Vol. 26, No. 2 Printed in Japan

Gastroenterologia Japonica Copyright 9 1991 by The Japanese Society of Gastroenterology

Detection of hepatitis C virus antibody in patients with autoimmune hepatitis and other chronic liver diseases Morikazu ONJI t, Takashi KIKUCHI 1, Kojiro MICHITAKA l, Izumu SAITO 2, Tatsuo MIYAMURA 2, and Yasuyuki OHTA 1 t Third Department of Internal Medicine, Ehime University School of Medicine, Ehime, Japan; and 2Department of Enteroviruses, National lnstitue of Health, Tokyo, Japan

Summary: To clarify the relationship between autoimmune hepatitis (AIH) and the hepatitis C virus (HCV), we investigated the prevalence of antibodies to HCV (anti-HCV) by an enzyme-linked immunosorbent assay in patients with AIH, primary biliary cirrhosis (PBC), rheumatoid arthritis and multiple myeloma. The antibody was detected in 9 out of 18 patients with AIH (50%), in 3 out of 23 with PBC (23%), in 2 out of 10 with rheumatoid arthritis (20%), and in 5 out of 9 with multiple myeloma (56%). However, the optical density values in these patients were lower than those observed in non-A, non-B hepatitis (NANBH). Anti-HCV became negative immediately after the initiation of glucocorticoid therapy in all four antibodypositive AIH patients tested. The extracted immunoglobulin G fraction from sera of 5 anti-HCV negative AIH patients became positive for the antibody. This phenomenon was not observed in 5 normal volunteer sera. The 9 family members of three anti-HCV positive AIH patients showed no anti-HCV positivity. These results suggest that autoantibodies in AIH patients may cross-react with the HCV -related antigen. Direct association of the HCV influencing the development of AIH is unlikely. Therefore, care should be taken in the evaluation of anti-HCV positivity in patients with autoimmune diseases and multiple myeloma.

Gastroenterol Jpn 1991;26:182-186 Key words:

autoimrnune hepatitis; crossreactivity; hepatitis C virus

Introduction

Viral infection has been considered as one of the possible causes of autoimmune disease ~. For example, hepatitis B virus infection was suggested to be a trigger for autoimmune hepatitis (AIH), although this could not be confirmed by a study in Japan. The genome of a blood borne non-A, nonB hepatitis virus, termed hepatitis C (HCV), was cloned recently and shown to contain a positivestranded RNA molecule 2. An assay for circulating viral antibodies was then developed using an antigen genetically expressed in recombinant yeast clones to capture circulating antibodies 3. The results obtained using this new assay has in-

dicated that HCV is a major cause of transfusionassociated non-A, non-B hepatitis (NANBH) 3. Such hepatitis is much more likely to become chronic than hepatitis B virus and often progresses to chronic liver diseases, including liver cirrhosis 4. It is also thought that HCV may affect immunological functions of hosts, during long persistent infections 4'5. In this paper, we investigated the relationship of AIH and HCV infection. Materials and Methods

Patients and serum samples Eighteen female patients with definite AIH,

Received May 2, 1990. Accepted September 21, 1990. Address for correspondence: Morikazu Onji, M.D., Third Department of Internal Medicine, Ehime University School of Meidicine, Shigcnobu, Ehime 791-02, Japan.

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A n t i - H C V in autoimmune hepatitis

and with a mean age of 55.3+_8.1 years (mean+_ SD; range, 45-76) were investigated. Three of the 18 patients had a history of blood transfusion. The diagnosis was made according to the standard clinical and laboratory criteria proposed by the Subcommittee on Autoimmune Hepatitis. All the serum samples showld raised concentrations of antinuclear antibodies and gammaglobulin. Histological examination of liver biospy specimen confirmed the clinical diagnosis in all cases. As a control group, we also investigated sera fro 90 patients with non-A, non-B chronic hepatitis (NANBH), 23 patients with primary biliary cirrhosis (PBC), 10 with rheumatoid arthritis, and 9 with multiple myeloma. Of 90 patients with N A N B H , 34 had a history of blood transfusion. There were no abnormalities in routine liver function tests in the patients with rheumatoid arthritis and multiple myeloma. Venous blood was obtained for standard liver function tests and HBsAg assay. An aliquot of serum was separated by centrifugation and was kept frozen at -20~ until the assay was performed.

