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Antimitochondrial antibodies in primary biliary cirrhosis
Journal of Hepatology, 1992; 15: 6-9 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 0168-8278/92/$05&O
6 HEPAT 01159
Leader ____
-
~ntimitochondrial
antibodies in primary
A clue to its etiopathogenesis?’ Peter
A. Berg and Reinhild
Klein
Department of Internal Medicine, Universityof Tiibingen, Tlibingen, Germany
In the last 20 years a mass of immunological data has been collected which strongly indicates that autoimmune processes play an important role in the pathogenesis of primary biliary cirrhosis (PBC). Furthermore, the close association of antimitochondrial antibodies (AMA) with PBC, and especially of anti-M2 which can be detected ; :I up to 96% of patients (1) seems to indicate the specific induction of these antibodies by a disease-precipitating agent expressing cross-reacting epitopes. In this article we discuss the significance of anti-M2 for the etiopathogenesis of PBC.
The M2/anti-M2 system and its relationship to the etiology of PBC In 1965, Walker and colleagues described a specific cytoplasmic fluorescence pattern in the immunofluorescence test on cryostat sections with sera from patients with PBC, and it was suggested that this reaction may be due to the presence of AMA in these sera (2). The association of the corresponding antigen with mitochondria was proven using purified mitochondria in the complement fixation test (3), and it could be shown that it was located within the inner membrane of mammalian mitochondria (4). This PBC-specific antigen, named M2, consists of 5 epitopes at molecular weights 70, 56, 53,45 and 36 kD (M2a-e) (5) which have been identified as subunits of the 2-oxo-dehydrogenase complexes located within mammalian mitochondria (6,7). Anti-M2 antibodies should be distinguished from naturally occurring mitochondrial antibodies (NOMA) which can also recognize several determinants in the MZfraction when tested by Western blotting (8). Three
epitopes have been described with a molecular weight of 65 kD (epsilon), and 60165 kD (zeta/eta). NOMA occur preferentially during infectious diseases such as Epstein-Barr, cytomegalovirus or E. coli infections but are rarely found in sera from PBC patients (8%) and healthy controls (10%). Interestingly, however, high titres of the antibodies against these three major NOMAg determinants have been detected in up to 70% in PBC contact persons not suffering from the disease. One could, therefore, postulate that a contagious agent circulat:ng in the blood of PBC patients may Slave induced the increased stuplulation of NOMA. A major function of natural autoantibodies is to protect individuals from an infection. The lack of NOMA production in PBC patients may, therefore, predispose these individuals to an infection with the ‘PBC-specific agent’. Several attempts have been made to identify this agent. It has been shown that sera from PBC patients react with a wide spectrum of prokaryotes including E. coli (5,9), Paracoccus denitri+zs, the thermophilic bacterium PS3, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus (9), and Enterobacteriaceae (10). Furthermore, the sera from patients with E. coli urinary tract infections reacted with ‘M2’ in the Western blot (9). In another study rabbits were immunized with rough mutants of Enterobacteriaceae and tested against mitochondria, hereby also showing a PBC-like reaction (10). It was concluded that bacteria, and especially r-forms of Enterobacteriaceae, may play a major role in the etiology of PBC. There are several findings, however, which strongly dispute this concept. For example, sera from patients with urinary tract infections did not react with the PBCspecific M2a-determinant using the purified pyruvate demajor
Correspondence:Prof. P.A. Berg, Departmentof Internal Medicine, University of Tiibingen, Otfried Miiller Str., W-7400 Tiibingen, Germany. ‘Dedicatedto Deborah Doniach on the occasion of her 80th birthday.
ANTIMTBCHONDRIAE
ANTIBODIES
IN BBC
hydrogenase complex (PDC = M2a) or the recombinant M2a antigen in the Western blot (personal obset vations). Furthermore, it has been demonstrated that sera from patients with E. coli and other infectious disorders recognize not the PBC-specific M2a antigen, but the NOMAg-epitope epsilon at 65 kD (11). As far as the reaction of sera from PBC patients with bacteria is concerned, there is clear-cut evidence that E. coli membranes, mycobacteria and other bacterial membranes may share both the M2a-determinant and NOMAg-epitopes (11). In another study it was observed that two distinct antibodies against PDC can be detected in PBC sera, one which reacts with mammalian and the other with prokaryotic PDC (12). Thus, affinity-purified anti-M2a antibodies to bovine PDC weakly reacted with E. coli PDC, and vice versa. These findings also argue against the etiological role of E. coli in PBC. One should also bear in mind that about 4% of patients with clinically, biochemically and histologically typical PBC are anti-M2-negative (1). Antibodies to the cytoplasmic enzyme, glycogen phosphorylase (= M9) (13), or to nuclear antigens such as ‘nuclear dots’ or nuclear membranes (14,15) have also been found in patients with classic but anti-MulZnegative PBC. Considering the strong B-cell activity associated with PBC, it is possible that the triggering agent is complex and expresses a wide spectrum of cross-reacting epitopes of varying immunologitial potencies which may account for the different autoantibodies observed in PBC. It is still unclear whether this B-cell stimulation is related to T-cell-dependent or -independent antigens.
