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Cross-reactivity of anti-Mycobacterium gordonae antibodies with the major mitochondrial autoantigens in primary biliary cirrhosis
Copyright © Journalof Hepatology 1994
Journal of Hepatology 1994; 21:673-677 Pr#lted hz Denmark. All rights reserved Munksgaard. Copenhagen
Journal of Hepatology ISSN 0168-8278
Rapid Publication
Cross-reactivity of anti-Mycobacteriumgordonae antibodies with the major mitochondrial autoantigens in primary biliary cirrhosis Lidia Vilagut l, Jordi Vila l, Odette Vifias 2, Albert Par6s 3, Angels Gin6s 3, M a r i a Teresa Jim6nez de A n t a l and Joan Rod6s 3 ILaboratorv o f Microbiology, 2Servei d'hnmunologia and 3Liver Unit, Hospital Clinic i Provincial, Universit.v o f Barcelona, Barcelona, Spain
(Received 30 December 1993)
Primary biliary cirrhosis is a chronic cholestatic liver disease associated with autoimmune disorders. Antimitochondrial autoantibodies and granulomatous portal lesions are characteristic in primary biliary cirrhosis. Since granuloma may be induced by Mycobacteria, and there is evidence implicating Mycobacteria as infectious agents capable of initiating autoimmunity, a study was performed to determine the presence of antibodies against 10 atypical Mycobacteria in 19 patients with primary biliary cirrhosis, and in 35 controls (25 patients with other chronic liver diseases and 10 healthy subjects). All primary biliary cirrhosis sera.and none of the controls reacted with the extract from Mycobacterium gordonae, showing identical recognition profiles with two polypeptides of 70-65 and 55 kDa. No other reaction was found in primary biliary cirrhosis patients and in controls with the extracts from the other nine atypical Mycobacteria tested. Eluted immunoglobulins which reacted with the 70-65 and 55 kDa polypeptides from M. gordonae, bound to the mitochondrial antigens PDH-E2 and BCKDH-E2. Furthermore, when the extract from M. gordonae was tested with eluted immunoglobulins from recognized PDH-E2 and BCKDH-E2 by primary biliary cirrhosis patients, we observed both 70-65 and,55 kDa polypeptides. These data indicate that antibodies to M. gordonae, found in all primary biliary cirrhosis patients, crossreact with the major mitochondrial targets of the disease. We suggest that M. gordonae may play a potential pathogenic role in primary biliary cirrhosis. © Journal of Hepatology.
Key words: Antimitochondrial autoantibodies; Autoimmunity; Molecular mimicry; Mycobacterial components
Primary biliary cirrhosis (PBC) is a chronic cholestastic liver disease of unknown etiology. The pathogenesis of PBC is considered autoimmune because of the presence of many immunological abnormalities, and its association with other autoimmune disorders. The most fascinating fact is that practically all PBC patients present antimitochondrial autoantibodies which, on immunoblots, recognize five antigens from the mitochondrial multienzymatic complex named alpha-ketoacid dehydrogenase complex, at apparent molecular weights of 75-70, 56, 51, 45 and 36 kDa (1). The 75-70 kDa antigen has been identified as the dihydrolipoamide acetyltransferase (E2) of the pyruvate dehydrogenase complex (PDH-E2), the 51 kDa antigen
corresponds to the E2 of the branched chain alpha-ketoacid dehydrogenase complex (BCKDH-E2), or to the E2 of the alpha-ketoglutarate dehydrogenase complex (aKGDH). The 56 kDa antigen is designated as protein X, a component of the PDH (PDH-X) and the remaining two antigens of 45 and 36 kDa are the alpha-subunit and the beta-subunit, respectively, of the pyruvate dehydrogenase (El) from the pyruvate dehydrogenase complex. Previous work showed that sera from PBC patients react with a wide spectrum of prokaryotes, including E. coli (2). Despite these studies, up to now there has been no definite evidence that a specific infectious agent may be the precipitating mechanism initiating bile duct lesions.
Correspondence to: Joan Rod6s MD, Liver Unit, Hospital Clinic i Provincial,C/Villarroel 170, 08036 Barcelona,Spain.
674
Mycobacteria are thought to play an etiological role in autoimmune and idiopathic diseases (3). Since granuloma, identifiable either as aggregates of histiocytes or as noncaseating lesions adjacent to damaged bile ducts, are characteristic in PBC, and since granuloma may be induced by Mycobacteria (4), the current study aimed to investigate whether sera of patients with PBC react against antigens from atypical Mycobacteria. Patients and Methods
Patients The study was performed in sera from 19 patients who met the clinical, biochemical and histological criteria for PBC, 25 patients with chronic liver diseases other than PBC (10 chronic active hepatitis B, 10 chronic active hepatitis C, five autoimmune chronic active hepatitis), and 10 healthy subjects.
