Abstract
Hepatitis C virus (HCV) infection has been found to be strikingly associated with autoimmune phenomena. The aim of the present study was to investigate the presence of various autoantibodies in patients with HCV infection. Anti-neutrophil cytoplamic antibody (ANCA), anti-dihydrolipoamide dehydrogenase (anti-E3), rheumatoid factor (RF), anti-dihydrolipoamide acetyltransferase (anti-E2), anti-SS-A/Ro (60 kD), anti-SS-A/Ro (52 kD), anti-SS-B/La, anti-topoisomerase II (anti-topo II), anti-cardiolipin (aCL), anti-dsDNA, anti-ssDNA, anti-nuclear antibodies (ANA), anti-proteinase 3 (anti-Pr3) and anti-myeloperoxidase (anti-MPO) were determined in sera from 516 patients with HCV infection, 11 with primary biliary cirrhosis (PBC) and 44 healthy controls. Assays employed were indirect immunofluoresence, the particle latex agglutination test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. ANCA, anti-E3 antibody and RF were positive in 278/516 (55·6%), 276/516 (53·3%) and 288/516 (56%) patients with HCV infection, respectively. Positivity for ANA was present in 15·8%, anti-ssDNA in 15·6%, anti-dsDNA in 8·5%, aCL in 5%, anti-SS-B/La in 4·1%, anti-SS-A/Ro (60 kD) in 3·9%, anti-E2 in 3·3% and anti-SSA/Ro (52 kD) in 1·2%, anti-MPO in 4·8%, anti-Topo II and anti-actinin in 0%. All sera with ANCA showed c-ANCA patterns and contained anti-PR3 specificity. HCV patients with ANCA showed a higher prevalence of skin involvement, anaemia, abnormal liver function and α-Fetoprotein (α-FP). HCV patients with anti-E3 antibodies showed a higher prevalence of liver cirrhosis, arthritis, abnormal liver function and elevated α-FP levels. The prevalence of autoantibodies was not affected by treatment with interferon-alpha (IFN-α). In conclusion, autoantibodies are commonly found in patients with HCV infection. There is a high prevalence of anti-E3, ANCA and RF in these patients. Proteinase 3 and E3 are the major target antigens in HCV infection. HCV may be regarded as a possible causative factor in ANCA-related vasculitis.
Keywords: ANCA, anti-E3 antibody, rheumatoid factor, hepatitis C virus, anti-proteinase 3
INTRODUCTION
Serum autoantibodies are a characteristic feature of autoimmune diseases [1]. The autoantibodies include anti-nuclear antibodies (ANA), anti-dsDNA, anti-ssDNA, anti-SS-A/Ro (60 kD), anti-SS-A/Ro (52 kD), anti-SS-B/La, anti-topoisomerase II (anti-topo II), anti-dihydrolipoamide dehydrogenase (anti-E3), anti- neutrophil cytoplasmic antibody (ANCA), anti-proteinase 3 (anti-Pr3), anti-myeloperoxidase (anti-MPO), anti-dihydrolipoamide acetyltransferase (anti-E2) and anti-cardiolipin (aCL).
Hepatitis C virus (HCV) infection is an important infection worldwide. The association of HCV infection and autoimmunity has been reported [2–7]. Several studies have suggested that chronic HCV may act as a trigger for the development of autoimmune rheumatic diseases [2–8]. The role of HCV in these disorders has not been established. HCV is a linear, single-stranded RNA virus of the Flaviviridae family, with extensive genomic variability associated with different autoimmune manifestations [2–7,9].
To assess whether HCV infection is associated with autoimmunity, we studied the prevalence of various autoantibodies in patients with chronic HCV infection. We also evaluated the clinical significance of positive autoantibodies in patients with HCV infection, and the effect of IFN-α treatment on autoantibody production in patients with HCV infection.
