hepatocyte plasma membrane that participates in calcium-dependent removal of serum desialylated glycoproteins.2-4 The subunit composition of asialoglycoprotein receptor (ASGPR), its cellular distribution, and function have been relatively well characterized for human and some other species.1-4 In this laboratory, the woodchuck ASGPR (WcASGPR) was identified.5 Its macromolecule contains two polypeptide subunits of 40 and 47 kd (woodchuck hepatic lectin 1 and 2, respectively), which occur at an approximate ratio of 2:1 in the purified receptor preparations. The highest incidence of anti-ASGPR was reported in individuals with active autoimmune hepatitis (about 80%),6,7 chronic liver diseases of undetermined etiology (67%),8 and in patients with varied stages of hepatitis B virus and hepatitis A virus infections (