Mycobacterial strahls The mycobacterial reference strains were Mycobacterium chelonei TMC 1542 (Trudeau Mycobacteria Collection), M. flavescens CCTM 1074 (Centre de Collection de Types Microbiens. Lausanne, Switzerland), M. fortuiturn TMC 1529, M. gordonae TMC 1318, M. intracellulare TMC 1403, M. kansasii TMC 1201, M. mahnoense CCTM 1635, M. scrofulaceum ATCC 1998 (American Type Culture Collection. Rockville, Md), M. xenopi NCTC 10042 (National Collection of Type Cultures. London, United Kingdom) and M. terrae. This latter strain was isolated from a clinical specimen and identified by standard biochemical procedures and high performance gas chromatography. All specimens were grown in Middlebrook 7H9 broth and were streaked onto Middlebrook 7 H l l agar medium plates to ensure pure cultures.
Preparation of membrane proteins of mycobacterial suspensions Growths from 100-ml cultures were pelleted, washed twice with 50 mM Tris-HCl, 2 mM EDTA, pH 8.5, containing 100/tM benzamidine hydrochloride and 100/lM phenylmethylsulfonyl fluoride, and suspended in 6 ml of the same buffer. Organisms were sonicated four times for 30 s while cooling. The cell debris were then centrifuged at 900 g for 20 min, and the supernatants were centrifuged at 12 300 g for 60 min. Finally, the membrane pellets were resuspended in 200/tl of 2 mM Tris-HCl, pH 7.7, containing 100/iM benzamidine hydrochloride and 100/~M phenylmethylsulfonyl fluoride.
L. VILAGUT et al.
Pyruvate dehydrogenase complex The purified pyruvate dehydrogenase complex from porcine heart, containing< 15% of the alpha-ketoglutarate dehydrogenase complex, was supplied by Sigma. hnmunoblotting studies One hundred and 300/tg of mycobacterial and mitochondrial antigens, respectively, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as previously described (5). The gels were electroblotted, using a trans-blot semi-dry apparatus (BioRad), onto nitrocellulose membranes. A control for the transfer of proteins was carried out using pre-stained molecular weight standards. The blots were blocked overnight at 4°C in phosphate-buffered saline (PBS) with 10% skim milk, 2% ovalbumin, 1% bovine serum albumin (BSA), and 0.2% Tween-20. They were then incubated with 1:50 diluted human sera in blocking solution for 1 h. After two 15-min washes with PBS-0.25% gelatine, the reacting antibodies were detected by peroxidase-conjugated rabbit anti-human immunoglobulins (Dako) at 1:200 dilution in blocking solution for 1 h. After two additional washes, a substrate solution containing 600 mg/ml of diaminobenzidine (Sigma) and 0.03% hydrogen peroxide in PBS was added to detect antigen-bound antibodies. Elution of antibodies After immunoblotting, antibodies bound to their specific antigen were eluted by cutting out the bands of the nitrocellulose membrane and placing them in a syringe, where a solution of 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.15 M NaC1, 0.5% Tween-20 and 100 pg/ml of BSA was forced twice. Afterwards, 3 ml of a solution of 0.2 M glycine-HC1 pH 2.8, 0.5 M NaCI, 0.5% Tween-20 and I00 pg/ml of BSA was added to the syringe and left for 2 min. This solution was also forced twice. The eluted material was immediately neutralized with NaOH, diluted in PBS and then concentrated using a Minicom-B clinical sample concentrator. This procedure was carried out at 4°C. The eluted antibodies were re-tested by the abovedescribed immunoblotting procedure. Informed consent was obtained from patients and healthy subjects, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Ethics Committee of the Hospital Clinic i Provincial. Results
The clinical, biochemical and histological features of patients with PBC are summarized in Table 1. In order to characterize the specific antigens recognized
MYCOBACTERIUMGORDONAEIN PBC
675
TABLE 1 Clinical, laboratory data and histological stages of PBC patients Age (years) Sex (F/M) Sj6gren's syndrome Biochemical tests ALT (u/l) AF (u/l) Bil (mg/dl) Immunoglobulins IgG (mg/dl) IgA (mg/dl) IgM (mg/dl)
54.9___1.8 17/2 14 (74%) 108.4± 11.4 1002± 130 2.1 ±0.9
Discussion
2326± 178 408±49 633±79
Histological stage I
2
II III IV
8 5 4
and 55 kDa polypeptid.es from M. gordonae were eluted and re-tested against the mitochondrial antigens. The two types of antibodies, bound to PDH-E2 and BCKDH-E2 (Fig. 2A). Conversely, antibodies eluted from PDH-E2 and BCKDH-E2 recognized by PBC patients, bound to the 70-65 and 55 kDa polypeptides from M. gordonae extract (Fig. 2B).
