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Frequency and significance of antibodies to P450IID6 protein in Japanese patients with chronic hepatitis C
Journal of Hepatology 1991; 26: 992-1000 Printed in Denmark All rights reserved Munksgaard . Copenhagen
Journal of Hepatology ISSN 0168-8278
protein in Japanese
Mikio Nishioka’,
Syed Ahmed Morshed’, Kazumi Kono ‘, Takashi Himoto’, Keiji Arima’, Seishiro Watanabe’ and Micheal I? Manns2
Salina Parveen’,
‘Third Department of Internal Medicine, Kagawa Medical University, Kagawa, Japan and ‘Department of Gastroenterology and Hepatology, Medizinische Hochschule. Hannover, Germany
BackgroudAims: The aims of the current study were to assess the frequency and the signiilcance of antibodies to cytochrome P45OIID6 protein (antiP45OIID6) in various diseases among Japanese patients. Metlrods: Sera from 541 patients were tested by indirect immunofluorescence, and the specificity of antiP45OIID6 was ascertained by either enzyme immunoassay (ELISA) or V&stern blot using recombinant antigen or rat liver microsomes. Results: Anti-P45OIID6 was found in only 6 of 235 patients (2.6%) with chronic active hepatitis (CAH) positive for hepatitis C virus (HCV) antibody and quantitative HCV-RNA with genotypes II and IV The predominant epitopes on immunoblots were 66 and 50KD, a 1OKD band being the newly undeflned microsomal antigen. Even in the patients negative for autoantibodies to nuclear antigens (ANA) by routine indirect immunofiuorescence test, various ANA were detected by the newly developed recombinant ELISA. These patients were younger, with lower y-globulin and IgG levels than patients with autoimmune hepa-
L
microsome type 1 antibody (antiLKM- 1) predominantly directed against cytochrome P450IID6 is accepted as a marker of autoimmune hepatitis (AIH) type II (l-3). AIH type II has been described mainly in Europe (4-6) and infrequently in the United States (7,8). In Japan, this disease seems to be very rare, although the presence of MB/KIDNEY
Received 21 December 1995; revised 15 October; accepted 6 November 1996
Correspondence: Dr. Mikio Nishioka, MD, Third Department of Internal Medicine, Kagawa Medical School, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa-ken, 76107 Japan. Tel: 81-878-98-5111 Ext 2640/2642. Fax: 81-878-91-0115. e-mail: [email protected].
992
Three of five patients with anti-P4!#OIID6 responded well to interferon therapy and one received prednisone when interferon was ineffective. Interestingly, only this patient was diagnosed as definite autoimmune hepatitis according to the criteria proposed by the International Autoimmune Hepatitis Group (LAHG). The other five patients who did not satisfy the IAHG criteria might be considered as CAH-C with autoimmune features. No autoimmune hepatitis patients positive for anti-P4!5OIID6 were identified in the current study, indicating that the variant is very rare in Japan. Conclusions: Anti-P45OIID6 in CAH-C patients in Japan is not as rare as expected. Anti-P45OIID6 among Japanese patients has uncertain significance and precludes further characterization of CAH-C with autoimmune features, which might require interferon therapy. MS
Key words: Anti-LKMl; Anti-P45OIID6; Autoimmune hepatitis; Hepatitis C; Japan.
either anti-LKMl or antibodies to P450IID6 (antiP450IID6) has been confirmed (9,lO). Recent reports have noted a high rate of antibodies to hepatitis C virus (HCV) in patients considered to have anti-P450IID6 (1 l-l 3). These findings, including amino acid sequence homology between P450IID6 and HCV polyproteins (molecular mimicry), suggest that cross-reactive antibody epitope(s) may be one of the triggering factors in these patients. Therefore, the subclassification of AIH as proposed on the basis of autoantibodies and HCV markers may be helpful for the diagnosis, therapy and prognosis in these patients (2). Unfortunately, the incidence of AIH type II, and the frequency of antGP450IID6 in patients with chronic hepatitis C, and the clinical spectrum, including ther-
Anti-P45OIID6
apy and prognosis, have not yet been well characterized in Japan, or in the rest of Asia. In the present report, we have attempted to assess the frequency and the significance of anti-P450IID6 seropositivity in adult patients with liver diseases. Characteristics of the patients positive for anti-P450IID6, including its predominant antibody epitopes, were analyzed in detail. AntiP450IID6 reactive sera were also investigated for the presence of ANA subtypes, using a battery of recombinant nuclear antigens in the ELISA tests.
Materials and Methods Study populations Serum samples were collected from 32 adult patients with acute viral hepatitis, 319 with chronic viral hepatitis, 20 with AIH, 10 with cryptogenic hepatitis, 5 with drug-induced hepatitis, 35 with primary biliary cirrhosis, 25 with liver cirrhosis, 95 with non-liver diseases (i.e., gallbladder stone, pancreatitis, chronic gastritis, peptic ulcer, nephritis, and collagen diseases) including 15 systemic lupus erythematosus, and 45 healthy blood donors serologically negative for viral markers. Four of six (66.6%) chronic active hepatitis (CAH) C patients positive for anti-P450IID6 had concurrent immunologic disease such as chronic thyroiditis. Four of 19 (21%) patients with AIH had concurrent immunological disease and two (10.5%) of them had chronic thyroiditis. Three of 50 (6%) CAH-C patients without anti-P450IID6 had concurrent immunologic disease, two (4%) with chronic thyroiditis. Diagnoses of AIH patients were done according to the criteria proposed by the International Autoimmune Hepatitis Group Scoring System (14). The European classification for liver histology was used to distinguish the characteristics of chronic liver diseases (15). CAH was categorized by chronic inflammatory infiltration involving portal tracts and extending into the parenchyma, with piecemeal necrosis and formation of interlobular septa. The architecture was destroyed but there was no nodular regeneration. Activity, seen as piecemeal necrosis and inflammation, varied from moderate (CAH-2A) to severe (CAH-2B). Informed consent was obtained from all patients who participated in the present study. Virological assessments Patients with acute and chronic viral hepatitis who satisfied the clinical, biochemical and histological criteria for hepatitis were diagnosed by the presence of virological and epidemiological markers. Seventy-four patients had hepatitis B (HBV) antigen (Abbott Laboratories, North Chicago, Illinois, USA). Fifty-one (68.9%) of these patients had histological criteria for CAH. Ten patients were seropositive for antibodies to
in Japanese AIH patients
hepatitis 6 virus (HDV) antigen (Abbott Laboratories, North Chicago, Illinois, USA). Two hundred and thirty-five patients had serological markers of HCV infection by second-generation enzyme immunoassay (anti-HCV-ELISA II, Ortho Diagnostic System, Inc., Raritan, New Jersey). HCV-RNA was detected by twostage polymerase chain reaction with two pairs of primers deduced from the 5’-non-coding region (16). HCV genotyping and the relative copy numbers were established according to previously described methods (17,18). One hundred and one (43%) of these HCVinfected patients showed CAH in their histologic findings. Identzjication of autoantibodies Anti-LKMl was detected by indirect immunofluorescence (IIF) using frozen sections of the kidney and the liver taken from rats (1). Sera were considered positive for anti-LKMl when they reacted at a minimal dilution of 1:40 with the cytoplasm of hepatocytes and proximal renal tubules of the kidney and with no staining of the distal renal tubules. In sera positive for antiLKM, Western blot assay was carried out using either P450IID6 fusion protein or purified rat liver microsomes (13). Autoantibodies to P450IID6, soluble liver antigen (SLA), pyruvate dehydrogenase-E2 subunit (PDH-E2) and branched-chain oxoacid dehydrogenase E2 subunit (BCKD-E2) were detected by ELISA, which determined whether test serum inhibited the reaction of reference serum. The target antigens fixed to the ELISA plates were microsomes (50KD, P450IID6), supernatant from rabbit liver homogenates (SLA), and mitochondrial (70KD PDH-E2 or 50KD BCKD-E2) proteins (13,19-21). ANA was detected by an IIF test using cultured human laryngeal tumor (HEp-2) cells as a target. Antibodies to smooth muscle (SMA), parietal cells (PCA), and mitochondrial antigens (AMA) were tested by the same method as ANA on rat kidney and stomach. Antibodies to nuclear antigens such as Cenp-B, U 1RNP-A, U 1RNP-70K, Ul RNP-B’/B, SS-A/Ro (52K and 6OK), SS-B/La, and Ki were detected using a battery of recombinant nuclear antigens in ELISA tests as previously described (22-26). Detection of antibodies to thyroid microsome and thyroglobulin in sera as performed using artificial gelatin particle carriers sensitized with either thyroid microsome or thyroglobulin, extracted and purified from human thyroid tissue, by the indirect agglutination test according to the instructions of the SERODIA-AMC kit (Fujirenbio Inc., Tokyo, Japan). Antibodies to both single-stranded DNA and double-stranded DNA were also investi993
M. Nishioka et al.
TABLE 1
Results
Prevelance of antibody to P450IID6 protein (anti-LKMl) diseases and controls Diagnosis
No. of patients
No. of anti-P450IID6 reactive patients (%)
Acute hepatitis Chronic viral hepatitis Hepatitis B virus (HBV) Hepatitis C virus (HCV) Hepatitis 6 virus (HDV) Total no. Autoimmune hepatitis Cryptogenic hepatitis Drug-induced hepatitis Primary biliary cirrhosis Liver cirrhosis Non-liver diseases Healthy blood donors
74 235 10 319 20 10 5 35 25 95 45
0 6 (2.6%) 0 6 (1.9%) 0 0 0 0 0 0 0
Total
586
6 (1%)
32
gated in these sera by commercially kits (MBL, Nagoya, Japan).
