Autoantibodies to Human Asialoglycoprotein Receptor in Autoimmune-type Chronic Hepatitis ULRICH TREICHEL, THOMASPORALLA, GEORGHESS,MICHAEL M A ” S AND KARL-HERMA” MEYERZUM BUSCHENFELDE I . Medizinische Klinik und Poliklinik der Johannes Gutenberg-Universitat Mainz, Federal Republic of Germany
Autoantibodies to the human asialoglycoprotein receptor (anti-h-ASGPR) were studied with a solidphase ELISA in the sera of 421 patients with inflammatory liver diseases, 288 patients with various other disorders and 3 1 controls. Anti-h-ASGPR were found predominantly in autoimmune chronic active hepatitis (44 of 88, 50%) and were closely related to inflammatory activity. In a subpopulation of these patients with untreated, biopsy-proven active disease or relapse, 15 of 17 were positive (88%). In contrast, only 11 of 204 patients (5.3%) with viral hepatitis were anti-h-ASGPR receptorepositive (9 analysis; p < 0.001). We also compared the occurrence of antih-ASGPR with antibodies to rabbit and rat asialoglycoprotein receptors in 352 sera. In contrast to the anti-human asialoglycoprotein receptor antibodies (3 of 107), anti-rabbit- or anti-rat-asialoglycoprotein receptor antibodies were found in 21 and 28 of 107 cases of viral hepatitis, indicating that different epitopes were recognized by these sera. In various other diseases anti-human asialoglycoprotein receptor antibodies were rarely found. Some sera from patients with connective-tissue diseases (8 of 73) and primary or secondary liver malignancies (6 of 55) exhibited anti-h-ASGPR. In autoimmune chronic active hepatitis the presence of anti-human asialoglycoprotein receptors did not correlate to other established autoantibody systems. Thus we conclude that anti-human asialoglycoprotein receptor antibodies can serve as diagnostic markers for inflammatory active cases of autoimmune chronic active hepatitis. Immune reactions to the asialoglycoprotein receptor, which is expressed on the hepatocellular membrane as a liver-specific antigen, might contribute to the pathogenesis of autoimmune chronic 1990;11:606-612.) active hepatitis. (HEPATOLOGY
Received January 9,1989;accepted October 10, 1989. This study was supported by the Deutsche Forschungsgemeinschaft PO-238/1-4. Address reprint requests to: Prof. Dr. Dr. K.-H. Meyer zum Busehenfelde, I. Medizinische Klinik und Poliklinik der Johannes Gutenberg-Universitat Mainz, Langenbeckstrasse I, 6500 Mainz, Federal Republic of Germany. 31/1/18721
Autoimmune phenomena in chronic hepatitis of autoimmune and viral origin are well known, as reviewed recently (1, 2). A number of autoantibodies (antinuclear antibodies (ANA), smooth muscle antibodies (SMA) and the recently described LKM and SLA) are established diagnostic markers for autoimmune chronic active hepatitis (ai-CAH) (3-5).Their target antigens, however, are apparently not expressed on tbe surfaces of hepatocytes and furthermore lack organ specificity. Thus their pathogenic relevance remains questionable. Liver membrane antibodies (LMA), first described by Hopf et al. in 1976 (6), also could not be established as mediators of the disease process because the corresponding antigens are not expressed on viable cells (7). Therefore other components of the hepatocellular plasma membrane were investigated as target candidates for immune reactions leading to hepatocellular injury in ai-CAH. The complex antigen preparation called liver-specific protein (LSP) (8) induced experimental hepatitis in rabbits after long-term immunization (9). Moreover, monoclonal antibodies to a liverspecific membrane component of rabbit LSP were shown to cause hepatocellular damage in uiuo and in uitro (10). The exact characterization of components of LSP with presumed pathogenic relevance in human liver diseases, however, is still the subject of ongoing studies (11-13). Thus the identification of the human asialoglycoprotein receptor (ASGPR) as a component of LSP by McFarlane et al. (14) in 1984 and the demonstration of antibodies reacting with rabbit ASGPR (r-ASGPR) in patients’ sera (15) were of promising importance. Hepatic ASGPR has been investigated in great detail, especially its important functions of hepatic clearance and cell-to-cell contact (16-18). We therefore investigated the occurrence and possible significance of autoantibodies to purified ASGPR of human, rabbit and rat origin in various acute and chronic inflammatory liver diseases, several nonhepatic disorders, malignancies, infectious diseases and normal controls.