ALT: alanine aminotransferase; AF: alkaline phosphatase; Bil: bili-
rubin. by antimitochondrial antibodies in PBC sera, an immunoblot was performed using the purified mitochondrial antigens. All sera from patients with PBC were antimitochondrial antibodies positive, showing five different patterns (Table 2 and Fig. 1A). The most common patterns were combinations of antigens PDH-E2/BCKDH-E2/PDHctE1/PDH-flE1 (42%) and PDH-E2/BCKDH-E2 (42%) which were found in 16 cases. In the remaining three sera the patterns of reactivity were PDH-E2/PDH-X/BCKDHE2/PDH-flE 1, PDH-E2/BCKDH-E2/PDH-flE 1 and BCKDH-E2. All samples recognized the 51 kDa antigen (BCKDH-E2), whilst the antigen of 75-70 kDa (PDHE2) was detected in sera of 18 patients. No reaction was observed in sera from the 25 patients with other chronic liver diseases and in the 10 healthy subjects. All PBC, but none of the control sera, reacted with the extract from M. gordonae, showing identical recognition profiles with two polypeptides of approximately 70-65 and 55 kDa (Fig. 1B). No reaction was found in PBC patients or control subjects with extracts from the other nine mycobacterial strains tested. In addition, immunoglobulins reacting with the 70-65 TABLE 2 Patterns of reactivity to mitochondrial antigens in primary biliary cirrhosis sera (n= 19) Antigens
No. of cases
PDH-E2, BCKDH-E2, PDH-aEI and PDH-flEI PDH-E2, PDH-X, BCKDH-E2 and PDH-flEI PDH-E2, BCKDH-E2 and PDH-flEI PDH-E2 and BCKDH-E2 BCKDH-E2
8 I 1 8 1
We observed fivecombinations of antigens, with molecular weights of 75-70 kDa (PDH-E2), 56 kDa (PDH-X), 51 kDa (BCKDH-E2), 45 kDa (PDH-txEI) and 36 kDa (PDH-pEI).
The results of this study clearly indicate that sera from all PBC patients are able to recognize mycobacterial antigens, a finding which has not been observed in patients with other liver diseases, including autoimmune and viral chronic active hapatitis and healthy subjects. In addition, antimycobacterial reactivity of PBC patients seems to be species-specific, since it was only found against two peptides of 70-65 and 55 kDa of M. gordonae, but not against polypeptides from the nine other atypical Mycobacteria tested. Moreover, the two main mitochondrial antigens identified by sera from PBC patients, PDH-E2 and BCKDH-E2, were recognized by the eluted antimycobacterial antibodies, indicating that there is cross-reactivity of these anti-M, gordonae antibodies with the mitochondrial antigens against which antimitochondrial antibodies react in PBC. We also observed that antibodies reacting to PDH-E2 and BCKDH-E2 were able to recognize the 70-65 and 55 kDa polypeptides of M. gordonae, which confirms the existence of a cross-reactive epitope between these polypeptides from M. gordonae and the two major autoantigens of PBC. It has been suggested that PBC may result from exposure of genetically susceptible individuals to a particular environmental agent. In this respect, although the mere presence of antibodies to M. gordonae in PBC patients does not prove that M. gordonae play a role in the etiology of PBC, the close antigenic association between antigens from M. gordonae and the major autoreactive targets in the disease, suggests a possible pathological role of this agent in predisposed subjects. The possible link between Mycobacteria and PBC has also been suggested by the fact that PBC-specific antibodies are particularly induced in patients suffering a mycobacterial infection (6). Autoimmune diseases appear to be influenced by multiple factors, including the genetic, hormonal and immunological status of the subject. Environmental agents have also been implicated in the initiation of some autoimmune diseases. The most important environmental factors are infecting agents, and the existence ofepitopes shared by pathogens and host may furnish a link between infection and autoimmunity. A cross-reactivity between antimitochondrial antibodies and PDH-E2 and BCKDH-E2 of a number of aer-