MATERIALS AND METHODS
Patients
Serum samples were obtained from 11 patients with primary biliary cirrhosis (PBC), 216 patients with chronic hepatitis B infection (HBV) and 516 patients with chronic hepatitis C infection (HCV). These were consecutive patients referred to the Department of Medicine, Chung Shan Medical and Dental College Hospital, Taichung, Taiwan. Diagnosis of chronic HBV or HCV infection is based on the presence of hepatitis B surface antigen (HBsAg) or anti-HCV antibodies (anti-HCV), respectively. Anti-HCV antibody was tested by a third-generation anti-HCV enzyme-linked immunosorbent assay (ELISA) kit (Axsym HCV 3·0, Abbott Laboratories, Chicago, IL, USA). HBsAg was also tested by ELISA (IMX-HbsAg assay, Abbott Laboratories). Quantitative measurements of serum HCV-RNA were performed by the AmpliSensor Assay (Biotronics Tech. Corp., Lowell, MA, USA). The diagnosis of PBC was based on accepted clinical and histological criteria [10]. Forty-four normal subjects were used as a control group. Patients with chronic HBV or HCV infection were evidence for the diagnosis of PBC. Patients with high ALT had elevated serum ALT levels one and a half times above the upper limit of the normal value (>60 U/l). Patients with skin disease were defined as those having rash, purpura, nodules, livedo reticularis and Raynaud’s phenomenon. Anaemia was arbitrarily defined as haemoglobin below 12·0 g/dl.
Indirect immunofluorescence (IIF)
IIF was used to test for ANCA on ethanol- or formalin-fixed human neutrophils (INOVA Dianostics, Inc., San Diego, CA, USA) according to the method described by Wilk [11]. For ANCA tests, titres ≥1:40 were considered positive. Fluorescence patterns were classified as classic cytoplasmic (c-ANCA) or perinuclear (p-ANCA) patterns. Anti-nuclear antibodies (ANA) were detected by IIF using a commercial Hep-2 cell system (Antibodies Inc., Davis, CA, USA) as described previously [12].
We used direct antigen-specific ELISA kits to detect anti-PR3 and anti-MPO (INOVA Diagnostics) as described [13,14]. All sera were assayed at a dilution of 1:100. Three positive and three negative control sera were included in each plate. Normal values of anti-Pr3 and anti-MPO antibodies were 50 IU/l. Anti-actin antibodies were detected by ELISA as described previously [15].
Preparation of SS-A/Ro (52kD), SS-A/Ro(60kD), SS-B/La, E3 and Topo II
Recombinant proteins of both SS-A/Ro and SS-B/La for ELISA were derived from complementary DNA clones obtained from W. M. Keck Autoimmune Disease Center, The Scripps Research Institute, La Jolla, CA, USA [16,17]. Recombinant proteins of E3 and Topo II for ELISA were also derived from complementary DNA as described previously [18,19]. Briefly, the E3 expression vector, pQE9-E3, was modified from pOTSU-E3 as described previously [18]. Using PCR, the mature E3-cDNA was amplified from pOTSU-E3, with the restriction site SalI and BamH1 at the N-terminal and c-terminal, respectively. Following SalI and BamHI digestion, the PCR fragment was sub-cloned into pQE-9, which was previously digested with SalI and BamHI, to complete the construction.
Anti-dsDNA and anti-ssDNA were prepared as described previously [20]. Anti-cardiolipin antibody was tested as described previously [21]. Rheumatoid factor was determined with a commercial kit (Avitex-RF test, Omega Diagnostics, UK).
Mitochondria from porcine liver and human placenta were prepared as described previously [22], except that all buffers contained the protease inhibitors phenylmethylfluorane sulphonate (1 mm) and dithiothreitol (5 mm).
ELISA
ELISA was performed according to the method of Rubin et al.[20]. All sera were assayed at a dilution of 1/100.