in various
0
available ELISA
Human leukocyte antigen (HLA) typing Patients positive for anti-P450IID6 and 25 of 50 CAHC patients without anti-P450IID6 were tested for HLA class I and II antigens. A standard microlymphocyte cytotoxicity assay was used as described elsewhere (27). Interferon therapy Five of six patients with CAH positive for antiP450IID6 were treated with human natural interferon (IFN-cr 2a, 6MU, three times weekly for 24 weeks) as recommended by the Ministry of Health and Welfare of Japan. Three had complete and one each had partial and no response to the therapy. Complete response was defined as the normalization of transaminase levels, HCV-RNA disappearance and improvement of histology; partial response indicates a decrease but no normalization of transaminase levels and low HCVRNA copies without histological improvement; and no response implies no changes in transaminase levels and sustained HCV-RNA copies without histological improvement. Statistical analysis Results were expressed as mean+SD (standard deviation). Statistical analysis was performed on a Power Macintosh using StatView 4 software. The MannWhitney test for continuous variables and Fisher’s exact test for dichotomous variables were used between various groups to examine the significant of differences, with p=O.O5 as the minimum level of significance. 994
Frequency of anti-P450IID6 Anti-P450IID6 was found in six of 3 19 patients (1.9%) with chronic viral hepatitis. None of 222 patients with acute hepatitis, primary biliary cirrhosis, liver cirrhosis, gallbladder stone, pancreatitis, chronic gastritis, peptic ulcer, nephritis, collagen diseases and 45 healthy blood donors was seropositive for anti-P450IID6 (Table 1). Anti-P45OIID6 in chronic hepatitis C Six of 235 patients (2.6%) with chronic hepatitis C were seropositive for anti-P450IID6 by both ELISA and immunoblot methods. Sera reactive for anti-P450IID6 on fusion peptide by immunoblot also reacted with purified rat liver microsomes, and recognized at least one band in each patient (KJ: 66 & 50KD; KK: 66KD; WT: 50KD; FK: 10KD; TK: 10KD; YO: 50KD). No sera recognized other corresponding bands such as 58 and 39KD. None of 74 patients with HBV and 10 patients with HDV was seropositive for anti-P450IID6 (Table 1). Autoantibody profiles in anti-P450ZZD6 reactive patients Three patients (KK, WT and FK) had anti-LKMl titers of 180 and others (KJ, TK, YO) had anti-LKMl titers of 1: 160 (Table 2). All of them have been diagnosed as CAH-C. All sera positive for anti-LKMl were also reactive to P450IID6 protein by ELISA. The presence of anti-P450IID6 in these sera was confirmed by immunoblot using recombinant P450IID6 protein (11) (figure not shown). None of these 6 sera was reactive to PDH-E2 or BCKD-E2. ANA was detected in five patients at a low titer of 20-80 times dilution by IIE Five of six anti-P450IID6 reactive patients had antibodies to several nuclear antigens, including UlRNPA, UlRNP-70K, UlRNP-B’/B, Cenp-B, 60KSS-A/Ro, and SS-B/La. None of them demonstrated any reactivities to either 52KSS-A/Ro or Ki nuclear antigens by the recombinant ELISA tests. All of them were also non-reactive to double-stranded or single-stranded DNA. One patient (YO) was nonreactive for ANA by both IIF and ELISA. Only one patient (WT) was seropositive for smooth muscle at 1:80 dilution by IIE Five patients had antibodies to thyroid microsome and four had antibodies to thyroglobulin. None of 6 sera had antibody reactivities to SLA, mitochondria nor parietal cell antigens (Table 2) by ELISA or IIE Characteristics of anti-P450IID6 reactive patients All six patients with anti-P450IID6 were adults (Table 3). Four were female and two were male. They had had
Anti-P45OIIDb in Japanese AIHpatients TABLE 2 Autoantibody profiles in patients with chronic active hepatitis C(CAH-C) positive for antibodies to P450IID6 protein Autoantibodies*
CAH-C positive for anti-P450IID6 KJ
KK
WT
FK
TK
YO
Anti-LKMI (IIF) Anti-Microsomes (WB:KD) AntGP450IID6 (WB:SOKD) Anti-P450IID6 (ELISA)# Anti-PDH-E2 (ELISA+WB) Anti-BCKD-E2 (ELISA+ WB) Anti-SLA (ELISA)
1:160 66, 50 + 90% -
1:80 66 + 57% _ -
1:80 50 + 45%
1:80 10 + 41% -
1:160 10 + 63% _ -
1:160 50 + 68% _
Anti-nuclear antibody (ANA-IIF) ANA subtypes (ELISA): UlRNP-A UIRNP-70K UlRNP-B’/B CENP-B 52K-SSAJRo 60K-SSAfRo SS-B/La Ki
1:80
1:80
I:40
1:4O
1:40
I:20
+ + + + + -
+ + -
+ + -
-
+ -
+ _ -
Anti-ssDNA (single-stranded) Anti-dsDNA (double-stranded)
-
-
-
-
Rheumatoid (RA) factor
+
-
-
-
Anti-SMA Anti-PCA Anti-thyroid microsome Anti-thyroglobulin
+ +
1:80 + +
+ -
+ +
-
* LKM: Liver/kidney microsome; IIF: Indirect immunofluorescent test; WB: Western blot; ELISA: Enzyme-linked immunosorbent assay; Antibodies to mitochondria (PDH-E2 and BCKD-EZ), soluble liver antigen @LA), smooth muscle antigen @MA), and parietal cell antigen (PCA). x Inhibition >40% selected as positive reactivity.
a long duration of illness (range: 2 to 9 years), and four of them had concurrent immunologic disease such as chronic thyroiditis. Five of them underwent interferon (IFN-o) trial. All six patients were positive for anti-HCV and HCV RNA. Five patients showed HCV genotype II (copy numbers ranged between 1. 104-1. 101o/ml, mean 1.107.“‘.‘) and the other (KK) genotype IV with viral copies of 1. 104/ml. They had high serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at presentation. One patient (KJ) had a higher level of serum y-globulin than the other five patients. There were some differences in laboratory findings between these six patients and 50 patients with CAH-C negative for anti-P450IID6 (Table 4). Patients positive for anti-P450IID6 were younger at presentation, without significant difference. Serum y-globulin and IgG levels were significantly less in AIH or CAHC negative for anti-P450IID6. Serum AST, y-globulin, IgG, IgM, IgA, HLA-DR4 distribution, and histopathological index (CAH-2B) were significantly higher in AIH patients than that of CAH-C without antiP450IID6. Serum ALT, AST, y-globulin, and IgG were significantly lower in CAH-C without anti-P450IID6,
as compared to CAH-C with anti-P450IID6 (Table 4). On histological analysis, FK was diagnosed as CAH-2B and the others were CAH-2A according to the European classification (15). One of these patients had a high score on the histological activity index (HAI) (28). All six LKMl-reactive patients were evaluated by IAHG scoring (14). One of the patients (WI) was diagnosed with definite AIH, because the aggregate scores were greater than 15. The other five did not have definite AIH by IAHG scoring. HLA typing of anti-P450IID6 reactive patients None of the patients showed positive reactivities for HLA-DR3. DR4, a characteristic HLA type found in Japanese prototype AIH patients (29), was detected in three of six (50%) patients. Outcome of treatment regimens Five patients were treated with human natural interferon-a (IFN-a). Three patients (KJ, YO and KK) are now in remission and are negative for HCV-RNA. Patient (KK) had been diagnosed as having chronic thyroiditis before IFN therapy and developed destructive thyroiditis after therapy, although she showed a com995
M. Nishioka
et cd.