676
L. VILAGUT et al.
A 1
2
3
i] 4
Fig. 1. (A) Immunoblot analysis of AMA in PBC patients: we observed five patterns of reactivity to antigens 75-70/51/45/36 kDa (lane I), 75-70/56/51/36 kDa (lane 2), 75-70/51/36 kDa (lane 3), 75-70/51 kDa (lane 4) and 51 kDa (lane 5). (13) Immunoblot analysis of extracted proteins from M. gordonaewith sera from patients with PBC (lanes 1-4) and patients with chronic hepatitis C (lane 5), chronic hepatitis B (lane 6), autoimmune chronic hepatitis (lane 7), and a healthy subject (lane 8). obic gram-negative and -positive bacteria has been observed since the P D H is very well conserved phylogenetically (7). However, it cannot be excluded that the shared epitope could also be contained in n o n - h o m o l o g o u s structures. Recently, antigenic similarities between molecules expressed by host cells and unrelated mycobacterial constituents have been established at the humoral level and have been extended to cellular mechanisms (8,9). It is therefore plausible that a shared epitope between mycobacterial proteins and h u m a n m i t o c h o n d r i a l antigens is involved in the induction o f reactive antibodies against the 70-65 and 55kDa polypeptides from M. gordonae and both h u m a n P D H - E 2 and B C K D H - E 2 . In fact, this is further supported by the recent identification o f a fouramino-acid homology between vimentin and a microbial protein as the molecular basis for a u t o i m m u n e glomerulonephritis (10). F r o m our results, we speculate that because o f the cross-reactivity with the m i t o c h o n d r i a l antigens, an induced humoral response to antigens from M. gordonae m a y evoke a sequence o f autoreactive events resulting in the existence o f antimitochondrial antibodies in genetically susceptible individuals. The existence o f a cross-reactive epitope is an i m p o r t a n t step in establishing a connection between a u t o i m m u n e reactions and the pathogenicity o f atypical Mycobacteria. F u r t h e r studies are in progress
5
kDa
1
23
45
6
78
-. 47
-' 33
to assess the role o f M. gordonae in the pathogenesis o f PBC.
Acknowledgements This work was supported in part by FISss grants 92/ 0282 to JV and 90/0206 to JR. We would like to thank Dr. E Cardellach for providing us with m i t o c h o n d r i a l extracts before using the purified P D H . This study was presented at the 28th A n n u a l Meeting o f the European Association for the Study o f the Liver.
References 1. Krawitt EL, Weisner RH. Autoimmune Liver Diseases. New York: Raven Press, 1991. 2. Hopf U, M611er B, Stemerowicz R, Lobeck H, Rodloff A, Freudenberg M, et al. Relation between Escherichia coli R (Rough)forms in gut, lipid A in liver, and primary biliary cirrhosis. Lancet 1989; ii: 1166-69. 3. Wayne LG, Hollander D, Anderson B, Sramek HA, Vadheim CM, Rotter Jl. Immunoglobulin A (IgA) and IgG serum antibodies to mycobacterial antigens in Crohn's disease patients and their relatives. J Clin Microbiol 1992; 30: 2013-18. 4. Kaufmann SHE. Immunity to Mycobacteria. Res Microbiol 1990; 141: 765-68. 5. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 68085. 6. Klein R, Wiebel M, Engelhart S, Berg PA. Sera from patients
MYCOBA CTERIUM GORDONAE IN PBC
677
7.
1A2 kDa li12
8.
9.
:4'.
" ".
:
10.
-.t33J,.- i. " Fig. 2. (A) Cross-reactivity of antibodies against 70-65 kDa (lane I) and 55 kDa (lane 2) from M. gordonae, eluted from PBC patients, towards the PDH-E2 and BCKDH-E2. (B) Cross-reactivity of antibodies against PDH-E2 (lane 1) and BCKDH-E2 (lane 2), eluted from PBC patients, towards the 70-65 kDa and 55 kDa polypeptides of M. gordonae.
with tuberculosis recognize the M2a-epitope (E2-subunit of pyruvate dehydrogenase) specific for primary biliary cirrhosis. Clin Exp Immunol 1993; 92: 308-16. Furrey SPM, Ali ST, Guest JR, James OFN, Bassendine MF, Yeaman SJ. Reactivity of primary biliary cirrhosis sera with E. coli dihydrolipoamide acetyltransferase (E2p): characterization of the main immunogenic region. Proc Natl Acad Sci USA 1990; 87: 3987-91. Esaguy N, Aguas AR, Van Embden JA, Silva MT. Mycobacteria and human autoimmune disease: direct evidence of cross-reactivity between human lactoferrin and the 65-KDa protein of tubercle and leprosy bacilli. Infect Immun 1991; 59:1117-25. Kaufmann SHE, Schoel B, Wand-Wtlttenberger A, Steinhoff U, Munk ME, Koga T. T-cells, stress proteins and pathogenesis of mycobacterial infections. Curr Top Micro Immunol 1990; 115: 125--41. Kraus W, Ohyama K, Snyder DS, Beachly EH. Autoimmune sequence of streptococcal M protein shared with the intermediate filament protein vimentin. J Exp Med 1989; 169: 481-92.