606
607
AUTOBODIES TO ASGPR IN AUTOIMMUNE CAH
Vol. 11, No. 4,1990
TABLE1. Patient characteristics Disease
Number
(male/female)
c 20
20-40
> 40 years
Acute viral hepatitis Chronic active B (Dcoinfection) Chronic hepatitis NANB Alcoholic liver disease Autoimmune hepatitis PHC PSC Hemochromatosis Nonhepatic autoimmune disorders" Malignancies (HCC) Infectious diseases (HIV infection) Chronic kidney failure Other chronic diseasesb Normal controls
14 107 (2) 83 59 88 65 2 2 74 55 (4) 30 (13) 77 53 31
8/6 74/33(lil) 51/32 33/26 21/67 9/56 210 210 23/51 34/21(3/1) 19/11(1013) 47/30 31/22 13/18
1 -
12 45 (2) 39 13 49 6 2 23 5 19 (10) 23 10 26
1 62 44 46 35 59
4 -
1 1 1
1
-
2 50 50 (4) 10 (3) 54 43 4
"Rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, systemic vasculitis. bChronic inflammatory bowel diseases, endocrine disorders, heart and pulmonary failure.
MATERIALS AND METHODS Sera. Sera from 709 patients and 31 healthy controls were
included in the study. Sera were taken between 1981 and 1989 and stored at - 20" C. Patient characteristics are summarized in Table 1. Diagnosis of the various liver diseases was based on internationally agreed criteria (19). HBsAg, HBeAg, antiHBsAg and anti-HBeAg were assessed by commercial RIAs (Abbott Laboratories, North Chicago, IL). Non-A, non-B (NANB) hepatitis was diagnosed after exclusion of HBV, hepatitis-A virus, cytomegalovirus, Epstein-Barr virus and herpes infections by appropriate serological tests in the absence of all the autoantibodies mentioned below and history of alcohol abuse. Most of these latter patients had received blood or blood products before contracting hepatitis. None of the patients suffering from viral hepatitis had received immunosuppressive or antiviral therapy. All patients diagnosed as having ai-CAH had autoantibody titers of ANA, SMA, LKM or SLA of at least 1:40. All these patients underwent liver biopsy at the time of diagnosis or later in the course of the disease. Patients with an initial diagnosis of ai-CAH (eight did not receive immunosuppressive therapy before the study), relapse of the disease despite low-dose immunosuppression with prednisone or azathioprine (five patients) or without treatment (four patients) at our institution during the last 2 yr, are summarized separately (Table 2). This is because blood samples for anti-h-ASGPR ELISA and liver biopsy samples were obtained at exactly the same time in this group of patients. Of the remaining 71 ai-CAH patients, 51 were in remission according to biochemical criteria (ALTvalues less than twice normal, y-globulins
Autoantibodies to the human asialoglycoprotein receptor (anti-h-ASGPR) were studied with a solidphase ELISA in the sera of 421 patients with inflammatory liver diseases, 288 patients with various other disorders and 3 1 controls. Anti-h-ASGPR were found predominantly in autoimmune chronic active hepatitis (44 of 88, 50%) and were closely related to inflammatory activity. In a subpopulation of these patients with untreated, biopsy-proven active disease or relapse, 15 of 17 were positive (88%). In contrast, only 11 of 204 patients (5.3%) with viral hepatitis were anti-h-ASGPR receptorepositive (9 analysis; p < 0.001). We also compared the occurrence of antih-ASGPR with antibodies to rabbit and rat asialoglycoprotein receptors in 352 sera. In contrast to the anti-human asialoglycoprotein receptor antibodies (3 of 107), anti-rabbit- or anti-rat-asialoglycoprotein receptor antibodies were found in 21 and 28 of 107 cases of viral hepatitis, indicating that different epitopes were recognized by these sera. In various other diseases anti-human asialoglycoprotein receptor antibodies were rarely found. Some sera from patients with connective-tissue diseases (8 of 73) and primary or secondary liver malignancies (6 of 55) exhibited anti-h-ASGPR. In autoimmune chronic active hepatitis the presence of anti-human asialoglycoprotein receptors did not correlate to other established autoantibody systems. Thus we conclude that anti-human asialoglycoprotein receptor antibodies can serve as diagnostic markers for inflammatory active cases of autoimmune chronic active hepatitis. Immune reactions to the asialoglycoprotein receptor, which is expressed on the hepatocellular membrane as a liver-specific antigen, might contribute to the pathogenesis of autoimmune chronic 1990;11:606-612.) active hepatitis. (HEPATOLOGY
Received January 9,1989;accepted October 10, 1989. This study was supported by the Deutsche Forschungsgemeinschaft PO-238/1-4. Address reprint requests to: Prof. Dr. Dr. K.-H. Meyer zum Busehenfelde, I. Medizinische Klinik und Poliklinik der Johannes Gutenberg-Universitat Mainz, Langenbeckstrasse I, 6500 Mainz, Federal Republic of Germany. 31/1/18721