stem
and its relationship
to the
Segmental and focal bile duct lesions showing T-cell infiltrations are typical in early PBC. Immunohistochemical studies have clearly indicated that these lymphocyte infiltrates are composed of cytotoxic CD8+ T-cells (1619). Based on these observations, it has been postulated that an antigen expressed on the biliary epithelium is the target of this T-cell response, and a bile-duct-associated protein or a component of the 2-oxo-dehydrogenase complex have been considered as candidates (20-23). The distribution of the EZcomponent of PDC was recently examined in the liver and lymph nodes of patients with PBC and other liver diseases using monoclonal or affinity-purified antibodies (23,24). Although the staining of bile ducts in. PBC patients was more intense than in normal or other livers, expression of EZ-components on the cell membranes of bile duct epithelial cells could not
be prove11 (24). In another study, strong expression of the mitochondrial and mycobacterial heat shock protein (hsp) 65 was found in PBC and PSC patients but not in patients with non-cholestatic liver disorders or in healthy controls (25). The increased presentation of intracellular proteins on cell membranes is a common phenomenon in cells exposed to infections or stress in general (26); due to their ability to express major histocompatibility complex (MHC) antigens class II on their surface (27-29), bile duct epithelial cells are known to function as antigenpresenting cells. However, it seems unlikely that these proteins would be major targets of the immune response, First, lymphocytes of PBC patients could not be stimulated in vitro with a hsp-containing mitochondrial antigen fractiocl (personal observation). Second, when sera from PBZ patients with high anti-M2 titers were tested against the cloned hsp 6j by Western blotting (kindly provided by Dr. Maufmann, Ulm), no specific reactions were observed. All these data provide littic evidence that mitochondrial autoantigens play a major role in the pathogenesis of the chronic inflammatory bile duct process. Anti-M2 antibodies do not seem to be involved in the pathogenesis of PBC either. Although immunoglobulins were found on the surface of biliary epithelium in liver biopsies of PBC patients, anti-M2 specificity could not be verified (30). Furthermore, immunization of experimental animals with the EZcomponent of PDC generated mitochondrial antibodies but no bile duct lesions (31). The observation that anti-M2 antibodies persist after orthotopic liver transplantation and that PBC did not reappear in these patients over an observation period of up to 10 years (32) is also intriguing. In recent publications an inhibition of mitochondrial function by antibodies against PDC has been demonstrated in vitro, and its role with respect to the pathogenesis of PBC was discussed (33-36). However, it seems unlikely that these antibodies could interfere with the mitochondrial functions of intact cells in vivo. Also the high and non-fluctuating AMA titers in PBC strongly argue against binding in vivo.
Although great progress has been made in identification of the mitochondrial antigen-antibody systems, the definition of the MZsystem at a molecular level has not really improved our understanding of the disease. This is not surprising since classical PBC can occur in the absence of anti-M2.