Journal of Hepatology 1994; 21:673-677 Pr#lted hz Denmark. All rights reserved Munksgaard. Copenhagen
Journal of Hepatology ISSN 0168-8278
Rapid Publication
Cross-reactivity of anti-Mycobacteriumgordonae antibodies with the major mitochondrial autoantigens in primary biliary cirrhosis Lidia Vilagut l, Jordi Vila l, Odette Vifias 2, Albert Par6s 3, Angels Gin6s 3, M a r i a Teresa Jim6nez de A n t a l and Joan Rod6s 3 ILaboratorv o f Microbiology, 2Servei d'hnmunologia and 3Liver Unit, Hospital Clinic i Provincial, Universit.v o f Barcelona, Barcelona, Spain
(Received 30 December 1993)
Primary biliary cirrhosis is a chronic cholestatic liver disease associated with autoimmune disorders. Antimitochondrial autoantibodies and granulomatous portal lesions are characteristic in primary biliary cirrhosis. Since granuloma may be induced by Mycobacteria, and there is evidence implicating Mycobacteria as infectious agents capable of initiating autoimmunity, a study was performed to determine the presence of antibodies against 10 atypical Mycobacteria in 19 patients with primary biliary cirrhosis, and in 35 controls (25 patients with other chronic liver diseases and 10 healthy subjects). All primary biliary cirrhosis sera.and none of the controls reacted with the extract from Mycobacterium gordonae, showing identical recognition profiles with two polypeptides of 70-65 and 55 kDa. No other reaction was found in primary biliary cirrhosis patients and in controls with the extracts from the other nine atypical Mycobacteria tested. Eluted immunoglobulins which reacted with the 70-65 and 55 kDa polypeptides from M. gordonae, bound to the mitochondrial antigens PDH-E2 and BCKDH-E2. Furthermore, when the extract from M. gordonae was tested with eluted immunoglobulins from recognized PDH-E2 and BCKDH-E2 by primary biliary cirrhosis patients, we observed both 70-65 and,55 kDa polypeptides. These data indicate that antibodies to M. gordonae, found in all primary biliary cirrhosis patients, crossreact with the major mitochondrial targets of the disease. We suggest that M. gordonae may play a potential pathogenic role in primary biliary cirrhosis. © Journal of Hepatology.
Key words: Antimitochondrial autoantibodies; Autoimmunity; Molecular mimicry; Mycobacterial components
Primary biliary cirrhosis (PBC) is a chronic cholestastic liver disease of unknown etiology. The pathogenesis of PBC is considered autoimmune because of the presence of many immunological abnormalities, and its association with other autoimmune disorders. The most fascinating fact is that practically all PBC patients present antimitochondrial autoantibodies which, on immunoblots, recognize five antigens from the mitochondrial multienzymatic complex named alpha-ketoacid dehydrogenase complex, at apparent molecular weights of 75-70, 56, 51, 45 and 36 kDa (1). The 75-70 kDa antigen has been identified as the dihydrolipoamide acetyltransferase (E2) of the pyruvate dehydrogenase complex (PDH-E2), the 51 kDa antigen
corresponds to the E2 of the branched chain alpha-ketoacid dehydrogenase complex (BCKDH-E2), or to the E2 of the alpha-ketoglutarate dehydrogenase complex (aKGDH). The 56 kDa antigen is designated as protein X, a component of the PDH (PDH-X) and the remaining two antigens of 45 and 36 kDa are the alpha-subunit and the beta-subunit, respectively, of the pyruvate dehydrogenase (El) from the pyruvate dehydrogenase complex. Previous work showed that sera from PBC patients react with a wide spectrum of prokaryotes, including E. coli (2). Despite these studies, up to now there has been no definite evidence that a specific infectious agent may be the precipitating mechanism initiating bile duct lesions.
Correspondence to: Joan Rod6s MD, Liver Unit, Hospital Clinic i Provincial,C/Villarroel 170, 08036 Barcelona,Spain.
674
Mycobacteria are thought to play an etiological role in autoimmune and idiopathic diseases (3). Since granuloma, identifiable either as aggregates of histiocytes or as noncaseating lesions adjacent to damaged bile ducts, are characteristic in PBC, and since granuloma may be induced by Mycobacteria (4), the current study aimed to investigate whether sera of patients with PBC react against antigens from atypical Mycobacteria. Patients and Methods
Patients The study was performed in sera from 19 patients who met the clinical, biochemical and histological criteria for PBC, 25 patients with chronic liver diseases other than PBC (10 chronic active hepatitis B, 10 chronic active hepatitis C, five autoimmune chronic active hepatitis), and 10 healthy subjects.