TABLE 3 Characteristics of chronic active hepatitis-C (CAH-C) patients with antibodies to P450IID6 protein at presentation Findings*
CAH-C patients positive for anti-P450IID6 KJ
Clinical features: Age (years)/sex 41/F Duration of illness (years) 8 Concurrent immunologic disease chronic thyroiditis Laboratory data: + Anti-HCV-II HCV-RNA (copies/ml) 1.10’ HCV-genotype II ALT (n<50 U/l) 80 AST (n<33 U/l) 48 y-globulin (n< 1.5 g/dl) 2.2 IgG (n< 1682 mg/dl) 2730 IgM (n<283 mg/dl) 322 IgA (n<340 mg/dl) 372 HLA-DR3IDR4 -lHistology: European types (CAH)** 2A HA1 score 6 IAHG a) Required parameters 12 b) Additional parameters 2 Interferon therapy CR
KK
WT
29/F 2 chronic thyroiditis
63/M 4 chronic thyroiditis
+ 1.104 IV 126 56 1.9 2290 218 257 -i+
+ 1.10’ II 261 113 1.8 2540 12 500 -i-
2A 4
2A 5
9 4 CR
13 3 NR
FK 22/M I -
TK
YO
62/F 9 chronic thyroiditis
+ + 1.10’0 1.108 II II 680 260 230 130 1.9 2.0 2310 2510 180 292 203 336 -i+ -/2B 15 10 4 PR
33/F 1 + 1.10’ II 230 140 1.5 2080 78 172 -i+
2A 10
2A 4
10 4 NT”
9 4 CR
* HCV: Hepatitis C virus; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HAI: Histologic activity score; IAHG: International Autoimmune Hepatitis Group; ** CAH: Chronic inflammatory infiltration involving portal tracts and extending into the parenchyma, with piecemeal necrosis moderate (CAH-2A) to severe (CAH-2B and formation of intralobular septa. CR: Complete response; NR: No response; PR: Partial response. t NT: Not treated
plete response to the therapy. Two patients in CAH-C without anti-P450IID6 had chronic thyroiditis; they had not received any therapy. One young male patient (FK, 22 years old) developed rapid progressive liver damage and ultimately liver cirrhosis. He had received a blood transfusion because of a severe traffic accident at the age of 22. Three months later, he was diagnosed as having posttransfusion hepatitis. He underwent intermittent IFN-a therapy for 2 years after the findings of fluctuating transaminase levels. Initially the patient showed a good response to IFN, but transaminase levels rose again when IFN therapy was withdrawn. HCV-RNA was persistently positive in his serum. Histopathology of the liver biopsy performed at the age of 26 demonstrated liver cirrhosis. Why the patient developed liver cirrhosis so quickly even at a young age is unclear, but he might have autoimmune liver destruction as seen in AIH patients. Interestingly, he showed HLA-DR4 positivity. In patient WI, who had definite AIH by IAHG scoring, serum ALT and AST levels increased rapidly after initiation of IFN therapy. One week later, ALT level increased four-fold and the IFN was withdrawn and replaced by steroids. The patient responded quickly, as shown by falling transaminase levels in his serum. Careful monitoring during therapy and regular 996
follow-up after therapy seem important with these variants.
in
patients
Discussion Our present study confirmed the presence of antiP450IID6 in patients with liver diseases, because the antibody was absent in diseases other than liver diseases, such as pancreatitis, chronic gastritis, peptic ulcer, nephritis and collagen diseases. The specificity of anti-P450IID6 was ascertained by ELISA, using purified microsomal protein and by immunoblot assay with recombinant P450IID6 (50 KD) or microsomes. Anti-P450IID6 was detectable in only six of 235 patients (2.6%) with chronic hepatitis C. The incidence of anti-P450IID6 in chronic HCV patients is not as low as our previous report suggested (9), although antiP450IID6 does not appear to be a common sequela of HCV infection (3). The seropositivity in patients with chronic hepatitis C appears to be different in various countries, and previous observations indicated a geographical difference in the frequency of HCV markers in patients with anti-P450IID6 (so-called AIH type IIb) @X,30-34). A high incidence of anti-P450IID6 in high HCV endemic areas in various countries suggests an association of HCV infection in patients positive for anti-P450IID6, as indicated by Vergani et al. (33).
Anti-P450IID6 in Japanese AIH patients TABLE 4 Comparison P450IID6
of characterestic findings between autoimmune hepatitis (AIH), and chronic active hepatitis C (CAH-C) with or without antiFindings*
Clinical features: Age (years) Sex (F/M) Immunologic disease Laboratory data: Anti-HCV-ELISA II (%) HCV-RNA (copies/ml) HCV genotypes (%) I II III IV mixed ALT (n<50 U/l) AST (n<33 VA) y-globulin (n< 1.5 g/d]) IgG (n< 1682 mg/dl) IgM (n<283 mg/dl) Ig,A (n<340 mg/dl)
AIH without anti-P450IID6 @,=19) p
CAH-C without anti-P4501ID6 (n=SO) P J
CAH-c with anti-P450IID6 (n=6)
--p-
56.82 15.7 NS 17f2 NS 4 NS
41.6217.2 412 4
NS NS NS
54.1~14.3 31/19 3
NS NS NS
NS NS
6(100) 1 lO,.“‘.s
NS NS
50 (100)
NS
-
221.42246 NS 238.35266 NS 3.020.8
NS
0
0
-
1.107.4’1.7
5 (83) 0 1 (16) 0 273.62210 120.3274 1.820.1 2355.32203 193.62 104 306.62121
NS NS NS NS <0.007 dO.05 CO.04 <0.009 NS NS
23/35 (65.7) 6135 (17.1) 6/35 (17.1) 3/35 (8.5) 110.1289 68.8241 1.620.3 2049.6?454 165.2?94 271.6’_111
NS NS NS NS NS
HLA-typing (%) DR3 DR4
O/l7 12/17 (70)
NS NS
0
NS
316 (50)
NS
Histology of liver European classification (%) CAH-2A CAH-2B
4 (21) 15 (79)
co.059
4 (66) 1 (16)
NS NS
41 (82) 9 (18)
Interferon-a therapy (%)’ Complete response Partial response No response
-
3 (60) 1 (20) 1 (20)
NS NS
lb25 (44) 6/25 (24) 9/25 (36)
NS NS
Steroid therapy (%) Complete response Partial response No response
13/16 2Jl6 1116
O/25 9125 (36)
NS CO.057
NS -
* HCV: Hepatitis C virus; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HAL Histologic activity score; ’ Complete response is defined as the normalization of transaminase levels with HCV-RNA disappearance in patients; partial response indicates decrease but no normalization of transaminase levels with sustained HCV-RNA; no response is no change in transaminase levels and presence of HCV-RNA continues. Statistical significant differences (p-value) were calculated by Mann-Whitney test for continuous variables and Fisher’s exact test for dichotomous variables.