P.A. BERG AND R. KLEIN
3
me outstanding immunological phenomenon in PBC is a StrongB-cell activation associated with the production of high-&red antibodies against a spectrum of autoantigens. This hyperreactivity may reflect a genetically determined disposition to respond to self- or non-self antigens in a subset of T-helper cells known to stimulate preferentially B-cells (Tm = T-helper cells, type 2) (37). The agentwhich triggers PBC is probably harboured in the epithelial cells of bile ducts. It is well documented that glandular ducts have a special property as reSeWOirS formicro-organisms (38); due to their ability to express MI-K class I and II molecules in high amounts (27-29) they can also act as antigen-presenting cells and become targets for cytotoxic T-cells. Analysis of portal infiltrates by immunohistochemical methods has revealed that the cellular composition of granuloma is similar to tuberculosis (39), and the histological pattern in mice inoculated with mycoplasma resembles the pattern in PBC (40). Early histological reactions include lymphocytic, eosinophilic and granulocytic infiltrates, morphological phenomena which are more compatible with an infection caused by bacteria or parasites than viruses. The exacerbation of latent intracellular infectious processes of some bile ducts due to insufficient control by the natural immune system (genetically determined specific B-cell defect related to NOMA production?) or other alterations in regulatory immunological mechanisms may be operative in the precipitation of the segmental or focal bile duct lesions. It is still unknown whether T-cell cytotoxicity is directed mainly against self or non-self epitopes. For the moment, there is no strong evidence for MZrelated T-cell cytotoxicity, although T-cells from PBC patients could be stimulated with the PDC-E2 complex to produce interleukin-2 (23). The strong activation of B-cells secreting disease-specific anti-M2 antibodies
could be due to either cross-reacting epitopes which activate T-cells, thereby stimu!ating B-cells to produce AMA, or merely a T-cell-independent activation of B-cells. Since an immune response may be triggered either by THI (responsible for controlling cytotoxic defense mechanisms) or T,,-cells, there is strong evidence for T,,activation in PBC. It is known that T,,-cells preferentially stimulate B-cells which secrete IgGl, IgG3, and IgM immunoglobulins. T,, cells also produce interleukin4, -5 and -6 (37,41), thus stimulating the release of bone marrow cells and especially eosinophils. Interestingly, anti-M2 antibodies have been shown to belong mainly to the IgGl, IgG3 or IgM class but rarely to the IgG2 class. The latter immunoglobulins are preferentially controlled by THL-cells. Furthermore, lymphocytes of PBC patients can spontaneously produce interleukin-6 compared to controls (unpublished observation), and in some patients PBC is accompanied by a rather strong eosinophilic reaction (42). These findings can also be explained by an activation of T,,-cells. The diagnostic exclusivity of antimitochondrial antibodies - so convincingly shown already 27 years ago (2) - makes research in this field fascinating. The statement made by Deborah Doniach at the time that an etiological or pathogenetic role in the perpetuation of liver disease cannot be demonstrated for mitochondrial antibodies or antigens (43) still holds true today.
Acknowledgements R.K. is supported by grants of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Be 417/19).
References 1 Berg PA, Klein R. Lindenborn-Fotinos J. Antimitochondrial antibodies in primary biiiary cirrhosis. J Hepatol 1986; 2: I2331. 2 Walker JG, Doniach D, Roitt IM, Sherlock S. Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965; i: 827-31. 3 Berg PA, Doniach D, Roitt IM. Mitochondrial antibodies in primary biliary cirrhosis. I. Localization of the antigen to mitochondrial membranes. J Exp Med 1967; 126: 277-90. 4 Berg PA, Muscztello U, Home RW, Roitt IM, Donia:h D. Mitochondrial antibodies in primary biliary cirrhosis, II. me complement fixing antigen as a component of mitochondrial inner membranes. Br J Exp Pathol 1969; 50: 200-g. 5 Lindenborn-Foiinos J, Baum H, Berg PA, Mitochondrial antibodies in primary biliary cirrhosis. Further characterizationof