HCV antibody assay The enzyme-linked immunosorbent assay (ELISA; provided by Ortho Diagnostics Japan) was used to detect anti-HCV. The assay uses a recombinant HCV polypeptide encoded by the NS3/4 region of the HCV genome (C100-3) 3. Anti-HCV in the serum binds to the solid-phase C100-3 antigen, and the specifically captured antibody is detected with an antibody-enzyme conjugate consisting of a murine monoclonal antibody against human IgG and the enzyme conjugated with horseradish peroxidase. The cut-off value was determined according to the manufacturer's instructions.

Immunoglobulin G fractionation from serum samples Serum samples from 5 normal volunteers and 5 A I H patients were separated into the immunoglobulin G (IgG) fraction and non-IgG fraction using IgG affinity chromatography (MAbTrap G, Pharmacia LKB Biotechnology, Uppsala

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Sweden). Separated fractions were then frozen, dried, and dissolved in phosphate-buffered saline immediately before the anti-HCV assay.

Detection of anti-HCV in family members of A I H patients Family members (3 husbands and 6 children) from some of the anti-HCV positive A I H patients were investigated for HCV infection by the antiHCV assay. None of them had clinical symptoms of hepatitis.

Anti-liver-kidney microsomal antibody The presence of liver-kidney microsomal antibody (anti-LKM 0 was confirmed by immunofluorescence, using rat liver and kidney cryostat sections and a serum dilution of 1:406.

Statistical analysis The significance of difference was determined using paired Student's t-test or the 22-test, and a value of P<0.05 was considered as significant. Results

The prevalence of anti-HCV in patients with AIH, PBC, N A N B H , rheumatoid arthritis and multiple myeloma is shown in Table 1. We could confirm that most of N A N B H patients had antibody to HCV (72/90:80%). However, 9 out of 18 patients (50%) with AIH, 3 of 23 (13%) with PBC, 2 out of 10 with rheumatoid arthritis, and 5 of 9 (56%) with multiple myeloma were also positive for anti-HCV. The optical density (O.D.) values of anti-HCV positive sera from A I H patients were less than 1.0 in 6 out of 8 cases (75%), whereas those of anti-HCV positive sera from N A N B H patients were more than 2.0 in 60 out of 72 patients (83.3%). The frequency of A I H patients with an O.D. value of <1.0 was significantly higher than in N A N B H (P<0.01). Few patients with PBC or rheumatoid arthritis had anti-HCV but their O.D. values were low. There was significant correlation between the serum IgG concentration and the O.D. values of anti-HCV in 18 patients with AIH (P<0.01). The changes of O.D. values of anti-HCV in

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Table 1 Anti-HCV status in patients with autoimmune diseases, non-A, non-B hepatitis, and multiple myeloma Number of cases tested AIH PBC NANBH RA MM

Percentage of antiHCV positive cases

18 23 90 10 9

50 13 80 20 56

(9) (3) (72) (2) (5)

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(7/9)* (3/3) (6/72)

11 0 8 100 40

(2/5)

_->2.0

(1/9)

11 0 83 0 20

(6/72) (2/2) (2/5)

(1/9) (60/72) (1/5)

Data shows percentage of cases. ( ) shows number of cases. *: Significant difference according to the x 2- test (P<0.001). AIH: autoimmune hepatitis, PBC: primary biliary cirrhosis, NANBH: non-A, non-B hepatitis, RA: rheumatoid arthritis, MM: mutiple myeloma

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four A I H patients during glucocorticoid hormone therapy are shown in Figure 1. Anti-HCV became negative after less than 1 year in all four cases. The typical course of anti-HCV titers and clinical progress during glucocorticoid hormone therapy was observed in the course of a 50-year-old female patient (Fig. 2). Anti-HCV became negative 3 weeks after the initiation of prednisolone administration. Remission of hepatitis was simultaneously observed. The anti-HCV reactivity in the IgG fraction of anti-HCV negative sera from 5 normal volunteers and 5 A I H patients are shown in Figure 3. In specimen from normal volunteers, although antiHCV O.D. values in the IgG fraction were significantly higher than those values. They were all

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within the negative range in the non-IgG fraction. On the other hand, the anti-HCV O.D. values in the i g G fraction were significantly higher than those in the non-IgG fraction in the A I H patients and all were over the cut-off values. There was no significant difference in the mean IgG concentration of 5 patients with AIH (1323+647mg/dl) and that of 5 normal controls (826+194mg/dl). Three husbands and 6 children of three antiHCV positive patients with AIH were negative for anti-HCV. Anti-LKM1 was not detected in any of the 5 A I H patients tested, suggesting that our patients had type 1 AIH.

