Acute hepatitis caused by alverine associated with anti-lamin A and C autoantibodies

Journal of Hepatology 1997; 27: 399-403 Printed in Denmark AN rights reserved Munksgaard Copenhagen

Copyright0 EuropeanAssociation for the Study o/‘fhe Liver 1997 Journal of Hepatology ISSN 011X3-8278

Case Report

Acute hepatitis caused by alverine associated with anti4mi.n A and C autoantibodies David Malka’, ‘Service d’Hkpatologie

Bach-Nga

Pham2, Jean-Claude Courvalin 3, Michele Corbic’, Serge Erlinger’

Dominique

Pessayre’ and

et Unit& de Recherche de Physiopathologie HL;oatique (INSERM Unite 24). 2Serviee d’Ht!matologie H6pital Beaujon, Clichy. and 31nstitut Jacques Monad, CNRS, UniversitP Paris VII, Paris, France

et Immunologic,

We report the case of a 67-year-old woman in whom onset and regression of acute hepatitis were closely related to the time of administration and withdrawal of the smooth muscle relaxant alverine. Antinuclear antibodies were positive, and their titer followed the course of hepatitis. They presented a smooth rim-like nuclear immunofluorescence staining pattern. Immunoblot assay showed that they were directed against

lamin A and lamin C. This suggests that alverine should be added to the list of drugs known to produce acute hepatitis, and that drug-induced liver injury is a possible cause of antinuclear antibodies specific for lamin A and lamin C.

D

However, there are very few data concerning antinuclear antibodies in drug-induced liver disease and even less about their specificity. We report here the case of a woman presenting with acute hepatitis, secondary to alverine administration, characterized by the occurrence of antinuclear antibodies directed against lamins A and C.

a common cause of acute liver disease. The severity of drug-induced hepatitis may vary from a mild elevation of liver enzyme levels without symptoms to fulminant hepatitis (1). Alverine is a chemical agent used as a smooth muscle relaxant. It is prescribed as an anti-spasmodic drug in patients with irritable bowel syndrome (2). In humans, the drug is completely absorbed by the gastrointestinal tract and mainly metabolized in the liver, with negligible amounts excreted in the urine (2). Hepatotoxicity of alverine has not been reported. Autoantibodies are present in some drug-induced hepatitis (3). Beside anti-smooth muscle antibodies, anti-mitochondrial antibodies type 6 and anti-liver kidney microsomal antibodies associated with drug-induced hepatitis (4-7) antinuclear antibodies have been described in patients with drug-induced autoimmune disease affecting the liver, such as lupoid hepatitis induced by oxyphenisatin (8) or similar disease induced by alpha-methyldopa (9) and nitrofurantoin (10). RUGS ARE

Received 4 February: revised 10 March; accepted 18 March 1997

Correspondence: Dr Bach-Nga Pham, Service d’Htmatologie et Immunologie, HGpital Beaujon, 100 Boulevard du General Leclerc, 92110 Clichy, France. Tel: (33) 1 40 87 55 40. Fax (33) 1 40 87 56 83.

Key words: Alverine; Antinuclear antibodies; Autoantibodies; Drug-induced hepatitis; Lamin.

Case Report A 67-year-old Caucasian woman was referred with a 2-month history of progressive painless jaundice and pruritus, without fever, rash, myalgia or arthralgia. The patient complained of anorexia and a 1O-kg weight loss. Five weeks before the onset of symptoms, she began a course of 180 mg/day alverine for irritable bowel syndrome. Alverine was added to psyllium, started 18 months previously She had had a blood transfusion 20 years before, during a hemicolectomy for a carcinomatous polyp (the last colonoscopy was normal). She had no previous history of jaundice or contact with jaundiced patients, intravenous drug use or alcohol misuse. Besides jaundice, her physical examination revealed only mild hepatomegaly, without signs of hepatic failure or portal hypertension. Laboratory tests disclosed severely impaired liver function (Table 1). Total bilirubin was 430 pmolll. As-

D. Malka TABLE

et (11

1

Results of laboratory

studies during

the course

of the disease

During

Serum alanine aminotransferase level Serum bilirubin level ($mol/l) Eosinophilia (X 109/1) Prothrombin time (%I activity of normal value) Antinuclear antibody titer’ Antinuclear

antibody

specificity’

alverine

therapy

Drug withdrawal Week 11

After withdrawal Day 15

Day 17

Day 60

Day 100

Week 6

Week 8

Week 10

43xN3 114 1.1 100%

25xN 242 N

ND ND ND ND

7.5 N 273 ND ND

8.7 N 257 0.12 86%

1.1 53 0.06 81%

N

100%

97xN 430 ND 62%

ANA not detected ND“

ND

ND

1:200

I:200

1:200

1:50

ND

ND

Lamin A Lamin C

ND

Lamin A Lamin C

Lamin /

ANA not detected ND

’ Antinuclear antibodies were titered by indirect immunofluorescence. The titer of antinuclear highest dilution of serum resulting in positive nuclear immunofluorescence. 2 The specificity of autoantibodies was assessed by immunoblotting. 3 N: Normal value. The upper limit of normal value was 40 IU/l. 4 ND: not determined.