Our study of immunoblot with rat liver microsome showed heterogeneity of anti-LKMl recognition with bands of 66KD and 1OKD; a 5OKD band was frequently observed. Recently, Muratori et al. (35) observed antibodies to 66KD, 58KD, 50KD and 39KD antigens in rat/human microsomes in patients with HCV infection positive for anti-CYP2D6 or antiP450IID6. Unfortunately, none of our sera recognized 58KD or 39KD peptide; instead they recognized a 1OKD band as the predominant antigen. It seems that antibody to 1OKD is a newly undefined antigen or a degradation product of common epitopes of P450 proteins in liver microsomes. Because the autoimmune re-
sponse to P450IID6 is directed to both linear and conformational antigenic sites (36), fine epitope mapping would be more feasible with overlapping short synthetic peptides using either ELISA or the immunoblot test. However, it is clear that the main target antigens in HCV patients with anti-P450IID6 are heterogeneous. Further study might reveal the molecular identity of the new antigens. Six patients with CAH-C positive for anti-P450IID6 in the present study satisfy the definition of “antiLKMl CAH or autoimmune hepatitis type II”, as first described by Homberg et al. (1). One (KI) of the six patients was diagnosed clinically as AIH before the de997
M. Nishioka et al
tection of anti-P450IID6 was carried out. Again, this patient was diagnosed as chronic hepatitis C, instead of AIH, because of: low titers of ANA; laboratory findings at presentation similar to two patients (WT and TK) with chronic hepatitis C positive for antiP450IID6; absence of the HLA-DR4 antigen that is considered to be a prototype HLA in Japanese AIH (29); and a good response to interferon therapy. Previous study reported that patients positive for anti-P450IID6 could be divided into two groups according to their positive HCV markers (2). All six patients in this study were considered as AIH type IIb as they showed a low titer of anti-P450IID6 and moderately increased levels of serum y-globulin. Although statistical comparisons are limited by small numbers, there were some significant differences in laboratory findings compared to either those with CAH-C or AIH patients negative for anti-P450IID6. Patients with antiP450IID6 had significantly lower y-globulin and IgG levels in their sera. It is interesting to know the genotypes of the HCV coding region as well as HCV copy numbers in these six patients. Five had genotype II and the other had genotype IV HCV infection with genotype II may be a dominant type for developing anti-P450IID6 (37), although HCV genotype II was found in almost 70% of CAN-C patients in our study population. In comparison to serum AST, ALT, y-globulin and IgG levels (Table 4), patients with CAH-C positive for antiP450IID6 were different from CAH-C without antiP450IID6. However, the present study clearly indicated that there were no obvious differences in HCV genotypes, viral copies and HLA phenotyping, between patients with or without anti-P450IID6, although few patients were examined. Our clinical and virological findings in six patients positive for anti-P450IID6 suggest that they belong to the patient group CAH-C. Incorporating the new IAHG criteria for diagnosis (14), only one patient (WT) in the current study could be considered as having definite AIH; this patient scored more than 15 points. The patient did not respond to IFN therapy from its counterpart, suggesting patients with a different variant reactive to antiP450IID6 might require an initial steroid treatment (for the improvement of liver function), followed by a further treatment with IFN (to kill persistent HCV infection). Future studies on double-therapy, such as steroid initially and IFN later, in this patient group are required to determine how this therapeutic regimen can help to eradicate both autoimmunity and HCV infections. A possible association between HCV infection and thyroiditis is intriguing. Chronic thyroiditis, a frequent
complication of AIH patients, was detected in patients with CAH-C positive for anti-P450IID6. Four of these six (66%) patients had chronic thyroiditis and one of them developed destructive thyroiditis during IFN therapy. Homberg et al. (1) also reported a high incidence of autoimmune thyroiditis in patients with antiP450IID6. Pateron et al. (38) found anti-thyroid antibodies in nine patients with chronic hepatitis C, and two patients developed overt thyroid dysfunction during IFN therapy. In our study, chronic thyroiditis seems to be the predominant concurrent immunologic disease among patients with AIH and CAH-C with or without anti-P450IID6. Chronic thyroiditis in CAHC patients with anti-P450IID6 may be a characteristic concurrent immunologic disease. Further study is needed to establish a temporal association between chronic thyroiditis and hepatitis C virus infection. In this study, anti-P450IID6-reactive patients showed autoantibodies to nuclear antigens by recombinant ELISA tests. Antibodies to UlRNP-70K were common in these patients. These autoantibodies usually occur among a selected group of patients with connective tissue diseases (39). Interestingly, the target antigen is cross-reactive with retroviral p30gag antigen (39). Furthermore, retesting of these sera for ANA subtypes after either IFN or steroid therapy revealed decreasing titers, and one patient (KK) showed ANA subtype titers below the cut-off values. These findings shed a new light on ANA behavior associated with HCV infection. The presence of various ANA subtypes has uncertain significance in these patients and may reflect the nature of the autoimmune phenomena. Finally, hosts rather than viral factors seem to be responsible for triggering an aberrant immune response in CAH-C (40).
References 1. Homberg JC, Abuaf N, Bernard 0, Islam S, Alvarez E Khalil SH, Poupon R, Darnis F, Levy VG, Grippon P Opolon P Bernuau J, Benhamou JP, Alagille D. Chronic active hepatitis associated with anti-liver/kidney microsome antibody type 1: a second type of “autoimmune” hepatitis. Hepatology 1987; 7: 1333-9. 2. Michel G, Ritter A, Gerken G, Meyer zum Biischenfelde KH, Decker R, Manns MI? Anti-GOR and hepatitis C virus in autoimmune liver diseases. Lancet 1992; 339: 267-9. 3. Lunel E Abuaf N, Frangeul L, Grippon E Perrin M, Le-Coz y, Valla D, Borotto E, Yamamoto AM, Huraux JM, Opolon P, Homberg JC. Liver/kidney microsome antibody type 1 and hepatitis C virus infection. Hepatology 1992; 16: 630-6. 4. Manns MF! Relevance of cytoplasmic autoantigens in autoimmune hepatitis. In: Nishioka M, Toda G, Zeniya M, eds. Autoimmune Hepatitis. Amsterdam: Elsevier, 1994: 16575. 5. Maggiore G, Bernard 0, Homberg JC, Hadchouel M, Alvarez F, Hadchouel P, Odievre M, Alagille D. Liver disease as-
Anti-P450IID6 sociated with anti-liver-kidney microsome antibody in children. J Pediatr 1986; 108: 399-404. 6. Manns MP Meyer zum Biischenfelde K-H. Nature of autoantigens and autoantibodies in autoimmune hepatitis. Springer Semin Immunopathol 1990; 12: 57-65. 7. Manns MI? Cytoplasmic autoantigens in autoimmune hepatitis: molecular analysis and clinical relevance. Semin Liver Dis 1991; 11: 205-14. 8. Czaja AJ, Manns MP Homberger HA . Frequency and significance of antibodies to liver/kidney microsome type 1 in adults with chronic active hepatitis. Gastroenterology 1992; 103: 1290-s. 9. Nishioka M, Morshed SA. Recent aspects of autoimmune hepatitis. In: Nishioka M, Toda G, Zeniya M, eds. Autoimmune Hepatitis. Amsterdam: Elsevier, 1994: 1319-36. 10. Miyakawa H, Kako M, Nagai K, Hino K, Iwabuchi S, Miyachi K, Okamoto H, Mayumi M. HCV RNA in type 2 autoimmune hepatitis. Am J Gastroenterol 1991; 86: 1688-g. 11. Lenzi M, Ballardini G, Fusconi M, Cassani F, Selleri L, Volta V, Zauli D, Bianchi FB. Type 2 autoimmune hepatitis and hepatitis C virus infection. Lancet 1990; 335: 258-9. 12. Todros L, Touscoz G, D’Urso N, Durazzo M, Albano E, Poli G, Baldi M, Rizzetto M. Hepatitis C virus-related chronic liver disease with autoantibodies to liver/kidney microsomes (LKM). Clinical characterization from idiopathic LKMpositive disorders. J Hepatol 1991; 13: 128-31. 13. Manns MP Griffin KJ, Sullivan KF, Johnson EE LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase. J Clin Invest 1991; 88: 1370-8. 14. Johnson PJ, McFarlane IG, Convenors, on behalf of the panel. Meeting report: International Autoimmune Hepatitis Group. Hepatology 1993; 18: 998-1005. 15. De Groote J, Desmet VJ, Gedigk P Korb G, Popper H, Poulsen H, Scheuer PJ, S&mid M, Thaler H, Uehlinger E, Wepler W. A classification of chronic hepatitis. Lancet 1968; ii: 626-8. 16. Okamoto H, Okada S, Sugiyama Y, Tanaka T, Sugai Y, Akahane Y, Machida A, Mishiro S, Yoshizawa H, Miyakawa Y, Mayumi M. Detection of hepatitis C virus RNA by a twostage polymerase chain reaction with two pairs of primers deduced from the 5’-noncoding region. Jpn J Exp Med 1990; 60: 215-22. 17. Okamoto H, Sugiyama Y, Okada S, Kurai K, Akahane Y, Sugai Y, Tanaka T, Sato K, Tsuda F, Miyakawa Y, Mayumi M. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J Gen Virol 1992; 73: 673-9. 18. Hagiwara H, Hayashi N, Mita E, Takehara T, Kasahara A, Fusamoto H, Kamada T. Quantitative analysis of hepatitis C virus RNA in serum during interferon alfa therapy. Gastroenterology 1993; 104: 877-83. 19. Manns M, Gerken G, Kyriatsoulis A, Staritz M, Meyer zum Btischenfelde K-H. Characterization of a new subgroup of autoimmune chronic active hepatitis by autoantibodies against a soluble liver antigen. Lancet 1987; i: 924. 20. Yoshida T, Bonkovsky H, Ansari A, Danner D. Antibodies against mitochondrial dehydrogenase complexes in primary biliary cirrhosis. Gastroenterology 1990; 99: 187-94. 21. Zurgil N, Bakimer R, Kaplan M, Youinou P Shoenfeld Y. Anti-pyruvate dehydrogenase autoantibodies in primary biliary cirrhosis. J Clin Immunol 1991; 11: 23945.