Mycobacterial strahls The mycobacterial reference strains were Mycobacterium chelonei TMC 1542 (Trudeau Mycobacteria Collection), M. flavescens CCTM 1074 (Centre de Collection de Types Microbiens. Lausanne, Switzerland), M. fortuiturn TMC 1529, M. gordonae TMC 1318, M. intracellulare TMC 1403, M. kansasii TMC 1201, M. mahnoense CCTM 1635, M. scrofulaceum ATCC 1998 (American Type Culture Collection. Rockville, Md), M. xenopi NCTC 10042 (National Collection of Type Cultures. London, United Kingdom) and M. terrae. This latter strain was isolated from a clinical specimen and identified by standard biochemical procedures and high performance gas chromatography. All specimens were grown in Middlebrook 7H9 broth and were streaked onto Middlebrook 7 H l l agar medium plates to ensure pure cultures.
Preparation of membrane proteins of mycobacterial suspensions Growths from 100-ml cultures were pelleted, washed twice with 50 mM Tris-HCl, 2 mM EDTA, pH 8.5, containing 100/tM benzamidine hydrochloride and 100/lM phenylmethylsulfonyl fluoride, and suspended in 6 ml of the same buffer. Organisms were sonicated four times for 30 s while cooling. The cell debris were then centrifuged at 900 g for 20 min, and the supernatants were centrifuged at 12 300 g for 60 min. Finally, the membrane pellets were resuspended in 200/tl of 2 mM Tris-HCl, pH 7.7, containing 100/iM benzamidine hydrochloride and 100/~M phenylmethylsulfonyl fluoride.
L. VILAGUT et al.
Pyruvate dehydrogenase complex The purified pyruvate dehydrogenase complex from porcine heart, containing< 15% of the alpha-ketoglutarate dehydrogenase complex, was supplied by Sigma. hnmunoblotting studies One hundred and 300/tg of mycobacterial and mitochondrial antigens, respectively, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as previously described (5). The gels were electroblotted, using a trans-blot semi-dry apparatus (BioRad), onto nitrocellulose membranes. A control for the transfer of proteins was carried out using pre-stained molecular weight standards. The blots were blocked overnight at 4°C in phosphate-buffered saline (PBS) with 10% skim milk, 2% ovalbumin, 1% bovine serum albumin (BSA), and 0.2% Tween-20. They were then incubated with 1:50 diluted human sera in blocking solution for 1 h. After two 15-min washes with PBS-0.25% gelatine, the reacting antibodies were detected by peroxidase-conjugated rabbit anti-human immunoglobulins (Dako) at 1:200 dilution in blocking solution for 1 h. After two additional washes, a substrate solution containing 600 mg/ml of diaminobenzidine (Sigma) and 0.03% hydrogen peroxide in PBS was added to detect antigen-bound antibodies. Elution of antibodies After immunoblotting, antibodies bound to their specific antigen were eluted by cutting out the bands of the nitrocellulose membrane and placing them in a syringe, where a solution of 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.15 M NaC1, 0.5% Tween-20 and 100 pg/ml of BSA was forced twice. Afterwards, 3 ml of a solution of 0.2 M glycine-HC1 pH 2.8, 0.5 M NaCI, 0.5% Tween-20 and I00 pg/ml of BSA was added to the syringe and left for 2 min. This solution was also forced twice. The eluted material was immediately neutralized with NaOH, diluted in PBS and then concentrated using a Minicom-B clinical sample concentrator. This procedure was carried out at 4°C. The eluted antibodies were re-tested by the abovedescribed immunoblotting procedure. Informed consent was obtained from patients and healthy subjects, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Ethics Committee of the Hospital Clinic i Provincial. Results
The clinical, biochemical and histological features of patients with PBC are summarized in Table 1. In order to characterize the specific antigens recognized
MYCOBACTERIUMGORDONAEIN PBC
675
TABLE 1 Clinical, laboratory data and histological stages of PBC patients Age (years) Sex (F/M) Sj6gren's syndrome Biochemical tests ALT (u/l) AF (u/l) Bil (mg/dl) Immunoglobulins IgG (mg/dl) IgA (mg/dl) IgM (mg/dl)
54.9___1.8 17/2 14 (74%) 108.4± 11.4 1002± 130 2.1 ±0.9
Discussion
2326± 178 408±49 633±79
Histological stage I
2
II III IV
8 5 4
and 55 kDa polypeptid.es from M. gordonae were eluted and re-tested against the mitochondrial antigens. The two types of antibodies, bound to PDH-E2 and BCKDH-E2 (Fig. 2A). Conversely, antibodies eluted from PDH-E2 and BCKDH-E2 recognized by PBC patients, bound to the 70-65 and 55 kDa polypeptides from M. gordonae extract (Fig. 2B).