partate aminotransferase (AST) was 3880 IU/l (the upper limit of normal values was 40 IUil), alanine aminotransferase (ALT) was 2320 III/l. Alkaline phosphatase was 325 IU/l (the upper limit of normal values was 130 IV/l). yGT was 326 IU/l (the upper limit of normal value was 40 III/l). Gammaglobulin concentrations were normal. The blood cell counts revealed hypereosinophilia at 1.06X 109/1. Coagulation tests, creatinine and blood urea nitrogen were normal. Serological viral markers were absent: IgM to hepatitis A virus, hepatitis B surface antigen (HBsAg), antibodies to HBsAg, IgM to hepatitis B core antigen (HBc), antibodies to hepatitis C virus (HCV) were not detected. Polymerase chain reactions for the detection of HCV RNA were repeatedly negative. Antibodies to human immunodeficiency virus, IgG to Epstein Barr virus, cytomegalovirus or herpes simplex virus, were not detected in serum when routine serologic tests were performed. Antinuclear antibodies exhibiting a smooth rim-like nuclear immunofluorescence staining pattern were detected (see below). The screening for antismooth muscle, anti-mitochondrial or anti-liver kidney microsome antibodies was negative. Abdominal ultrasonography disclosed mild hepatomegaly and slight ascites. A transvenous liver biopsy was performed 17 days after drug withdrawal (almost 3 months after beginning drug intake). Hepatic hemodynamic study showed portal hypertension, with a wedge hepatic pressure gradient of 14 mmHg. Histological examination showed numerous foci of collapse, inflammatory infiltrates containing eosinophils, centrilobular cholestasis and hepatocellular necrosis, without fibrosis or architectural disarray, all findings consistent with druginduced hepatitis. Less than 2 weeks after drug withdrawal, AST, ALT, alkaline phosphatase and yGT de-

antibodies

was expressed

19 ND 100%

A

as the reciprocal

of the

creased markedly. Jaundice and pruritus were improved. Three months after drug withdrawal, the patient was symptom-free and her examination was unremarkable. The liver tests (AST, ALT, alkaline phosphatase and yGT) were normal. Antinuclear antibodies were absent. Abdominal ultrasonography showed regression of the hepatomegaly and ascites. No repeat biopsy was performed.

Materials and Methods Serum samples were collected and divided into aliquots and stored at -20°C until analyzed. HBsAg, antibodies to HBsAg and HBcAg were analyzed using commercially available radioimmunoassays (Abbott Laboratories, Chicago, IL, USA). Serum samples were tested for anti-hepatitis A virus IgM using the IMX assay (Abbott Laboratories). Serum samples were tested for anti-HCV antibodies using a third-generation enzyme immunoassay (Abbott Laboratories). HCV RNA detection was performed with reverse transcriptase “nested” polymerase chain reaction (PCR) as previously described (11). Briefly, RNA was extracted from 100 ~1 of serum stored at -20°C. We used two sets of synthetic oligonucleotide primers derived from the 5’ non-coding region of the HCV genome. These primers directed the amplification of 277 bp and 177 bp fragments, respectively Antinuclear antibodies were detected by standard routine indirect immunofluorescence and immunoblotting. Indirect immunofluorescence was performed using fixed sections of rat tissue (12) and HeLa cells as substrates. Binding of autoantibodies was detected with affinity-purified goat F(ab’)2 anti-human immunoglobulins antibodies labeled with FITC (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France)

Drug-induced hepatitis bvith anti-kumin antibodies

drug intake (Table 1). In contrast, antinuclear antibodies displaying a smooth rim-like nuclear immunofluorescence staining pattern were detected in sera collected at the time of drug withdrawal and until 60 days after drug withdrawal (Fig. 1). The titer of antinuclear antibodies initially increased up to 1:200 and then decreased. At day 100 after drug withdrawal, antinuclear antibodies were not detected. Sera with antinuclear antibodies were tested by immunoblotting using total nuclear proteins. The serum samples recognized on immunoblots two bands at 74 kDa and 64 kDa representing lamin A and lamin C, respectively (Fig 2, lanes l-3). While signals for both lamins were strong at the time of drug withdrawal (Fig. 2, lane l), they decreased after withdrawal of the drug,

