Autoimmune hepatitis

Background & objectives: Hepatitis C virus (HCV) infection is associated with several autoimmune markers. Despite HCV being common in India, no information on this aspect is available. This study was undertaken to ascertain the frequency and clinical significance of autoimmune markers like rheumatoid factor (RF), antinuclear antibodies (ANA), antibodies to double stranded deoxyribonucleic acid (dsDNA), anti neutrophil cytoplasmic antibody (ANCA), anti smooth muscle antibodies (ASMA), anti liver kidney microsomal I antibodies (anti LKM1), anti gastric parietal cell antibodies (anti GPCA), anti mitochondrial antibodies (AMA), anti cardiolipin antibodies (ACL) and cryoglobulins in HCV infection and to determine the effect of treatment on these markers.

Methods: Twenty five patients with chronic hepatitis C and 25 healthy controls were studied. Cryoglobulins were detected by cryoprecipitation, RF by latex agglutination, anti dsDNA and ACL by ELISA while indirect immunofluorescence was used to detect all other autoantibodies.

Results: Eighteen patients (72%) demonstrated autoimmune markers. RF, cryoglobulins and anti LKMI antibodies were the most frequently detected markers (in 32% patients each). ASMA, perinuclear ANCA (pANCA), ANA and anti GPCA were seen in 24, 20, 12 and 4 per cent patients respectively. None of the patients exhibited ACL, AMA or antibodies to dsDNA. No antibodies were detected in healthy controls. Sixty per cent of the patients had rheumatological symptoms. Of the seven patients followed up after treatment with alpha interferon, only two exhibited persistence of RF, while symptoms and other markers disappeared.

Interpretation cf conclusion: Rheumatological symptoms and autoimmune markers are common in HCV infection and are usually overlooked. Patients with unexplained joint pains and/or palpable purpura should be screened for HCV. Further studies are needed to delineate fully the link between infection and autoimmunity.

Key words Autoimmune markers - hepatitis C - rheumatological symptoms

Hepatitis C virus (HCV) infection affects nearly 1 per cent of the world population1. It is also reported to be common in India, accounting for 4.8-9 per cent cases of acute viral hepatitis and 13.8-25.6 per cent patients with chronic liver disease2,3. The frequency in healthy blood donors in India ranges from 1.8-15.9 per cent3,4. Extra hepatic clinical manifestations are often seen in HCV patients and involve primarily the joints, muscles and skin. Apart from these rheumatologic manifestations, several autoimmune markers like antinuclear antibodies (ANA), anti smooth muscle antibodies (ASMA), rheumatoid factor (RF), cryoglobulins etc., have been demonstrated in HCV infection5-8. Despite numerous reports of HCV infection from India there have been no studies on the rheumatologic manifestations or autoimmune markers in HCV infection from the country. Taking in view the significant prevalence of HCV in India coupled with lack of information about autoantibodies/rheumatological features in Indian patients with HCV, a prospective study was planned to ascertain the frequency and clinical significance of autoimmune markers in HCV infection and to determine the effect of treatment on these markers.

Material & Methods

Study design and patient population: This prospective study was carried out at the All India Institute of Medical Sciences (AIIMS), New Delhi from June 1997 to June 1999. The study group included 25 adult patients with chronic HCV infection selected by systematic random sampling from patients with HCV attending the liver clinic. The study was approved by the departmental committee and patients were recruited into the study after obtaining informed consent. HCV antibodies were detected by third generation ELISA technique. An in-house, validated, third generation, ELISA kit developed at the AIIMS, New Delhi was used. Patients with concomitant hepatitis B virus (HBV) infection or other forms of associated liver disease like alcoholic liver disease were excluded from the study.

Control group: The control group included 25 age and sex matched healthy individuals drawn from blood bank donors.

Clinical examination: All patients and control individuals were subjected to a detailed history and systemic examination, which included a comprehensive examination of the musculoskeletal system. Clinical features that were specifically looked for included arthralgias, arthritis, purpura, livedo reticularis, Raynaud's phenomenon and muscle weakness.

Autoimmune markers: The autoimmune markers studied included cryoglobulins, RF, ANA, ASMA, anti liver kidney microsomal 1 (LKM 1) antibodies, anti gastric parietal cell antibodies (anti GPCA), anti mitochondrial antibodies (AMA), antibodies to double stranded deoxyribonucleic acid (ds DNA), anti cardiolipin antibodies (ACL) and anti neutrophil cytoplasmic antibodies (ANCA).

Serum cryoglobulins were detected by cryoprecipitation. Briefly 20 ml of venous blood was taken from subjects in a prewarmed (37 deg C) syringe, allowed to clot at 370C and the serum separated by means of centrifugation, The supernatant was incubated at 4 deg C for 8 days and examined daily for cryoprecipitate.

RF was detected by latex agglutination10 test (Avitex-RF kit, Omega Diagnostics Limited, UK). Detection of ANA, ASMA, anti LKM1 antibodies, anti GPCA and AMA was done by indirect immunofluorescence (IIF) assay using air dried cryostat sections from rat liver, kidney and stomach as substrates11-13. ANA was interpreted to be positive when antibodies were found to be reacting with the nuclear antigen in the hepatocytes. ASMA was detected when antibodies were found to be reacting with gastric smooth muscle cells. Anti LKM I antibody reactivity was interpreted by positive immunofluorescence of the proximal kidney tubules and negative staining of the distal kidney tubules, with fine granular and uniform staining of liver sections. AMA was detected when antibodies reacted with distal kidney tubules as well as gastric parietal cells with the liver sections staining coarse granular. Presence of anti GPCA was interpreted by reactivity of antibodies with only gastric parietal cells. IF was also used to detect ANCA using neutrophils as a substrate14.