Autoimmune phenomena in chronic hepatitis of autoimmune and viral origin are well known, as reviewed recently (1, 2). A number of autoantibodies (antinuclear antibodies (ANA), smooth muscle antibodies (SMA) and the recently described LKM and SLA) are established diagnostic markers for autoimmune chronic active hepatitis (ai-CAH) (3-5).Their target antigens, however, are apparently not expressed on tbe surfaces of hepatocytes and furthermore lack organ specificity. Thus their pathogenic relevance remains questionable. Liver membrane antibodies (LMA), first described by Hopf et al. in 1976 (6), also could not be established as mediators of the disease process because the corresponding antigens are not expressed on viable cells (7). Therefore other components of the hepatocellular plasma membrane were investigated as target candidates for immune reactions leading to hepatocellular injury in ai-CAH. The complex antigen preparation called liver-specific protein (LSP) (8) induced experimental hepatitis in rabbits after long-term immunization (9). Moreover, monoclonal antibodies to a liverspecific membrane component of rabbit LSP were shown to cause hepatocellular damage in uiuo and in uitro (10). The exact characterization of components of LSP with presumed pathogenic relevance in human liver diseases, however, is still the subject of ongoing studies (11-13). Thus the identification of the human asialoglycoprotein receptor (ASGPR) as a component of LSP by McFarlane et al. (14) in 1984 and the demonstration of antibodies reacting with rabbit ASGPR (r-ASGPR) in patients’ sera (15) were of promising importance. Hepatic ASGPR has been investigated in great detail, especially its important functions of hepatic clearance and cell-to-cell contact (16-18). We therefore investigated the occurrence and possible significance of autoantibodies to purified ASGPR of human, rabbit and rat origin in various acute and chronic inflammatory liver diseases, several nonhepatic disorders, malignancies, infectious diseases and normal controls.
606
607
AUTOBODIES TO ASGPR IN AUTOIMMUNE CAH
Vol. 11, No. 4,1990
TABLE1. Patient characteristics Disease
Number
(male/female)
c 20
20-40
> 40 years
Acute viral hepatitis Chronic active B (Dcoinfection) Chronic hepatitis NANB Alcoholic liver disease Autoimmune hepatitis PHC PSC Hemochromatosis Nonhepatic autoimmune disorders" Malignancies (HCC) Infectious diseases (HIV infection) Chronic kidney failure Other chronic diseasesb Normal controls
14 107 (2) 83 59 88 65 2 2 74 55 (4) 30 (13) 77 53 31
8/6 74/33(lil) 51/32 33/26 21/67 9/56 210 210 23/51 34/21(3/1) 19/11(1013) 47/30 31/22 13/18
1 -
12 45 (2) 39 13 49 6 2 23 5 19 (10) 23 10 26
1 62 44 46 35 59
4 -
1 1 1
1
-
2 50 50 (4) 10 (3) 54 43 4
"Rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, systemic vasculitis. bChronic inflammatory bowel diseases, endocrine disorders, heart and pulmonary failure.
MATERIALS AND METHODS Sera. Sera from 709 patients and 31 healthy controls were
included in the study. Sera were taken between 1981 and 1989 and stored at - 20" C. Patient characteristics are summarized in Table 1. Diagnosis of the various liver diseases was based on internationally agreed criteria (19). HBsAg, HBeAg, antiHBsAg and anti-HBeAg were assessed by commercial RIAs (Abbott Laboratories, North Chicago, IL). Non-A, non-B (NANB) hepatitis was diagnosed after exclusion of HBV, hepatitis-A virus, cytomegalovirus, Epstein-Barr virus and herpes infections by appropriate serological tests in the absence of all the autoantibodies mentioned below and history of alcohol abuse. Most of these latter patients had received blood or blood products before contracting hepatitis. None of the patients suffering from viral hepatitis had received immunosuppressive or antiviral therapy. All patients diagnosed as having ai-CAH had autoantibody titers of ANA, SMA, LKM or SLA of at least 1:40. All these patients underwent liver biopsy at the time of diagnosis or later in the course of the disease. Patients with an initial diagnosis of ai-CAH (eight did not receive immunosuppressive therapy before the study), relapse of the disease despite low-dose immunosuppression with prednisone or azathioprine (five patients) or without treatment (four patients) at our institution during the last 2 yr, are summarized separately (Table 2). This is because blood samples for anti-h-ASGPR ELISA and liver biopsy samples were obtained at exactly the same time in this group of patients. Of the remaining 71 ai-CAH patients, 51 were in remission according to biochemical criteria (ALTvalues less than twice normal, y-globulins