the W-antigen by immunoblottingrevealing species and nonspeciesSp‘XifiC determinants. Hepatology 1985; 5: 763-g. 6 Bassendine MF, Fussey SPM. Mutimer DJ, James OFW, Yeaman SJ. Identification and characterization of four M2 mite-
7
8
9
10
11 12
chondrial autoantigens in primary biliary cirrhosis. Semin Liver Dis 1989; 9: 124-31. van de Water J, Surh CD, Leung PSC et al. Molecular definitions, autoepitopes, and enzymatic activities of the mitochondrial autoantigens of primary biliary cirrhosis. Semin Liver Dis 1989; 9: 132-7. Klein R, Berg PA. Demonstration of ‘naturally occurring mitochondrial antibodies’ in family members of patients with primary biliary cirrhosis. Hepatology 1990; 12: 335-41. Baum H. Nature of the mitochondrial antigens of primary biliary cirrhosis and their probable relationship to the etiology of the disease. Semin Liver Dis 1989; 9: 117-23. Stemerowicz R, Hopf U, Miiller B. Are mitochondrial antibodies in primary biliary cirrhosis induced by r(rough) mutants of Enterobacteriaceae? Lancet 1988; ii: 1166-70. Berg PA, Klein R. Mitochondrial M2 autoantigens and primary biliary cirrhosis. Lancet 1989; i: 447. Fussey SPM, Lindsay JG, Fuller C et al. Autoantibodies in primary biliary cirrhosis: analysis of reactivity against eukaryotic and prokaryotic 2-oxoacid dehydrogenase complexes. Hepatol-
ANTIMITOCHONDRIAL
ANTIBODIES
IN PBC
ogy 1991; 13: 467-74. 13 Klein R, Berg PA. Anti-M9 antibodies in sera from patients with primary biliary cirrhosis recognize an epitope of glycogen phosphorylase. Clin Exp Immunol 1990: 81: 65-71. 14 Pisi E. Bianchi FB. Ballnrdini 6, Cassani F, Bottazzo GE New immunopathologic features in primary biliary cirrhosis. Front Gastroenterol Res 1986; 9: 132-42. 15 Lozano F, Pares A, Borche L, Gallart T, Rod& J. Autoantibodies against nuclear envelope associated proteins in primary biliary cirrhosis. Hepatology 1988; 8: 93,0-3. 16 Pape GR, Rieber EP, Eisenburg J, Hoffman R, Paumgartner 6. Riethmiiller G. Involvement of the cytotoxic/suppressor T-cell subset in liver tissue injury of patients with acute and chronic liver diseases. Gastroenterology 1983; 85: 657-62. 17 van den Oord JJ, Fevery J, De Groote J, Desmet VJ. Immunohistochemical characterization of inflammatory infiltrates in primary biliary cirrhosis. Liver 1984; 4: 264-74. 18 Yamada G. Hyodo I, Tobe K, Mizuno M, Nishihore T, Kobayashi T, Nagashima H. Ultrastructural immunocytochemical analysis of lymphocytes infiltrating bile duct epithelia in primary biliary cirrhosis. Hepatology 1986: 6: 355-91. 19 Hoffman RM, Pape GE, Spengler U et al. Clonal analysis of liver-derived T-cells of patients with primary biliary cirrhosis. Clin Exp Immunol 1989; 76: 210-5. 20 Wojcicka-McFarlane BM, McFarlane IG, Amoroso P, Williams R. Differential in vitro immune response to biliary tract antigens in primary biliary cirrhosis and chronic active hepatitis. Liver 1981; I: 268-79. 21 Onishi S. Maeda T, Iwasaki S et al. A bi? ary protein identllied by immunoblotting stimulates proliferation of peripheral blood T-lymphocytes in primary biliary cirrhosis. Liver 1991; 11: 32128. 22 Ghadiminejad I. Baum H. Evidence for the cell-surface localization of antigens cross-reacting with the ‘mitochondrial antibodies’ of primary biliary cirrhosis. Hepatology 1987; 7: 743-9. 23 van de Water J, Ansari A, Surh CD et al. Evidence for the targeting by 2-oxo-dehydrogenase enzymes in the T-cell response of primary biliary cirrhosis. J Immunol 1991; 146: 89-94. 24 Joplin R, Lindsay JG. Hubacher SG et al. Distribution of dihydrolipoamide acetyltransferase (E2) in the liver and portal lymph nodes of patients with primary biliary cirrhosis: an immunohistochemical study. Hepatology l’*Y1: 14: 342-7. 25 Broome U, Scheynius A, Hultcrantz R. Induced expression of heat shock protein (hsp) on bile ducts in patients with primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Hepatology 1991; 14: 134A. 26 Kaufmann SHE. Heat shock proteins and the immune response. Immunol Today 1990: 11: 129-36. 27 Ballardini G. Mirakian R, Bianchi FB. Pisi F. Doniach D. Botazzo GE Aberrant expression of HLA-DR expression on bile duct epithelium in primary biliary cirrhosis: relevance to pathogenesis. Lancet 1984; ii: 1009-13. 28 van den Oord JJ, Sciot P, Desmet VJ. Expression of MHC products by normal and abnormal bile duct epithelium. J Hepatol 1986; 3: 310-7. 29 Spengler U, Pape GR, Hoffman RM et al. Differential expression of MHC class II subregion products on bile duct epithelial