ALT: alanine aminotransferase; AF: alkaline phosphatase; Bil: bili-
rubin. by antimitochondrial antibodies in PBC sera, an immunoblot was performed using the purified mitochondrial antigens. All sera from patients with PBC were antimitochondrial antibodies positive, showing five different patterns (Table 2 and Fig. 1A). The most common patterns were combinations of antigens PDH-E2/BCKDH-E2/PDHctE1/PDH-flE1 (42%) and PDH-E2/BCKDH-E2 (42%) which were found in 16 cases. In the remaining three sera the patterns of reactivity were PDH-E2/PDH-X/BCKDHE2/PDH-flE 1, PDH-E2/BCKDH-E2/PDH-flE 1 and BCKDH-E2. All samples recognized the 51 kDa antigen (BCKDH-E2), whilst the antigen of 75-70 kDa (PDHE2) was detected in sera of 18 patients. No reaction was observed in sera from the 25 patients with other chronic liver diseases and in the 10 healthy subjects. All PBC, but none of the control sera, reacted with the extract from M. gordonae, showing identical recognition profiles with two polypeptides of approximately 70-65 and 55 kDa (Fig. 1B). No reaction was found in PBC patients or control subjects with extracts from the other nine mycobacterial strains tested. In addition, immunoglobulins reacting with the 70-65 TABLE 2 Patterns of reactivity to mitochondrial antigens in primary biliary cirrhosis sera (n= 19) Antigens
No. of cases
PDH-E2, BCKDH-E2, PDH-aEI and PDH-flEI PDH-E2, PDH-X, BCKDH-E2 and PDH-flEI PDH-E2, BCKDH-E2 and PDH-flEI PDH-E2 and BCKDH-E2 BCKDH-E2
8 I 1 8 1
We observed fivecombinations of antigens, with molecular weights of 75-70 kDa (PDH-E2), 56 kDa (PDH-X), 51 kDa (BCKDH-E2), 45 kDa (PDH-txEI) and 36 kDa (PDH-pEI).
The results of this study clearly indicate that sera from all PBC patients are able to recognize mycobacterial antigens, a finding which has not been observed in patients with other liver diseases, including autoimmune and viral chronic active hapatitis and healthy subjects. In addition, antimycobacterial reactivity of PBC patients seems to be species-specific, since it was only found against two peptides of 70-65 and 55 kDa of M. gordonae, but not against polypeptides from the nine other atypical Mycobacteria tested. Moreover, the two main mitochondrial antigens identified by sera from PBC patients, PDH-E2 and BCKDH-E2, were recognized by the eluted antimycobacterial antibodies, indicating that there is cross-reactivity of these anti-M, gordonae antibodies with the mitochondrial antigens against which antimitochondrial antibodies react in PBC. We also observed that antibodies reacting to PDH-E2 and BCKDH-E2 were able to recognize the 70-65 and 55 kDa polypeptides of M. gordonae, which confirms the existence of a cross-reactive epitope between these polypeptides from M. gordonae and the two major autoantigens of PBC. It has been suggested that PBC may result from exposure of genetically susceptible individuals to a particular environmental agent. In this respect, although the mere presence of antibodies to M. gordonae in PBC patients does not prove that M. gordonae play a role in the etiology of PBC, the close antigenic association between antigens from M. gordonae and the major autoreactive targets in the disease, suggests a possible pathological role of this agent in predisposed subjects. The possible link between Mycobacteria and PBC has also been suggested by the fact that PBC-specific antibodies are particularly induced in patients suffering a mycobacterial infection (6). Autoimmune diseases appear to be influenced by multiple factors, including the genetic, hormonal and immunological status of the subject. Environmental agents have also been implicated in the initiation of some autoimmune diseases. The most important environmental factors are infecting agents, and the existence ofepitopes shared by pathogens and host may furnish a link between infection and autoimmunity. A cross-reactivity between antimitochondrial antibodies and PDH-E2 and BCKDH-E2 of a number of aer-