Immunoblotting
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), using a 12·5% acrylamide slab gel with a 5% acrylamide stacking gel, was performed according to the method of Laemmli [23]. Samples were reduced for 5 min in boiling water with 0·0625 m Tris-HCl buffer, pH 6·8, containing 2·3% SDS, 5% 2-mercaptoethanol and 10% glycerol. Samples applied to the gel were run at 100–150 V for 1·5 h. They were then electrophoretically transferred to nitrocellulose, according to the method of Towbin et al.[24]. The nitrocellulose-transferred proteins were cut into strips and soaked in 5% non-fat powered milk in phosphate-buffered saline (PBS), for 30 min at room temperature, to saturate irrelevant protein binding sites. Anti-serum diluted with 5% non-fat powered milk in PBS was reacted with the nitrocellulose strips and incubated for 1·5 h at room temperature (25°C). The strips were washed twice with PBS-Tween for 1 h, and then a secondary antibody, consisting of alkaline phosphatase conjugated goat anti-human or mouse IgG antibodies, was added. The substrate NBT/BCIP (nitroblue tetrazolium/5-bromo-4-chloro-3 indolyl phosphate) was used to detect antigen- antibody complexes. All sera were assayed at a dilution of 1/200 at 25°C.
Statistical analyses
Statistical analysis was done with the χ2 test.
RESULTS
Table 1 shows the frequency of various autoantibodies in patients with HCV infection. ANCA were detected in 278 of 516 sera (55·6%) by IIF. All sera with ANCA showed c-ANCA patterns. None of the sera from patients with HCV infection had p-ANCA. ANCA was not detected in 44 sera from normal subjects and 26 sera from patients with hepatitis B infection. Therefore, sera from patients with HBV infection were not analysed further for anti-MPO or anti-Pr3 antibodies. Sera with ANCA were tested for anti-PR3 and anti-MPO antibodies by ELISA. Anti-Pr3 antibodies were detected in all sera with ANCA. Anti-MPO antibodies were detected in only 25 of 516 (4·8%) sera from patients with HCV infection, and most anti-MPO antibodies were at low titres (Fig. 1). No anti-actin antibody was detected in sera from patients with HCV infection. Table 2 shows the results of anti-Pr3 and anti-E3 antibodies in HCV patients grouped according to PCR positivity and PCR negativity. Anti-Pr3 antibodies were detected in 239 of the 405 (59%) sera from HCV patients with PCR positivity, whereas 39 of the 111 (35%) sera from PCV patients with PCR negativity were found to have anti-PR3 antibody. The frequency of anti-Pr3 antibody in HCV patients with PCR positivity was higher than those with PCR negativity (P < 0·01). The mean titres of anti-Pr3 in HCV patients with PCR positivity and PCR negativity were 142·6IU/l and 64·3IU/l, respectively. The results indicate that the major antigen of ANCA in HCV infection is proteinase 3.
Table 1.
Prevalence of autoantibodies in patients with HCV infection
| Autoantibodies | HCV patients (n = 516) |
|---|---|
| ANCA | 278 (55·6) |
| Anti-PR3 (mean titre=118·7IU/l) | 278 (55·6) |
| Anti-MPO | 25 (4·8) |
| Anti-actin | 0 (0) |
| Anti-E3 (mean absorbance=1·57) | 276 (53·5) |
| Anti-E2 | 17 (3·3) |
| RF | 288 (56) |
| ANA | 82 (15·8) |
| aCL | 53 (10·3) |
| Anti-Topo II | 0 (0) |
| Anti-dsDNA | 44 (8·5) |
| Anti-ssDNA | 81 (15·6) |
| Anti-SS-A/Ro (60kD) | 6 (1·2) |
| Anti-SS-A/Ro (52kD) | 20 (3·9) |
| Anti-SS-B/La | 21 (4·1) |
Numbers in parentheses are percentages. Normal values of anti-PR3=50IU/l. Normal absorbance of anti-E3 antibody=0·91. ANCA, anti-neutrophil cytoplasmic antibody; PR3, proteinase 3; MPO, myelo-peroxidase; ANA, anti-nuclear antibody; RF, Rheumatoid factor; aCLA, anti-cardiolipin antibody; E3, dihydrolipoamide dehydrogenase; E2, dihydrolipoamide acetyltransferase; Topo II, topoisomerase II.
Fig. 1.
Anti-proteinase 3 (anti-Pr3) and anti-myeloperoxidase (anti-MPO) antibody levels in 516 sera form patients with hepatitis C virus (HCV) infection by ELISA.