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22. Nishioka M. Nuclear antigens in autoimmune hepatitis. In: Meyer zum Bttschenfelde K-H, Hoofnagle J, Manns MP, eds. Immunology and Liver. Dordrecht: Kluwer Academic, 1992: 193-205. 23. Czaja A, Nishioka M, Morshed SA, Hachiya T. Patterns of nuclear immunofluorescence and reactivities to recombinant nuclear antigens in autoimmune hepatitis. Gastroenterology 1994; 107: 20&7. 24. Lutz-Freyermuth C, Query CC, Keene JD. Quantitative determination that one of two potential RNA-binding domains of the A protein component of the Ul small nuclear ribonucleoprotein complex binds with high affinity to stem-loop II of UlRNA. Proc Nat1 Acad Sci USA 1990; 87: 6393-7. 25. Nikaido T, Shimada K, Shibata M, Hata M, Sakamoto M, Takasaki Y, Sato C, Takahashi T, Nishida Y. Cloning and nucleotide sequence of cDNA for Ki antigen, a highly conserved nuclear protein detected with sera from patients with systemic lupus erythematosus. Clin Exp Immunol 1990; 79: 209-14. 26. Deutscher SL, Harley JB, Keene JD. Molecular analysis of the 60-kDa human ribonucleoprotein. Proc Nat1 Acad Sci USA 1988; 85: 9479-83. 27. Terasaki PI, Bernoco D, Park MS, Ozturk G, Iwaki Y. Microdroplet testing for HLA-A, -B, -C and -D antigens. Am J Clin Path01 1978; 69: 103-20. 28. Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan Tw, Wollman J. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1981; 1: 431-5. 29. Seki T, Kiyosawa K, Inoko H, Ota M. Association of autoimmune hepatitis with HLA-Bw54 and DR4 in Japanese patients. Hepatology 1990; 12: 1300-4. 30. Lenzi M, Johnson PJ, McFarlane IG, Ballardini G, Smith HM, McFarlane BM, Bridger C, Vergani D, Bianchi FB, Williams R. Antibodies to hepatitis C virus in autoimmune liver disease: evidence for geographical heterogeneity Lancet 1991; 338: 277-80. 31. Czaja AJ, Carpenter HA, Manns Ml? Antibodies to soluble liver antigen, P450IID6, and mitochondrial complexes in chronic hepatitis. Gastroenterology 1993; 105: 1522-8. 32. Fried MW, Draguesku JO, Shindo M, Simpson LH, Banks SM, Hoofnagle JH, Di-Bisceglie AM. Clinical and serological differentiation of autoimmune and hepatitis C virus-related chronic hepatitis. Dig Dis Sci 1993; 38: 631-6. 33. Vergani D, Mieli-Vergani G. Type II autoimmune hepatitis. What is the role of the hepatitis C virus ? Gastroenterology 1993; 104: 1762-7. 34. Abuaf N, Lunel E Giral P Borotto E, Laperche S, Poupon R, Opolon P Huraux JM, Homberg JC. Non-organ specific autoantibodies associated with chronic C virus hepatitis. J Hepatol 1993; 18: 359-64. 35. Muratori L, Lenzi M, Ma Y, Cataleta M, Mieli-Vergani G, Vergani D, Bianchi FB. Heterogeneity of liver/kidney microsomal antibody type I in autoimmune hepatitis and hepatitis C virus related liver disease. Gut 1995; 37: 406-12. 36. Duclos-Vallee JC, Hajoui 0, Yamamoto AM, Jacqz-Aigrain E, Alvarez E Conformational epitopes on CYP2D6 are recognized by liver/kidney microsomal antibodies. Gastroenterology 1995; 108: 470-6. 37. Seelig R, Renz M, Bunger G, Schroter H, Seelig HP AntiLKM-1 antibodies determined by use of recombinant P4502D6 in ELISA and Western blot and their association
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M. Nishioka et al. with anti-HCV and HCV-RNA. Clin Exp Immunol 1993; 92: 373-80. 38. Pateron D, Hartmann DJ, Duclos-Vallee JC, Jouanolle H, Beaugrand M. Latent autoimmune thyroid disease in patients with chronic HCV hepatitis. J Hepatol 1993; 17: 417-9. 39. Query CC, Keene JD. A human autoimmune protein associated with UlRNA contains a region of homology that is
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cross-reactive with retroviral p30gag antigen. Cell 1987; 51: 21 l-20. 40. Michitaka K, Durazzo-M, Tillmann-HL, Walker D, Philipp T, Manns MI? Analysis of hepatitis C virus genome in patients with autoimmune hepatitis type 2. Gastroenterology 1994; 106: 1603-10.
Journal of Hepatology ISSN 0168-8278
protein in Japanese
Mikio Nishioka’,
Syed Ahmed Morshed’, Kazumi Kono ‘, Takashi Himoto’, Keiji Arima’, Seishiro Watanabe’ and Micheal I? Manns2
Salina Parveen’,
‘Third Department of Internal Medicine, Kagawa Medical University, Kagawa, Japan and ‘Department of Gastroenterology and Hepatology, Medizinische Hochschule. Hannover, Germany
BackgroudAims: The aims of the current study were to assess the frequency and the signiilcance of antibodies to cytochrome P45OIID6 protein (antiP45OIID6) in various diseases among Japanese patients. Metlrods: Sera from 541 patients were tested by indirect immunofluorescence, and the specificity of antiP45OIID6 was ascertained by either enzyme immunoassay (ELISA) or V&stern blot using recombinant antigen or rat liver microsomes. Results: Anti-P45OIID6 was found in only 6 of 235 patients (2.6%) with chronic active hepatitis (CAH) positive for hepatitis C virus (HCV) antibody and quantitative HCV-RNA with genotypes II and IV The predominant epitopes on immunoblots were 66 and 50KD, a 1OKD band being the newly undeflned microsomal antigen. Even in the patients negative for autoantibodies to nuclear antigens (ANA) by routine indirect immunofiuorescence test, various ANA were detected by the newly developed recombinant ELISA. These patients were younger, with lower y-globulin and IgG levels than patients with autoimmune hepa-
L
microsome type 1 antibody (antiLKM- 1) predominantly directed against cytochrome P450IID6 is accepted as a marker of autoimmune hepatitis (AIH) type II (l-3). AIH type II has been described mainly in Europe (4-6) and infrequently in the United States (7,8). In Japan, this disease seems to be very rare, although the presence of MB/KIDNEY
Received 21 December 1995; revised 15 October; accepted 6 November 1996
Correspondence: Dr. Mikio Nishioka, MD, Third Department of Internal Medicine, Kagawa Medical School, 1750-1, Ikenobe, Miki-cho, Kita-gun, Kagawa-ken, 76107 Japan. Tel: 81-878-98-5111 Ext 2640/2642. Fax: 81-878-91-0115. e-mail: [email protected].
992
Three of five patients with anti-P4!#OIID6 responded well to interferon therapy and one received prednisone when interferon was ineffective. Interestingly, only this patient was diagnosed as definite autoimmune hepatitis according to the criteria proposed by the International Autoimmune Hepatitis Group (LAHG). The other five patients who did not satisfy the IAHG criteria might be considered as CAH-C with autoimmune features. No autoimmune hepatitis patients positive for anti-P4!5OIID6 were identified in the current study, indicating that the variant is very rare in Japan. Conclusions: Anti-P45OIID6 in CAH-C patients in Japan is not as rare as expected. Anti-P45OIID6 among Japanese patients has uncertain significance and precludes further characterization of CAH-C with autoimmune features, which might require interferon therapy. MS
Key words: Anti-LKMl; Anti-P45OIID6; Autoimmune hepatitis; Hepatitis C; Japan.
either anti-LKMl or antibodies to P450IID6 (antiP450IID6) has been confirmed (9,lO). Recent reports have noted a high rate of antibodies to hepatitis C virus (HCV) in patients considered to have anti-P450IID6 (1 l-l 3). These findings, including amino acid sequence homology between P450IID6 and HCV polyproteins (molecular mimicry), suggest that cross-reactive antibody epitope(s) may be one of the triggering factors in these patients. Therefore, the subclassification of AIH as proposed on the basis of autoantibodies and HCV markers may be helpful for the diagnosis, therapy and prognosis in these patients (2). Unfortunately, the incidence of AIH type II, and the frequency of antGP450IID6 in patients with chronic hepatitis C, and the clinical spectrum, including ther-
Anti-P45OIID6
apy and prognosis, have not yet been well characterized in Japan, or in the rest of Asia. In the present report, we have attempted to assess the frequency and the significance of anti-P450IID6 seropositivity in adult patients with liver diseases. Characteristics of the patients positive for anti-P450IID6, including its predominant antibody epitopes, were analyzed in detail. AntiP450IID6 reactive sera were also investigated for the presence of ANA subtypes, using a battery of recombinant nuclear antigens in the ELISA tests.