676
L. VILAGUT et al.
A 1
2
3
i] 4
Fig. 1. (A) Immunoblot analysis of AMA in PBC patients: we observed five patterns of reactivity to antigens 75-70/51/45/36 kDa (lane I), 75-70/56/51/36 kDa (lane 2), 75-70/51/36 kDa (lane 3), 75-70/51 kDa (lane 4) and 51 kDa (lane 5). (13) Immunoblot analysis of extracted proteins from M. gordonaewith sera from patients with PBC (lanes 1-4) and patients with chronic hepatitis C (lane 5), chronic hepatitis B (lane 6), autoimmune chronic hepatitis (lane 7), and a healthy subject (lane 8). obic gram-negative and -positive bacteria has been observed since the P D H is very well conserved phylogenetically (7). However, it cannot be excluded that the shared epitope could also be contained in n o n - h o m o l o g o u s structures. Recently, antigenic similarities between molecules expressed by host cells and unrelated mycobacterial constituents have been established at the humoral level and have been extended to cellular mechanisms (8,9). It is therefore plausible that a shared epitope between mycobacterial proteins and h u m a n m i t o c h o n d r i a l antigens is involved in the induction o f reactive antibodies against the 70-65 and 55kDa polypeptides from M. gordonae and both h u m a n P D H - E 2 and B C K D H - E 2 . In fact, this is further supported by the recent identification o f a fouramino-acid homology between vimentin and a microbial protein as the molecular basis for a u t o i m m u n e glomerulonephritis (10). F r o m our results, we speculate that because o f the cross-reactivity with the m i t o c h o n d r i a l antigens, an induced humoral response to antigens from M. gordonae m a y evoke a sequence o f autoreactive events resulting in the existence o f antimitochondrial antibodies in genetically susceptible individuals. The existence o f a cross-reactive epitope is an i m p o r t a n t step in establishing a connection between a u t o i m m u n e reactions and the pathogenicity o f atypical Mycobacteria. F u r t h e r studies are in progress
5
kDa
1
23
45
6
78
-. 47
-' 33
to assess the role o f M. gordonae in the pathogenesis o f PBC.
Acknowledgements This work was supported in part by FISss grants 92/ 0282 to JV and 90/0206 to JR. We would like to thank Dr. E Cardellach for providing us with m i t o c h o n d r i a l extracts before using the purified P D H . This study was presented at the 28th A n n u a l Meeting o f the European Association for the Study o f the Liver.
References 1. Krawitt EL, Weisner RH. Autoimmune Liver Diseases. New York: Raven Press, 1991. 2. Hopf U, M611er B, Stemerowicz R, Lobeck H, Rodloff A, Freudenberg M, et al. Relation between Escherichia coli R (Rough)forms in gut, lipid A in liver, and primary biliary cirrhosis. Lancet 1989; ii: 1166-69. 3. Wayne LG, Hollander D, Anderson B, Sramek HA, Vadheim CM, Rotter Jl. Immunoglobulin A (IgA) and IgG serum antibodies to mycobacterial antigens in Crohn's disease patients and their relatives. J Clin Microbiol 1992; 30: 2013-18. 4. Kaufmann SHE. Immunity to Mycobacteria. Res Microbiol 1990; 141: 765-68. 5. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 68085. 6. Klein R, Wiebel M, Engelhart S, Berg PA. Sera from patients
MYCOBA CTERIUM GORDONAE IN PBC
677
7.
1A2 kDa li12
8.
9.
:4'.
" ".
:
10.
-.t33J,.- i. " Fig. 2. (A) Cross-reactivity of antibodies against 70-65 kDa (lane I) and 55 kDa (lane 2) from M. gordonae, eluted from PBC patients, towards the PDH-E2 and BCKDH-E2. (B) Cross-reactivity of antibodies against PDH-E2 (lane 1) and BCKDH-E2 (lane 2), eluted from PBC patients, towards the 70-65 kDa and 55 kDa polypeptides of M. gordonae.
with tuberculosis recognize the M2a-epitope (E2-subunit of pyruvate dehydrogenase) specific for primary biliary cirrhosis. Clin Exp Immunol 1993; 92: 308-16. Furrey SPM, Ali ST, Guest JR, James OFN, Bassendine MF, Yeaman SJ. Reactivity of primary biliary cirrhosis sera with E. coli dihydrolipoamide acetyltransferase (E2p): characterization of the main immunogenic region. Proc Natl Acad Sci USA 1990; 87: 3987-91. Esaguy N, Aguas AR, Van Embden JA, Silva MT. Mycobacteria and human autoimmune disease: direct evidence of cross-reactivity between human lactoferrin and the 65-KDa protein of tubercle and leprosy bacilli. Infect Immun 1991; 59:1117-25. Kaufmann SHE, Schoel B, Wand-Wtlttenberger A, Steinhoff U, Munk ME, Koga T. T-cells, stress proteins and pathogenesis of mycobacterial infections. Curr Top Micro Immunol 1990; 115: 125--41. Kraus W, Ohyama K, Snyder DS, Beachly EH. Autoimmune sequence of streptococcal M protein shared with the intermediate filament protein vimentin. J Exp Med 1989; 169: 481-92.