Table 2.
Anti-proteinase 3 and anti-E3 antibodies in HCV patients grouped according to PCR positivity and PCR negativity
| PCR positive n = 405 | PCR negative n = 111 | P-value | |
|---|---|---|---|
| Anti-proteinase 3 | 239 (59) | 39 (35) | <0·01 |
| (titre (50IU/l) | mean titre=142·6IU/l | Mean titre=64·3IU/l | |
| Anti-E3 | 190 (46·9) | 36 (32·4) | <0·01 |
| (absorbance ≥ 0·91) | mean absorbance=1·81 | Mean absorbance=1·29 |
Numbers in parentheses are percentages.
Figure 2 shows the reactivities of anti-E3 on immunoblotting using recombinant E3 proteins as antigens. Lanes 1 and 2 are sera from normal controls. Lanes 3–12 are sera from patients with HCV infection. Sera from patients with HCV infection reacted with a band with a molecular weight of 55 kD of E3 protein.
Fig. 2.
Reactivities of anti-E3 on immunoblotting using recombinant E3 proteins as antigens. Lanes 1 and 2: sera from normal controls. Lanes 3–12: sera from patients with HCV infection. Sera from patients with HCV infection reacted with a band of molecular weight 55 kD of E3 protein.
Figure 3 shows the results of ELISA using recombinant E3 as antigen in sera from patients with HCV infection, PBC and normal controls. The normal value for absorbance was based on the results from 40 healthy controls. The normal value of the absorbance was 0·478 ± 0·2187 (mean ± 2 s.d.). Values above 0·9154 were regarded as increased anti-E3 antibody. The frequency of increased anti-E3 antibody in patients with HCV infection and PBC was 276/516 (53·5%) and 3/11 (27%), respectively. Only one of the 40 normal controls had low titres of anti-E3 antibody. Anti-E3 antibodies were detected in only five of the 216 (2·3%) sera from patients with HBV infection. Anti-E3 antibodies were detected in 190 of the 405 (46·9%) sera from HCV patients with PCR positivity, whereas 36 of the 111 (32·4%) sera from HCV patients with PCR negativity were found to have anti-E3 antibodies. The frequency of anti-E3 antibody in HCV patients with PCR positivity was higher than those with PCR negativity (P < 0·01). The mean absorbance of anti-E3 antibody in HCV patients with PCR positivity and PCR negativity was 1·81 and 1·29, respectively.
Fig. 3.
Anti-E3 antibody levels in patients with hepatitis C virus infection, primary biliary cirrhosis (PBC) and normal controls. Purified recombinant dihydrolipoamide dehydrogenase (E3) was used as antigen and determined by ELISA. Values above 0·9154 were regarded as increased anti-E3. Anti-E3 antibody was present in patients with HCV infection and PBC.
The prevalence of anti-SSA/Ro (52kD), anti-SSA/Ro (60kD), anti-SSB/La, aCLA, anti-dsDNA, anti-ssDNA and anti-E2 in patients with HCV infection was 3·9% (20 of 516), 1·2% (six of 516), 4·1% (21 of 516), 10·3% (53 of 516), 8·5% (44 of 516), 15·6% (81 of 516) and 3·3% (17 of 516), respectively. RF and ANA were detected in 56% (288 of 516) and 15·8% (82 of 516), respectively, of these patients. RF has a high prevalence in patientsce with HCV infection. These data indicate that various autoantibodies are produced in HCV infection.
Table 3 shows the frequency of laboratory findings and clinical manifestations in patients with and without ANCA. The frequency of arthritis and liver cirrhosis in patients with and without ANCA was similar. However, 185 of the 278 (66·5%) patients with ANCA had high ALT, whereas 106 of the 238 (44·5%) without ANCA were found to have high ALT (P < 0·01). Similarly, 62 of the 278 (22·3%) patients with ANCA had high α-FT, and 26 of the 238 (10·9%) patients without ANCA had high α-FT (P < 0·01). Ten of the 278 (3·6%) patients with ANCA and none of the 238 patients without ANCA had skin disease (P < 0·01). Twenty-five of the 238 (9·0%) patients with ANCA and none of the 238 patients without ANCA had anaemia (P < 0·01). HCV patients with ANCA showed a higher prevalence of skin involvement, anaemia, abnormal liver functions and α-FP elevation.