Fig. 1. Indirect immunojuorescence performed with the serum of the patient. HeLa cells grown attached on coverslips were-fixed in methanol, then processed for indirect immunojluorescence, using the serum at a l/100 dilution. Three nuclei of cells in interphase are shown with a homogeneous labeling and a peripheral rim that correspond to the intranuclear polymerized lamins. Arrowhead indicates a cell in metaphase of mitosis. The dark central region in this cell corresponds to the spindle and the chromosomes mass and the peripheral bright area to the cytoplasm containing the solubilized lamins. Bar 5 pm.

used at 1:lOO dilution. Sera given positive staining at a 1:20 dilution were further examined in serial dilutions. The titer of antinuclear antibodies was expressed as the reciprocal of the highest dilution of serum resulting in nuclear immunofluorescence. Nuclei, nuclear envelope, and nuclear envelope subfractions were prepared from rat liver as previously described (13). SDS-solubilized proteins were subjected to electrophoresis under reducing conditions on polyacrylamide gels according to Laemmli (14). Proteins were transferred to nitrocellulose sheets by electrophoresis using a semi-dry method. All the following steps were carried out in 10 mM Tris-HCl (pH 7.4) 0.15 M NaCl, 0.1% Tween-20, and 50 g/l non-fat milk (Carnation, Los Angeles, CA, USA). Nitrocellulose strips were blocked for 1 h, incubated for 1 h with a 1:100 dilution of serum, washed four times, incubated for 1 h with a 1:7500 dilution of anti-human IgG conjugated to alkaline phosphatase (Promega Madison, WI, USA) and finally revealed using the BCIP/NBT reaction.

Results No antinuclear antibody was detected in the 1:20 diluted serum collected 6 weeks after the beginning of

gp210m

lam Ah lam Blam C-

_.

1

2

3

4

5

6

Fig. 2. Immunoblotting of nuclear envelope proteins with human autoantibodies. Rat liver nuclear envelopes were isolated as indicated (see Methods). Proteins were separated by electrophoresis on a 8% denaturing polyacrylamide gel, then transferred on nitrocellulose and finally revealed by incubation with several human autoantibodies and secondary antibodies coupled to alkaline phosphatase. Serum samples were tested at a I:100 dilution. Lane I, patient serum at the time of alverine withdrawal, lanes 2 and 3, same patient after 17 days and 60 days of withdrawal, respectively. Lanes 4 to 6, control human autoimmune sera directed against all 3 lamins (lane 4), lamin B (lane 5), and nuclear pore protein gp210 (lane 6), respectively. 401

D. Malka

et al

with disappearance of the signal for lamin C after 2 months (Fig. 2, lane 3).

Discussion Acute hepatitis in this patient was probably related to administration of alverine, using the imputation criteria defined by Consensus Meetings on the causality assessment of drug-induced liver injury (15). Firstly, there was no previous history of liver or biliary disease or alcohol intake. Secondly, there were no serological arguments in favor of viral infection. Thirdly, classical markers for primary biliary cirrhosis or autoimmune hepatitis were negative. Fourthly, there was no ultrasonographic evidence of underlying biliary tract disease. Fifthly, other types of liver disease could reasonably be ruled out: there was no evidence of extra-hepatic auto-immune or systemic manifestations, malignant infiltration of the liver, heart disease, or previous history of hypotension episodes; the usual criteria for diagnosis of systemic lupus erythematosus, particularly the detection of antibody to double-stranded DNA, were not fulfilled; and the levels of serum iron were normal. Sixthly, although alverine readministration was not performed, a close chronological relationship was found between alverine intake and hepatic injury, and between withdrawal of the drug and regression of clinical and biochemical disorders. Seventhly, except for psyllium, taken by the patient 18 months before the onset of hepatitis, no other drug was taken simultaneously, nor previously; psyllium is not thought to cause drug-induced liver disease because it is not absorbed by the gastro-intestinal tract. Lastly, the histological features were consistent with drug-induced hepatitis (15). We report here the first case of alverine hepatotoxicity. Alverine belongs to a group of drugs with papaverine-like effects, although its chemical structure is different from that of papaverine. Several cases of acute and chronic hepatotoxicity due to papaverine have been reported (1619). Papaverine hepatotoxicity has been suggested to be related to an immune mechanism. This hypothesis was supported by the short interval between the rechallenge with the drug and the recurrence of symptoms. In this case, several arguments support the hypothesis that alverine hepatotoxicity is also related to an immune mechanism: a) the transient hypereosinophilia: b) the presence of eosinophi1 polymorphonuclear cells in the liver inflammatory infiltrates; and c) the presence of antinuclear autoantibodies. This case of drug-induced hepatitis is remarkable because of the detection in patient serum of antinuclear antibodies directed against lamins A and C, an 402