Materials and Methods Study populations Serum samples were collected from 32 adult patients with acute viral hepatitis, 319 with chronic viral hepatitis, 20 with AIH, 10 with cryptogenic hepatitis, 5 with drug-induced hepatitis, 35 with primary biliary cirrhosis, 25 with liver cirrhosis, 95 with non-liver diseases (i.e., gallbladder stone, pancreatitis, chronic gastritis, peptic ulcer, nephritis, and collagen diseases) including 15 systemic lupus erythematosus, and 45 healthy blood donors serologically negative for viral markers. Four of six (66.6%) chronic active hepatitis (CAH) C patients positive for anti-P450IID6 had concurrent immunologic disease such as chronic thyroiditis. Four of 19 (21%) patients with AIH had concurrent immunological disease and two (10.5%) of them had chronic thyroiditis. Three of 50 (6%) CAH-C patients without anti-P450IID6 had concurrent immunologic disease, two (4%) with chronic thyroiditis. Diagnoses of AIH patients were done according to the criteria proposed by the International Autoimmune Hepatitis Group Scoring System (14). The European classification for liver histology was used to distinguish the characteristics of chronic liver diseases (15). CAH was categorized by chronic inflammatory infiltration involving portal tracts and extending into the parenchyma, with piecemeal necrosis and formation of interlobular septa. The architecture was destroyed but there was no nodular regeneration. Activity, seen as piecemeal necrosis and inflammation, varied from moderate (CAH-2A) to severe (CAH-2B). Informed consent was obtained from all patients who participated in the present study. Virological assessments Patients with acute and chronic viral hepatitis who satisfied the clinical, biochemical and histological criteria for hepatitis were diagnosed by the presence of virological and epidemiological markers. Seventy-four patients had hepatitis B (HBV) antigen (Abbott Laboratories, North Chicago, Illinois, USA). Fifty-one (68.9%) of these patients had histological criteria for CAH. Ten patients were seropositive for antibodies to
in Japanese AIH patients
hepatitis 6 virus (HDV) antigen (Abbott Laboratories, North Chicago, Illinois, USA). Two hundred and thirty-five patients had serological markers of HCV infection by second-generation enzyme immunoassay (anti-HCV-ELISA II, Ortho Diagnostic System, Inc., Raritan, New Jersey). HCV-RNA was detected by twostage polymerase chain reaction with two pairs of primers deduced from the 5’-non-coding region (16). HCV genotyping and the relative copy numbers were established according to previously described methods (17,18). One hundred and one (43%) of these HCVinfected patients showed CAH in their histologic findings. Identzjication of autoantibodies Anti-LKMl was detected by indirect immunofluorescence (IIF) using frozen sections of the kidney and the liver taken from rats (1). Sera were considered positive for anti-LKMl when they reacted at a minimal dilution of 1:40 with the cytoplasm of hepatocytes and proximal renal tubules of the kidney and with no staining of the distal renal tubules. In sera positive for antiLKM, Western blot assay was carried out using either P450IID6 fusion protein or purified rat liver microsomes (13). Autoantibodies to P450IID6, soluble liver antigen (SLA), pyruvate dehydrogenase-E2 subunit (PDH-E2) and branched-chain oxoacid dehydrogenase E2 subunit (BCKD-E2) were detected by ELISA, which determined whether test serum inhibited the reaction of reference serum. The target antigens fixed to the ELISA plates were microsomes (50KD, P450IID6), supernatant from rabbit liver homogenates (SLA), and mitochondrial (70KD PDH-E2 or 50KD BCKD-E2) proteins (13,19-21). ANA was detected by an IIF test using cultured human laryngeal tumor (HEp-2) cells as a target. Antibodies to smooth muscle (SMA), parietal cells (PCA), and mitochondrial antigens (AMA) were tested by the same method as ANA on rat kidney and stomach. Antibodies to nuclear antigens such as Cenp-B, U 1RNP-A, U 1RNP-70K, Ul RNP-B’/B, SS-A/Ro (52K and 6OK), SS-B/La, and Ki were detected using a battery of recombinant nuclear antigens in ELISA tests as previously described (22-26). Detection of antibodies to thyroid microsome and thyroglobulin in sera as performed using artificial gelatin particle carriers sensitized with either thyroid microsome or thyroglobulin, extracted and purified from human thyroid tissue, by the indirect agglutination test according to the instructions of the SERODIA-AMC kit (Fujirenbio Inc., Tokyo, Japan). Antibodies to both single-stranded DNA and double-stranded DNA were also investi993
M. Nishioka et al.
TABLE 1
Results
Prevelance of antibody to P450IID6 protein (anti-LKMl) diseases and controls Diagnosis
No. of patients
No. of anti-P450IID6 reactive patients (%)
Acute hepatitis Chronic viral hepatitis Hepatitis B virus (HBV) Hepatitis C virus (HCV) Hepatitis 6 virus (HDV) Total no. Autoimmune hepatitis Cryptogenic hepatitis Drug-induced hepatitis Primary biliary cirrhosis Liver cirrhosis Non-liver diseases Healthy blood donors
74 235 10 319 20 10 5 35 25 95 45
0 6 (2.6%) 0 6 (1.9%) 0 0 0 0 0 0 0
Total
586
6 (1%)
32
gated in these sera by commercially kits (MBL, Nagoya, Japan).