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A n t i - H C V in autoimmune luTmtitis

April 1991

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Discussion

The present study showed that half of the serum specimens from AIH patients were positive for anti-HCV, and became negative during glucocorticoid hormone therapy. The O.D. values of anti-HCV in these AIH patients were very low compared with those in N A N B H patients. Serum from anti-HCV negative AIH patients became positive after the IgG fraction of serum was assayed. All A I H patients tested were negative for anti-LKM1. Previous reports have indicated that the HCV antibody assay using C100-3 antigen is specific for the diagnosis of N A N A B H . The prevalence of anti-HCV in patients with A I H described here is evidently higher than the 1.2% in Japanese healthy blood donors 7. HCV infection in adulthood can persist for more than ten years and cause chronic liver disease including chronic hepatitis and liver cirrhosis. There are many reports on the relationship between viral infections and autoimmune diseases ~. Epstein-Barr virus infection induced by polypeptides, for example, react with sera from patients with rheumatoid arthritis or systemic lupus erythematosis. There are also some reports of a high prevalence of anti-HCV in AIH patients s. Lenzi et al recently reported that anti-LKMl positive type 2 AIH patients were also positive for anti-HCV 9. Thus the present data, together with previous reports, suggest that there is a possibility that HCV infection influences AIH in some manner. However, it cannot be concluded that HCV infection is a direct cause of

non-I9G fraction

]gG fraction

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Anti-HCV antibody in the IgG fraction of anti-HCV antibody negative sera from normal volunteers and autoimmune hepatitis patients, In the 5 normal volunteers (left figure), anti-HCV antibody in the IgG fraction did not become positive despite an increase in optical density (O.D.). However, anti-HCV antibody became positive in sera from the 5 patients with autoimmune hepatitis (right figure).

AIH, because of the following data regarding anti-HCV: 1) the low O.D. value of anti-HCV in AIH, 2) the disappearance of anti-HCV during glucocorticoid hormone therapy, 3) the high prevalence of anti-HCV in rheumatoid arthritis and multiple myeloma, in which serum IgG usually increases, and 4) the low frequency of antiHCV detection by radioimmunoassay using the same C100-3 antigen. In fact, 4 specimens which were positive for anti-HCV by the ELISA method were negative by the RIA (G. Kuo; personal communication). Patients with autoimmune disease have a number of different circulating autoantibodies. In most cases, antibodies with low titers are attributable to their cross-reactivity. Taken these findings together, we suggest that the observed high prevalence of anti-HCV in AIH patients were from cross-reaction of C100-3 antigen and circulating autoantibodies. Therefore, care must be exercised in the evaluation of anti-HCV in patients with autoimmune diseases and multiple myeloma. In future studies should be performed to attempt the direct detection of the HCV genome in liver tissue of hepatitis patients. These analyses will provide more detailed information about the relationship of HCV and AIH. AIH; autoimmunc hepatitis. PBC; primary bfliary cirrhosis. HCV; hepatitis C virus, anti-HCV; antibody to HCV. NANBH; non-A non-B hcpatitis, anti-LKM~; liver-kidney microsomal antibody type 1. This study was supportcd by the Ministry of Health, Scicncc and Wclfarc of Japan.

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6.

7.

8. 9.

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hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis. N Engl J Med 1989;324:1494-1500 Homberg JC, Abuaf N, Bermard O, et al: Chronic active hepatitis associated with antiliver/kidney microsomal antibody type 1: a second type of "autoimmune'" hepatitis. Hepatology 1987;7: 1333-1339 Katayama T, Kikuchi S, Tanaka Y, et al: Blood screening for non-A, non-B hepatitis by anti-hepatitis C virus antibody assay. Transfusion 1990;30:374-376 Esteeban JI, Esteban R, Viladomiu L, et al: Hepatitis C virus antibodies among risk groups in Spain. Lancet 1989;ii:294-296 Lenzi M, Ballardini G, Fusconi M, et al: Type 2 autoimmune hepatitis and hepatitis C virus infection. Lancet 1990;i:258-259