Table 3.
Clinical manifestations of HCV patients with and without ANCA
| Clinical feature | With ANCA n = 278 | Without ANCA n = 238 | P-value |
|---|---|---|---|
| High ALT | 185 (66·5) | 106 (44·5) | <0·01 |
| High α-FP | 62 (22·3) | 26 (10·9) | <0·01 |
| Arthritis | 37 (13·3) | 40 (16·8) | |
| Skin disease | 10 (3·6) | 0 (0) | <0·01 |
| Liver cirrhosis | 49 (17·6) | 26 (10·9) | |
| Anaemia | 25 (9·0) | 0 (0) | <0·01 |
Numbers in parentheses are percentages. ALT=alanine aminotransferase; α-FP=alpha-Fetoprotein.
The correlation between the presence of anti-E3 antibody and clinical manifestations and laboratory findings was studied. As shown in Table 4, the frequency of high ALT, high ALP, high α-FT, arthritis and liver cirrhosis was higher in HCV patients with anti-E3 antibody than in those patients without anti-E3 antibody.
Table 4.
Clinical manifestations of HCV patients with and without E3 autoantibody
| Clinical feature | With anti-E3 n = 276 | Without anti-E3 n = 240 | P-value |
|---|---|---|---|
| Mean age (years) | 49·08 | 50·05 | |
| Sex (female/male) | 83/193 | 88/152 | |
| HCV-RNA positive | 190 (68·8) | 215 (89·6) | |
| High ALT | 205 (74·3) | 138 (57·5) | <0·01 |
| High ALP | 74 (26·8) | 19 (8·0) | <0·01 |
| High α-FP | 59 (21·4) | 19 (8·0) | <0·01 |
| Arthritis | 59 (21·4) | 19 (8·0) | <0·01 |
| Liver cirrhosis | 92 (33·3) | 41 (17·1) | <0·01 |
Numbers in parentheses are percentages. ALT=alanine aminotransferase; ALP=alkaline phosphate; α-FP=alpha-Fetoprotein.
The effect of interferon-α (IFN-α) treatment on the production of autoantibodies is shown in Table 5. Fifty-six of the 106 (52·8%) patients with IFN-α were anti-E3 positive whereas 220 of the 410 (53·7%) patients without IFN-α treatment were anti-E3 positive (P > 0·01). Similarly, 76 of the 106 (71·7%) patients with IFN-α treatment and 212 of the 410 (51·7%) patients without IFN-α treatment were RF positive. Twenty of the 106 (18·9%) patients with IFN-α treatment and 62 of the 410 (15·1%) patients without IFN-α treatment were ANA positive. Twelve of the 106 (11·3%) patients with IFN-α treatment and 41 of the 410 (10%) patients without IFN-α treatment were anti-cardiolipin antibody positive. Twenty-four of the 106 (22·6%) patients with IFN-α treatment and 254 of the 410 (62%) patients without IFN-α treatment were ANCA positive. The occurrence of autoantibodies in patients with HCV infection was not affected by the treatment of IFN-α.
Table 5.
The association between autoantibody production and interferon-alpha (IFN-α) therapy
| With IFN-α treatment n = 106 | Without IFN-Iα treatment n = 410 | P-value | |
|---|---|---|---|
| Anti-E3 Ab positive | 56 (52·8) | 220 (53·7) | >0·01 |
| RF positive | 76 (71·7) | 212 (51·7) | >0·01 |
| ANA positive | 20 (18·9) | 62 (15·1) | >0·01 |
| Anti-cardiolipin Ab positive | 12 (11·3) | 41 (10) | >0·01 |
| ANCA positive | 24 (22·6) | 254 (62) | >0·01 |
Numbers in parentheses are percentages. IFN-α=interferon-α; E3=dihydrolipoamide dehydrogenase; ANA=anti-nuclear antibody; ANCA=anti-neutrophil cytoplasmic antibody.