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38
39
40
41
42
43
Ceils alld hepatocytes in patients with primary biliary cirrhosis, Hepatology 1988: 8: 459-62. Krams SM, van de Water J, Coppel RL et al. Analysis of hepatic T-lymphocyte and immunoglobulin deposits in patients wirh primary biliary cirrhosis. Hepatology 1990; 12: 306-13. Krams SM. Surh SD, Coppel RL et al. Immunization of experimental animals with dihydrolipoamide acetyltransferasc. as a purified recombinant polypeptide, generates mitochondrial autoantibodies but not primary biliary cirrhosis. Hepatology 1989: 9: 411-6. Haagsma EB, Manns M, Klein R et al. Subtypes of antimitochondrial antibodies in primary biliary cirrhosis before and after orthotopic liver transplantation. Hepatology 1987; 7: 129-33. van de Water j, Fregeau D, Davis PA et al. Autoantibodies of primary biliary cirrhosis recognize dihydrolipoamide acetyltransferase and inhibit enzyme function. J Immunol 1988; 141: 2321-4. Fregeau D, Rochc TE, Davis PA, Coppel R, Gershwin ME. Primary biliary cirrhosis. Inhibition of pyruvate dehydrogenase complex activity by autoantibodies speciEc for Elalpha, a nonlipoic acid contairiing mitochondrial enzyme. J Immunol 1990; 144: 1671-6. Fregeau D. Priniville T. Coppel RL. Kaplan M. Dickson ER. Gershwin ME. Inhibition of alpha-ketogiutarate dehdrogcnase activity by a distinct population of autoantibodies recognizing dihydrolipoamide succinyltransferase in primary bihary cirrhosis. Hepatology 1990; 11: 975-81. Begley JP, Barry RE. Danton RM, Stansbie D. Inhibition of the activity of the 2-oxoacid dehydrogenase complexes by sera from patients with primary biliary cirrhosis. Eur J Gastroenterol Hepatol 1991; 3: 365-9. Mosmann TR, Coffman RL. Two types of mouse helper T-cell clone. Implications for immune regulation. Immunol Today 1987: 8: 223-7. Green JE, Hinrichs SH, Vogel J et al. Exocrinopathy resembling Sj8gren’s syndrome in 4TLV-I tax transgenic mice. Nature 1989: 341: 72-4. van den Oord JJ, Wolf-Peeters CD. Facchetti F. Desmet V. Cellular composition of hypersensitivity-type granuloma: immunohistochemical analysis of tuberculosis and sarcoidal lymphadenitis. Hum Pathol 1984: 15: 559-68. Johnson L. Wirostko E. Wirsotko W. Primary biliary cirrhosis in the mouse: induction by human mycoplasma-like organisms. Int J Exp Pathol 1990; 71: 701-12. Coffman RL, Seymour BWP, Lebman DA et al. The role of helper T cell products in mouse B cell differentiation and isotype regulation. Immunol Rev 1988; 102: 5-28. Morito T, Kuroda M, Saito K, Takagi T. Nisimaki T. Kasukawa R. Clinical features of primary biliary cirrhosis: abnormal complement system and eosinophilia. In: Tsuchiya M. Nagura H. Hibi T, Mori I, eds. Frontiers in Mucosal Immunology, Vol. 2. Amsterdam: Elsevier, 1991; 125-8. Doniach D. Roitt IM. Walker JG, Sherlock S. Tissue antibodies in primary biliary cirrhosis, active chronic (lupoid) hepatitis, cryptogenic cirrhosis and other liver diseases and their clinical relevance. Clin Exp Immunol 1966; 1: 237-62.
6 HEPAT 01159
Leader ____
-
~ntimitochondrial
antibodies in primary
A clue to its etiopathogenesis?’ Peter
A. Berg and Reinhild
Klein
Department of Internal Medicine, Universityof Tiibingen, Tlibingen, Germany
In the last 20 years a mass of immunological data has been collected which strongly indicates that autoimmune processes play an important role in the pathogenesis of primary biliary cirrhosis (PBC). Furthermore, the close association of antimitochondrial antibodies (AMA) with PBC, and especially of anti-M2 which can be detected ; :I up to 96% of patients (1) seems to indicate the specific induction of these antibodies by a disease-precipitating agent expressing cross-reacting epitopes. In this article we discuss the significance of anti-M2 for the etiopathogenesis of PBC.