ELISA15,16 was used to detect anti dsDNA antibodies and IgG ACL (Varelisa dsDNA and Cardiolipin IgG antibody kits, Pharmacia & Upjohn, Germany).

Treatment: Seven of the 25 patients were able to afford specific treatment of HCV in form of alpha interferon therapy (a-IFN) given subcutaneously (1-3 million units, thrice weekly). These patients were re-evaluated at the completion of 6 months of treatment with alpha-IFN treatment and all the autoimmune markers were again measured.

Statistical analysis: Fisher's exact test was used to compare the patient and control groups.

Results

The study group of 25 patients with chronic HCV infection had 16 males and 9 females (mean age SD; 3913 yr). The source of infection was blood transfusion in 23 patients and intravenous drug use in two. The control group consisted of 15 males and 10 females (mean age +/- SD; 38 +/- 11.8 yr).

Rheumatological manifestations were seen in 15 (60%) HCV patients (10 males, 5 females). These included arthralgias (56%), arthritis involving small and large joints((%), palpable purpura (44%), myalgias (36%),.and Raynaud's phenomenon (4%). Autoimmune markers were seen in 18 (72%) patients [cryoglobulins, RF and anti LKM 1 antibodies in 8 patients each (32%), ASMA in 6 (24%) pANCA in 5 (20%), ANA in 3 (12%), and anti GPCA in one (4%) patient]. None of the patients had anti dsDNA antibodies, AMA or ACL antibodies. All controls tested negative for autoimmune markers. Statistically significant (P

Discussion

It is being increasingly recognised that the spectrum of HCV infection extends beyond the liver5-8. Apart from extrahepatic manifestations such as Sjogren's syndrome, membranoproliferative glomerulonephritis, arthritis etc., several autoimmune markers viz., ANA, RF, ASMA, ACL have been reported in HCV infection6-8. Humoral autoimmune mechanisms are said to be the connecting link between arthritis and hepatitis. A high proportion (60%) of our patients had rheumatic symptoms. The clinical picture of arthritis (8%lo patients) was indistinguishable from rheumatoid arthritis as has been reported in the literature17,18. The clinical importance of this lies in the fact that patients with anicteric hepatitis C and arthritis may inadvertently get treated with methotrexate with worsening of liver disease. This can be obviated by high index of suspicion and obtaining aminotransferases in all patients with arthritis. Elevated base line aminotransferases should be considered as an indication for hepatitis C screening. Antikeratin antibodies, if present, lend support to the diagnosis of rheumatoid arthritis rather than hepatitis C arthritis19.

No data are available from India regarding the presence of autoimmune markers in hepatitis C. Studies in different population groups from all over the world20-25 reveal that autoimmune markers are frequent in hepatitis C though the frequency varies widely. Cryoglobulins have been reported in 11-36 per cent patients, RF in 44-76 per cent, anti LKM 1 antibodies in 2.4-5 per cent, ASMA 5-66 per cent, ANA 6.2-63 per cent, AMA 1-4 per cent and ACL antibodies in 0-22 per cent patients with HCV. The common autoantibodies detected in our patients were cryoglobulins, RF and anti LKM1 antibodies. However, none of the patients had renal or neurologic disease suggestive of cryoglobulinemic vasculitis syndrome. One drawback of our study is that we could not do the isotyping and clonality analysis of cryoglobulins in our patients. The disappearance of autoimmune markers with treatment suggests a causal link between the markers and HCV. The clinical course and response to treatment in our patients with or without autoimmune markers was similar in keeping with the observations in the literature6,25.

The presence of autoantibodies in hepatitis C appears to be an expression of generalized immune activation by cytokines, although the exact mechanisms are not clearly understood26. The potential of HCV to induce autoreactive CD8(+) cytotoxic T lymphocytes by molecular mimicry may be responsible27. It has also been demonstrated that CD4+ T cells expressing CD45RO and CD95 are increased in HCV. These CD95+ CD45RO+ primed T cells most likely reflect a continuous antigen specific or nonspecific activation of T lymphocytes, and/or the persistent presence of activated lymphocytes as a consequence of abnormalities in the peripheral deletion of activated lymphocytes. These persistently activated lymphocytes may be playing a role in the induction of autoimmunity28.

In conclusion, our study indicates that musculoskeletal symptoms and autoimmune markers are fairly common in HCV infection. Patients presenting with new rheumatic symptoms and risk factors for HCV should be screened for hepatitis C. Further studies are required to establish a link between HCV infection and autoimmunity.

Acknowledgment

The authors thank Prof. R.N. Misra for providing anti LKMI antibody positive serum as control. Expert laboratory work of Sriyuts Titus Varghese, Sube Singh, Gopal Singh and Chandrasenan is gratefully acknowledged.

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N. Agarwal, R. Handa, S.K. Acharya*, J.P. Wali, A.K. Dinda** & P. Aggarwal

Departments of Medicine, *Gastroenrology & **Pathology, All India Insitute of Medical Sciences, New Delhi, India

Received February 5, 2001

Reprint requests: Dr R. Handa, Associate Professor, Department of Medicine, All India Institute of Medical Sciences Ansari Nagar, New Delhi 110029, India

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