immunological feature not yet ascribed to drug-induced hepatitis. Antinuclear antibodies are useful diagnostic markers for autoimmune diseases (20). Antinuclear antibodies are directed against different components of cell nuclei that generate different immunofluorescence staining patterns described as homogeneous, speckled, nucleolar or rim-like. Antinuclear antibodies with a rim-like staining pattern usually recognize nuclear envelope proteins (21). The nuclear envelope is composed of the nuclear membranes, the nuclear pore complexes and the nuclear lamina. The nuclear pore complex contains two proteins called gp 210 and p 62, which are recognized by autoantibodies found in patients with primary biliary cirrhosis (13,22-24). The nuclear lamina is composed of a family of proteins called lamins. Lamins A, B, and C are recognized by autoantibodies in a variety of diseases. Anti-lamin antibodies have been described in patients with systemic lupus erythematosus (25), rheumatic diseases (26,27), autoimmune hepatitis (28) and other diseases (28-32). To our knowledge, anti-lamin antibodies have not previously been reported in drug-induced hepatitis. Our patient did not have antinuclear antibodies prior to alverine therapy, and they disappeared 100 days after alverine withdrawal. This suggests that they are closely related to alverine intake. However, the detection of anti-lamin antibodies was delayed compared to the rise in liver enzyme and bilirubin levels. This suggests that the occurrence of anti-lamin antibodies is an epiphenomenon related to liver injury rather than a causative event. This patient initially developed autoantibodies directed against lamins A and C. Later, only autoantibodies directed against lamin A could be detected. Autoantibodies against lamin A generally also recognize lamin C because of structural similarities between lamins A and C, although autoantibodies directed only against lamin A have been described (33). In this case, the autoantibody profile suggests that autoantibodies are polyclonal and that they recognize multiple epitopes. In conclusion, we report the first case of acute hepatitis probably caused by alverine. In addition, druginduced liver injury should be considered as a possible cause of anti-lamin autoantibodies.

Acknowledgements We thank Mrs A. Castot, P. Castex and C. Quilichini for their help.

References 1. Pessayre D, Larrey D. Drug-induced liver injury. In: McIntyre N, Benhamou JP, Bircher J, Rizetto M, Rodes J, editors. Ox-

Drug-induced hepatitis with anti-lamin antibodies ford Textbook of Clinical Hepatology. Oxford: Oxford University Press; 1992. p. 875-902. 2. Vidal 1996. 72nd Edn. Paris: Editions Vidal; 1996. 3. Farrell GC. Drug-Induced Liver Disease. Edinburgh, London, Madrid, Melbourne, New York and Tokyo: Churchill Livingstone; 1994. 4. Homberg JC, Abuaf N, Helmy-Khalil S, Biour M, Poupon R, Islam S, et al. Drug-induced hepatitis associated with anticytoplasmic organelle autoantibodies. Hepatology 1985; 5: 722-7. 5. Homberg JC, Stelly N, Andreis I, Abuaf N, Saadoun F, Andre J. A new antimitochondrial antibody (anti-M6) in iproniazid-induced hepatitis. Clin Exp Immunol 1982; 47: 93102. 6. Bourdi M, Larrey D, Nataf J, Pessayre D, Iwasaki M, Guengerich FP et al. Anti-liver endoplasmic reticulum autoantibodies are directed against human cytochrome P-450IA2. J Clin Invest 1990; 85: 1967-73. 7. Beaune P Dansette PM, Mansuy D, Kiffel L, Finck M, Amar C, et al. Human anti-endoplasmic reticulum autoantibodies appearing in a drug-induced hepatitis are directed against a human liver cytochrome P-450 that hydroxylates the drug. Proc Nat1 Acad Sci USA 1987; 84: 551-5. 8. Reynolds TB, Peters RL, Yamada S. Chronic active and lupoid hepatitis caused by a laxative, oxyphenisatin. N Engl J Med 1971; 280: 813-20. 9. Toghill PJ, Smith PG, Benton P Brown RC, Matthews HL. Methyldopa liver damage. Br Med J 1974; 3: 545-8. 10. Iwarson S, Lindberg J, Lunden P Nitrofurantoin-induced chronic liver disease. Clinical course and outcome of five cases. Stand J Gastroenterol 1979; 14: 497-50 11. Martinot-Peignoux M, Marcellin P Xu LZ, Benhamou JP Larzul D. Reactivity to c33c antigen as a marker of hepatitis C virus multiplication. J Infect Dis 1992; 19: 133741. 12. Homberg JC, Abuaf N, Bernard 0, Islam S, Alvarez F, Khadil SH, et al. Chronic active hepatitis associated with antiliver/kidney microsome antibody type 1: a second type of “autoimmune” hepatitis. Hepatology 1987; 7: 1333-9. 13. Courvalin JC, Lassoued K, Bartnik E, Blobel G, Wozniak RW. The 210 kilodalton nuclear envelope polypeptide recognized by human autoantibodies in primary biliary cirrhosis is the major glycoprotein of the nuclear pore. J Clin Invest 1990; 85: 279-85. 14. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5. 15. Danan G. Consensus meetings on causality assessment of drug-induced liver injury. J Hepatol 1988; 7: 132-6. 16. Ronnov-Jessen V, Tjernlund A. Hepatotoxicity due to treatment with papaverine: report of four cases. N Engl J Med 1969; 281: 133335. 17. Kiaer HW, Olsen S, Ronnov-Jessen V Hepatotoxicity of papaverine. Arch Path01 1974; 98: 292-6. 18. Poupon R, Longchal C, Darnis F. Hepatite chronique assoc-