in various
0
available ELISA
Human leukocyte antigen (HLA) typing Patients positive for anti-P450IID6 and 25 of 50 CAHC patients without anti-P450IID6 were tested for HLA class I and II antigens. A standard microlymphocyte cytotoxicity assay was used as described elsewhere (27). Interferon therapy Five of six patients with CAH positive for antiP450IID6 were treated with human natural interferon (IFN-cr 2a, 6MU, three times weekly for 24 weeks) as recommended by the Ministry of Health and Welfare of Japan. Three had complete and one each had partial and no response to the therapy. Complete response was defined as the normalization of transaminase levels, HCV-RNA disappearance and improvement of histology; partial response indicates a decrease but no normalization of transaminase levels and low HCVRNA copies without histological improvement; and no response implies no changes in transaminase levels and sustained HCV-RNA copies without histological improvement. Statistical analysis Results were expressed as mean+SD (standard deviation). Statistical analysis was performed on a Power Macintosh using StatView 4 software. The MannWhitney test for continuous variables and Fisher’s exact test for dichotomous variables were used between various groups to examine the significant of differences, with p=O.O5 as the minimum level of significance. 994
Frequency of anti-P450IID6 Anti-P450IID6 was found in six of 3 19 patients (1.9%) with chronic viral hepatitis. None of 222 patients with acute hepatitis, primary biliary cirrhosis, liver cirrhosis, gallbladder stone, pancreatitis, chronic gastritis, peptic ulcer, nephritis, collagen diseases and 45 healthy blood donors was seropositive for anti-P450IID6 (Table 1). Anti-P45OIID6 in chronic hepatitis C Six of 235 patients (2.6%) with chronic hepatitis C were seropositive for anti-P450IID6 by both ELISA and immunoblot methods. Sera reactive for anti-P450IID6 on fusion peptide by immunoblot also reacted with purified rat liver microsomes, and recognized at least one band in each patient (KJ: 66 & 50KD; KK: 66KD; WT: 50KD; FK: 10KD; TK: 10KD; YO: 50KD). No sera recognized other corresponding bands such as 58 and 39KD. None of 74 patients with HBV and 10 patients with HDV was seropositive for anti-P450IID6 (Table 1). Autoantibody profiles in anti-P450ZZD6 reactive patients Three patients (KK, WT and FK) had anti-LKMl titers of 180 and others (KJ, TK, YO) had anti-LKMl titers of 1: 160 (Table 2). All of them have been diagnosed as CAH-C. All sera positive for anti-LKMl were also reactive to P450IID6 protein by ELISA. The presence of anti-P450IID6 in these sera was confirmed by immunoblot using recombinant P450IID6 protein (11) (figure not shown). None of these 6 sera was reactive to PDH-E2 or BCKD-E2. ANA was detected in five patients at a low titer of 20-80 times dilution by IIE Five of six anti-P450IID6 reactive patients had antibodies to several nuclear antigens, including UlRNPA, UlRNP-70K, UlRNP-B’/B, Cenp-B, 60KSS-A/Ro, and SS-B/La. None of them demonstrated any reactivities to either 52KSS-A/Ro or Ki nuclear antigens by the recombinant ELISA tests. All of them were also non-reactive to double-stranded or single-stranded DNA. One patient (YO) was nonreactive for ANA by both IIF and ELISA. Only one patient (WT) was seropositive for smooth muscle at 1:80 dilution by IIE Five patients had antibodies to thyroid microsome and four had antibodies to thyroglobulin. None of 6 sera had antibody reactivities to SLA, mitochondria nor parietal cell antigens (Table 2) by ELISA or IIE Characteristics of anti-P450IID6 reactive patients All six patients with anti-P450IID6 were adults (Table 3). Four were female and two were male. They had had
Anti-P45OIIDb in Japanese AIHpatients TABLE 2 Autoantibody profiles in patients with chronic active hepatitis C(CAH-C) positive for antibodies to P450IID6 protein Autoantibodies*
CAH-C positive for anti-P450IID6 KJ
KK
WT
FK
TK
YO
Anti-LKMI (IIF) Anti-Microsomes (WB:KD) AntGP450IID6 (WB:SOKD) Anti-P450IID6 (ELISA)# Anti-PDH-E2 (ELISA+WB) Anti-BCKD-E2 (ELISA+ WB) Anti-SLA (ELISA)
1:160 66, 50 + 90% -
1:80 66 + 57% _ -
1:80 50 + 45%
1:80 10 + 41% -
1:160 10 + 63% _ -
1:160 50 + 68% _
Anti-nuclear antibody (ANA-IIF) ANA subtypes (ELISA): UlRNP-A UIRNP-70K UlRNP-B’/B CENP-B 52K-SSAJRo 60K-SSAfRo SS-B/La Ki
1:80
1:80
I:40
1:4O
1:40
I:20
+ + + + + -
+ + -
+ + -
-
+ -
+ _ -
Anti-ssDNA (single-stranded) Anti-dsDNA (double-stranded)
-
-
-
-
Rheumatoid (RA) factor
+
-
-
-
Anti-SMA Anti-PCA Anti-thyroid microsome Anti-thyroglobulin
+ +
1:80 + +
+ -
+ +
-
* LKM: Liver/kidney microsome; IIF: Indirect immunofluorescent test; WB: Western blot; ELISA: Enzyme-linked immunosorbent assay; Antibodies to mitochondria (PDH-E2 and BCKD-EZ), soluble liver antigen @LA), smooth muscle antigen @MA), and parietal cell antigen (PCA). x Inhibition >40% selected as positive reactivity.
a long duration of illness (range: 2 to 9 years), and four of them had concurrent immunologic disease such as chronic thyroiditis. Five of them underwent interferon (IFN-o) trial. All six patients were positive for anti-HCV and HCV RNA. Five patients showed HCV genotype II (copy numbers ranged between 1. 104-1. 101o/ml, mean 1.107.“‘.‘) and the other (KK) genotype IV with viral copies of 1. 104/ml. They had high serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at presentation. One patient (KJ) had a higher level of serum y-globulin than the other five patients. There were some differences in laboratory findings between these six patients and 50 patients with CAH-C negative for anti-P450IID6 (Table 4). Patients positive for anti-P450IID6 were younger at presentation, without significant difference. Serum y-globulin and IgG levels were significantly less in AIH or CAHC negative for anti-P450IID6. Serum AST, y-globulin, IgG, IgM, IgA, HLA-DR4 distribution, and histopathological index (CAH-2B) were significantly higher in AIH patients than that of CAH-C without antiP450IID6. Serum ALT, AST, y-globulin, and IgG were significantly lower in CAH-C without anti-P450IID6,
as compared to CAH-C with anti-P450IID6 (Table 4). On histological analysis, FK was diagnosed as CAH-2B and the others were CAH-2A according to the European classification (15). One of these patients had a high score on the histological activity index (HAI) (28). All six LKMl-reactive patients were evaluated by IAHG scoring (14). One of the patients (WI) was diagnosed with definite AIH, because the aggregate scores were greater than 15. The other five did not have definite AIH by IAHG scoring. HLA typing of anti-P450IID6 reactive patients None of the patients showed positive reactivities for HLA-DR3. DR4, a characteristic HLA type found in Japanese prototype AIH patients (29), was detected in three of six (50%) patients. Outcome of treatment regimens Five patients were treated with human natural interferon-a (IFN-a). Three patients (KJ, YO and KK) are now in remission and are negative for HCV-RNA. Patient (KK) had been diagnosed as having chronic thyroiditis before IFN therapy and developed destructive thyroiditis after therapy, although she showed a com995
M. Nishioka
et cd.
TABLE 3 Characteristics of chronic active hepatitis-C (CAH-C) patients with antibodies to P450IID6 protein at presentation Findings*
CAH-C patients positive for anti-P450IID6 KJ
Clinical features: Age (years)/sex 41/F Duration of illness (years) 8 Concurrent immunologic disease chronic thyroiditis Laboratory data: + Anti-HCV-II HCV-RNA (copies/ml) 1.10’ HCV-genotype II ALT (n<50 U/l) 80 AST (n<33 U/l) 48 y-globulin (n< 1.5 g/dl) 2.2 IgG (n< 1682 mg/dl) 2730 IgM (n<283 mg/dl) 322 IgA (n<340 mg/dl) 372 HLA-DR3IDR4 -lHistology: European types (CAH)** 2A HA1 score 6 IAHG a) Required parameters 12 b) Additional parameters 2 Interferon therapy CR
KK
WT
29/F 2 chronic thyroiditis
63/M 4 chronic thyroiditis
+ 1.104 IV 126 56 1.9 2290 218 257 -i+
+ 1.10’ II 261 113 1.8 2540 12 500 -i-
2A 4
2A 5
9 4 CR
13 3 NR
FK 22/M I -
TK
YO
62/F 9 chronic thyroiditis
+ + 1.10’0 1.108 II II 680 260 230 130 1.9 2.0 2310 2510 180 292 203 336 -i+ -/2B 15 10 4 PR
33/F 1 + 1.10’ II 230 140 1.5 2080 78 172 -i+
2A 10
2A 4
10 4 NT”
9 4 CR
* HCV: Hepatitis C virus; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HAI: Histologic activity score; IAHG: International Autoimmune Hepatitis Group; ** CAH: Chronic inflammatory infiltration involving portal tracts and extending into the parenchyma, with piecemeal necrosis moderate (CAH-2A) to severe (CAH-2B and formation of intralobular septa. CR: Complete response; NR: No response; PR: Partial response. t NT: Not treated
plete response to the therapy. Two patients in CAH-C without anti-P450IID6 had chronic thyroiditis; they had not received any therapy. One young male patient (FK, 22 years old) developed rapid progressive liver damage and ultimately liver cirrhosis. He had received a blood transfusion because of a severe traffic accident at the age of 22. Three months later, he was diagnosed as having posttransfusion hepatitis. He underwent intermittent IFN-a therapy for 2 years after the findings of fluctuating transaminase levels. Initially the patient showed a good response to IFN, but transaminase levels rose again when IFN therapy was withdrawn. HCV-RNA was persistently positive in his serum. Histopathology of the liver biopsy performed at the age of 26 demonstrated liver cirrhosis. Why the patient developed liver cirrhosis so quickly even at a young age is unclear, but he might have autoimmune liver destruction as seen in AIH patients. Interestingly, he showed HLA-DR4 positivity. In patient WI, who had definite AIH by IAHG scoring, serum ALT and AST levels increased rapidly after initiation of IFN therapy. One week later, ALT level increased four-fold and the IFN was withdrawn and replaced by steroids. The patient responded quickly, as shown by falling transaminase levels in his serum. Careful monitoring during therapy and regular 996
follow-up after therapy seem important with these variants.
in
patients
Discussion Our present study confirmed the presence of antiP450IID6 in patients with liver diseases, because the antibody was absent in diseases other than liver diseases, such as pancreatitis, chronic gastritis, peptic ulcer, nephritis and collagen diseases. The specificity of anti-P450IID6 was ascertained by ELISA, using purified microsomal protein and by immunoblot assay with recombinant P450IID6 (50 KD) or microsomes. Anti-P450IID6 was detectable in only six of 235 patients (2.6%) with chronic hepatitis C. The incidence of anti-P450IID6 in chronic HCV patients is not as low as our previous report suggested (9), although antiP450IID6 does not appear to be a common sequela of HCV infection (3). The seropositivity in patients with chronic hepatitis C appears to be different in various countries, and previous observations indicated a geographical difference in the frequency of HCV markers in patients with anti-P450IID6 (so-called AIH type IIb) @X,30-34). A high incidence of anti-P450IID6 in high HCV endemic areas in various countries suggests an association of HCV infection in patients positive for anti-P450IID6, as indicated by Vergani et al. (33).