DISCUSSION
In the present study, we investigated the presence of autoantibodies, directed against various autoantigens in the sera of patients with HCV infection, using purified antoantigens and recombinant proteins as antigens. These autoantibodies were chosen for the study because they are common antibodies in systemic autoimmune diseases such as systemic lupus erythematosus (SLE), Sjogren’s syndrome (SS) and systemic sclerosis (SSc). Due to its lymphotropism, HCV has been reported to be associated with a broad spectrum of autoimmune manifestations, including cryoglobulinaemia, Sicca syndrome, vasculitis and lymphoproliferative disorders [2–8], but there remains some controversy as to whether it plays a role in the pathogenesis of autoimmune diseases.
In this study, we found that autoantibodies resulting from HCV infection were common, and a high prevalence of ANCA with anti-proteinase 3 (anti-Pr3) specificity, anti-E3 antibody and RF was found in patients with HCV infection. Anti-Pr3 and anti-E3 antibodies were in high titre in sera from patients with HCV infection. Using PCR to detect HCV-RNA, anti-Pr3 and anti-E3 antibodies were found more frequently in HCV patients with PCR positivity than those who were PCR negative. In patients with PCR positivity, titres of anti-Pr3 and anti-E3 antibodies were also higher. Anti-Pr3 and anti-E3 were rarely found in patients with HBV infection in the present study. The results indicate that proteinase 3 and E3 are the major antigens in patients with HCV infection.
We also found that HCV patients with ANCA had a higher prevalence of skin involvement, anaemia, abnormal liver functions and elevated α-FP levels. ANCA are present in a high percentage of patients with systemic vasculitis, who often have associated skin involvement and anaemia. The association of HCV infection with autoantibody production and autoimmune phenomenon was further confirmed.
This is the first report identifying proteinase 3 as an ANCA major antigen in patients with HCV infection. Although the presence of ANCA has already been described, it is still a rare occurrence [25]. Anti-Pr3 antibody is a characteristic antibody for vasculitis in Wegener’s granulomatosis [26,27]. HCV-induced ANCA production may provide a clue towards understanding the pathogenesis of autoimmune disease. ANCA testing should be considered in patients with HCV infection.
Anti-E3 antibody was detected in patients with PBC [28,29]. The significance of anti-E3 antibody in these patients is not clear. We have provided evidence for the presence of anti-E3 antibodies in the sera of patients with HCV infection. The prevalence of anti-E3 in patients with HCV infection was higher than that in PBC patients. Evidence for the presence of anti-E3 in these patients was tested by ELISA and immunoblotting. The HCV patients with anti-E3 antibodies showed a higher prevalence of liver cirrhosis, arthritis, abnormal liver function and α-FP levels. The association of anti-E3 with HCV infection in the present study has also provided a clue to the understanding of a possible connection between viral infection and autoimmunity.
The possibility of an association between autoantibody production in HCV patients and IFN-α therapy has been reported [30,31]. In our 516 patients with HCV infection (108 treated with IFN-α, 410 untreated), aCLA, ANCA, ANA, anti-E3 and RF were prevalent at similar levels in treated and untreated patients (P > 0·01). The prevalence of autoantibodies was not affected by treatment with IFN-α.
In conclusion, the present study shows that autoantibody production in patients with HCV infection is common and is not secondary to IFN-α treatment. ANCA, especially for anti-Pr3 antibody, anti-E3 antibody and RF, were highly prevalent in patients with HCV infection. HCV patients with ANCA and anti-E3 antibody showed a higher prevalence of clinical manifestations and abnormal liver functions. HCV may be regarded as a possible causative factor in ANCA-related vasculitis. Proteinase 3 and E3 are the major target antigens in HCV infection.
Acknowledgments
This study was supported by grants NSC 86-2314-B040-012 and NSC 89-2314-B040-021 from the National Science Council, Taiwan. The manuscript review by Professor William R. Bell, Johns Hopkins University, is greatly appreciated.
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