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

ibe a la prise prolongee de papadrine. Gastroenterol Clin Biol 1978; 2: 305-8. Poncin E, Sylvain C, Touchard G, Barbier J, Beauchant M. Papaverine-induced chronic liver disease. Gastroenterology 1986; 90: 1051-3. Tan EM. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immuno1 1989; 144: 93-151. Worman HJ, Courvalin JC. Autoantibodies against nuclear envelope proteins in liver disease. Hepatology 1991; 14: 126979. Lassoued K, Guilly MN, Andre C, Paintrand M, Dhumeaux D, Danon E et al. Autoantibodies to 200 kD polypeptide(s) of the nuclear envelope: a new serologic marker of primary biliary cirrhosis. Clin Exp Immunol 1988; 74: 283-8. Lassoued K, Brenard R, Degos E Courvalin JC, Andre C, Danon E et al. Antinuclear antibodies directed to a 200-kilodalton polypeptide of the nuclear envelope in primary biliary cirrhosis. Gastroenterology 1990; 99: 181-6. Wesierska-Gadek J, Hohenauer H, Hitchman E, Penner E. Autoantibodies against nucleoporin p62 constitute a novel marker of primary biliary cirrhosis. Gastroenterology 1996; 110: 840-7. Reeves WH, Chaudhary N, Salerno A, Blobel G. Lamin B autoantibodies in sera of certain patients with systemic lupus erythematosus. J Exp Med 1987; 165: 750-62 Konstantinov K, Halberg P Wiik A, Moier-Madsen M, Wantzin P Ullman S, et al. Clinical manifestations in patients with autoantibodies specific for nuclear lamin proteins. Clin Immunol Immunopatholl992; 62: 112-8. Lassoued S, Oksman E, Fournie B, Danon E Fournie A, Lassoued K. Autoantibodies to lamins in rheumatoid arthritis. Arthritis Rheum 1990; 33: 877-9. Wesierska-Gadek J, Penner E, Hitchman E, Sauermann G. Antibodies to nuclear lamins in autoimmune liver disease. Clin Immunol Immunopathol 1988; 49: 107-15. Lassoued K, Guilly MN, Danon F, Andre C, Dhumeaux D, Clauvel JP et al. Antinuclear autoantibodies specific for lamins. Characterization and clinical significance. Ann Intern Med 1988; 108: 829-33. McKeon FD, Tuffanelli DL, Fukuyama K, Kirschner MW. Autoimmune response directed against conserved determinants of nuclear envelope proteins in a patient with linear scleroderma. Proc Nat1 Acad Sci USA 1983; 80: 4374-8. Guilly MN, Danon F, Brouet JC, Bornens M, Courvalin JC. Autoantibodies to nuclear lamin B in a patient with thrombocytopenia. Eur J Cell Biol 1987; 43: 26672. Wesierska-Gadek J, Penner E, Hitchman E, Sauermann G. Antibodies to nuclear lamin C in chronic hepatitis delta virus infection. Hepatology 1990; 12: 1129-33. Courvalin JC, Chaudhary N, Danon E Brouet JC, Lassoued K. Characterization of human autoantibodies specific for lamin A. Biol Cell 1990; 69: 93-7.

403