Anti-P450IID6 in Japanese AIH patients TABLE 4 Comparison P450IID6
of characterestic findings between autoimmune hepatitis (AIH), and chronic active hepatitis C (CAH-C) with or without antiFindings*
Clinical features: Age (years) Sex (F/M) Immunologic disease Laboratory data: Anti-HCV-ELISA II (%) HCV-RNA (copies/ml) HCV genotypes (%) I II III IV mixed ALT (n<50 U/l) AST (n<33 VA) y-globulin (n< 1.5 g/d]) IgG (n< 1682 mg/dl) IgM (n<283 mg/dl) Ig,A (n<340 mg/dl)
AIH without anti-P450IID6 @,=19) p
CAH-C without anti-P4501ID6 (n=SO) P J
CAH-c with anti-P450IID6 (n=6)
--p-
56.82 15.7 NS 17f2 NS 4 NS
41.6217.2 412 4
NS NS NS
54.1~14.3 31/19 3
NS NS NS
NS NS
6(100) 1 lO,.“‘.s
NS NS
50 (100)
NS
-
221.42246 NS 238.35266 NS 3.020.8
NS
0
0
-
1.107.4’1.7
5 (83) 0 1 (16) 0 273.62210 120.3274 1.820.1 2355.32203 193.62 104 306.62121
NS NS NS NS <0.007 dO.05 CO.04 <0.009 NS NS
23/35 (65.7) 6135 (17.1) 6/35 (17.1) 3/35 (8.5) 110.1289 68.8241 1.620.3 2049.6?454 165.2?94 271.6’_111
NS NS NS NS NS
HLA-typing (%) DR3 DR4
O/l7 12/17 (70)
NS NS
0
NS
316 (50)
NS
Histology of liver European classification (%) CAH-2A CAH-2B
4 (21) 15 (79)
co.059
4 (66) 1 (16)
NS NS
41 (82) 9 (18)
Interferon-a therapy (%)’ Complete response Partial response No response
-
3 (60) 1 (20) 1 (20)
NS NS
lb25 (44) 6/25 (24) 9/25 (36)
NS NS
Steroid therapy (%) Complete response Partial response No response
13/16 2Jl6 1116
O/25 9125 (36)
NS CO.057
NS -
* HCV: Hepatitis C virus; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HAL Histologic activity score; ’ Complete response is defined as the normalization of transaminase levels with HCV-RNA disappearance in patients; partial response indicates decrease but no normalization of transaminase levels with sustained HCV-RNA; no response is no change in transaminase levels and presence of HCV-RNA continues. Statistical significant differences (p-value) were calculated by Mann-Whitney test for continuous variables and Fisher’s exact test for dichotomous variables.
Our study of immunoblot with rat liver microsome showed heterogeneity of anti-LKMl recognition with bands of 66KD and 1OKD; a 5OKD band was frequently observed. Recently, Muratori et al. (35) observed antibodies to 66KD, 58KD, 50KD and 39KD antigens in rat/human microsomes in patients with HCV infection positive for anti-CYP2D6 or antiP450IID6. Unfortunately, none of our sera recognized 58KD or 39KD peptide; instead they recognized a 1OKD band as the predominant antigen. It seems that antibody to 1OKD is a newly undefined antigen or a degradation product of common epitopes of P450 proteins in liver microsomes. Because the autoimmune re-
sponse to P450IID6 is directed to both linear and conformational antigenic sites (36), fine epitope mapping would be more feasible with overlapping short synthetic peptides using either ELISA or the immunoblot test. However, it is clear that the main target antigens in HCV patients with anti-P450IID6 are heterogeneous. Further study might reveal the molecular identity of the new antigens. Six patients with CAH-C positive for anti-P450IID6 in the present study satisfy the definition of “antiLKMl CAH or autoimmune hepatitis type II”, as first described by Homberg et al. (1). One (KI) of the six patients was diagnosed clinically as AIH before the de997
M. Nishioka et al
tection of anti-P450IID6 was carried out. Again, this patient was diagnosed as chronic hepatitis C, instead of AIH, because of: low titers of ANA; laboratory findings at presentation similar to two patients (WT and TK) with chronic hepatitis C positive for antiP450IID6; absence of the HLA-DR4 antigen that is considered to be a prototype HLA in Japanese AIH (29); and a good response to interferon therapy. Previous study reported that patients positive for anti-P450IID6 could be divided into two groups according to their positive HCV markers (2). All six patients in this study were considered as AIH type IIb as they showed a low titer of anti-P450IID6 and moderately increased levels of serum y-globulin. Although statistical comparisons are limited by small numbers, there were some significant differences in laboratory findings compared to either those with CAH-C or AIH patients negative for anti-P450IID6. Patients with antiP450IID6 had significantly lower y-globulin and IgG levels in their sera. It is interesting to know the genotypes of the HCV coding region as well as HCV copy numbers in these six patients. Five had genotype II and the other had genotype IV HCV infection with genotype II may be a dominant type for developing anti-P450IID6 (37), although HCV genotype II was found in almost 70% of CAN-C patients in our study population. In comparison to serum AST, ALT, y-globulin and IgG levels (Table 4), patients with CAH-C positive for antiP450IID6 were different from CAH-C without antiP450IID6. However, the present study clearly indicated that there were no obvious differences in HCV genotypes, viral copies and HLA phenotyping, between patients with or without anti-P450IID6, although few patients were examined. Our clinical and virological findings in six patients positive for anti-P450IID6 suggest that they belong to the patient group CAH-C. Incorporating the new IAHG criteria for diagnosis (14), only one patient (WT) in the current study could be considered as having definite AIH; this patient scored more than 15 points. The patient did not respond to IFN therapy from its counterpart, suggesting patients with a different variant reactive to antiP450IID6 might require an initial steroid treatment (for the improvement of liver function), followed by a further treatment with IFN (to kill persistent HCV infection). Future studies on double-therapy, such as steroid initially and IFN later, in this patient group are required to determine how this therapeutic regimen can help to eradicate both autoimmunity and HCV infections. A possible association between HCV infection and thyroiditis is intriguing. Chronic thyroiditis, a frequent
complication of AIH patients, was detected in patients with CAH-C positive for anti-P450IID6. Four of these six (66%) patients had chronic thyroiditis and one of them developed destructive thyroiditis during IFN therapy. Homberg et al. (1) also reported a high incidence of autoimmune thyroiditis in patients with antiP450IID6. Pateron et al. (38) found anti-thyroid antibodies in nine patients with chronic hepatitis C, and two patients developed overt thyroid dysfunction during IFN therapy. In our study, chronic thyroiditis seems to be the predominant concurrent immunologic disease among patients with AIH and CAH-C with or without anti-P450IID6. Chronic thyroiditis in CAHC patients with anti-P450IID6 may be a characteristic concurrent immunologic disease. Further study is needed to establish a temporal association between chronic thyroiditis and hepatitis C virus infection. In this study, anti-P450IID6-reactive patients showed autoantibodies to nuclear antigens by recombinant ELISA tests. Antibodies to UlRNP-70K were common in these patients. These autoantibodies usually occur among a selected group of patients with connective tissue diseases (39). Interestingly, the target antigen is cross-reactive with retroviral p30gag antigen (39). Furthermore, retesting of these sera for ANA subtypes after either IFN or steroid therapy revealed decreasing titers, and one patient (KK) showed ANA subtype titers below the cut-off values. These findings shed a new light on ANA behavior associated with HCV infection. The presence of various ANA subtypes has uncertain significance in these patients and may reflect the nature of the autoimmune phenomena. Finally, hosts rather than viral factors seem to be responsible for triggering an aberrant immune response in CAH-C (40).
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