The M2/anti-M2 system and its relationship to the etiology of PBC In 1965, Walker and colleagues described a specific cytoplasmic fluorescence pattern in the immunofluorescence test on cryostat sections with sera from patients with PBC, and it was suggested that this reaction may be due to the presence of AMA in these sera (2). The association of the corresponding antigen with mitochondria was proven using purified mitochondria in the complement fixation test (3), and it could be shown that it was located within the inner membrane of mammalian mitochondria (4). This PBC-specific antigen, named M2, consists of 5 epitopes at molecular weights 70, 56, 53,45 and 36 kD (M2a-e) (5) which have been identified as subunits of the 2-oxo-dehydrogenase complexes located within mammalian mitochondria (6,7). Anti-M2 antibodies should be distinguished from naturally occurring mitochondrial antibodies (NOMA) which can also recognize several determinants in the MZfraction when tested by Western blotting (8). Three
epitopes have been described with a molecular weight of 65 kD (epsilon), and 60165 kD (zeta/eta). NOMA occur preferentially during infectious diseases such as Epstein-Barr, cytomegalovirus or E. coli infections but are rarely found in sera from PBC patients (8%) and healthy controls (10%). Interestingly, however, high titres of the antibodies against these three major NOMAg determinants have been detected in up to 70% in PBC contact persons not suffering from the disease. One could, therefore, postulate that a contagious agent circulat:ng in the blood of PBC patients may Slave induced the increased stuplulation of NOMA. A major function of natural autoantibodies is to protect individuals from an infection. The lack of NOMA production in PBC patients may, therefore, predispose these individuals to an infection with the ‘PBC-specific agent’. Several attempts have been made to identify this agent. It has been shown that sera from PBC patients react with a wide spectrum of prokaryotes including E. coli (5,9), Paracoccus denitri+zs, the thermophilic bacterium PS3, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus (9), and Enterobacteriaceae (10). Furthermore, the sera from patients with E. coli urinary tract infections reacted with ‘M2’ in the Western blot (9). In another study rabbits were immunized with rough mutants of Enterobacteriaceae and tested against mitochondria, hereby also showing a PBC-like reaction (10). It was concluded that bacteria, and especially r-forms of Enterobacteriaceae, may play a major role in the etiology of PBC. There are several findings, however, which strongly dispute this concept. For example, sera from patients with urinary tract infections did not react with the PBCspecific M2a-determinant using the purified pyruvate demajor
Correspondence:Prof. P.A. Berg, Departmentof Internal Medicine, University of Tiibingen, Otfried Miiller Str., W-7400 Tiibingen, Germany. ‘Dedicatedto Deborah Doniach on the occasion of her 80th birthday.
ANTIMTBCHONDRIAE
ANTIBODIES
IN BBC
hydrogenase complex (PDC = M2a) or the recombinant M2a antigen in the Western blot (personal obset vations). Furthermore, it has been demonstrated that sera from patients with E. coli and other infectious disorders recognize not the PBC-specific M2a antigen, but the NOMAg-epitope epsilon at 65 kD (11). As far as the reaction of sera from PBC patients with bacteria is concerned, there is clear-cut evidence that E. coli membranes, mycobacteria and other bacterial membranes may share both the M2a-determinant and NOMAg-epitopes (11). In another study it was observed that two distinct antibodies against PDC can be detected in PBC sera, one which reacts with mammalian and the other with prokaryotic PDC (12). Thus, affinity-purified anti-M2a antibodies to bovine PDC weakly reacted with E. coli PDC, and vice versa. These findings also argue against the etiological role of E. coli in PBC. One should also bear in mind that about 4% of patients with clinically, biochemically and histologically typical PBC are anti-M2-negative (1). Antibodies to the cytoplasmic enzyme, glycogen phosphorylase (= M9) (13), or to nuclear antigens such as ‘nuclear dots’ or nuclear membranes (14,15) have also been found in patients with classic but anti-MulZnegative PBC. Considering the strong B-cell activity associated with PBC, it is possible that the triggering agent is complex and expresses a wide spectrum of cross-reacting epitopes of varying immunologitial potencies which may account for the different autoantibodies observed in PBC. It is still unclear whether this B-cell stimulation is related to T-cell-dependent or -independent antigens.
stem
and its relationship
to the
Segmental and focal bile duct lesions showing T-cell infiltrations are typical in early PBC. Immunohistochemical studies have clearly indicated that these lymphocyte infiltrates are composed of cytotoxic CD8+ T-cells (1619). Based on these observations, it has been postulated that an antigen expressed on the biliary epithelium is the target of this T-cell response, and a bile-duct-associated protein or a component of the 2-oxo-dehydrogenase complex have been considered as candidates (20-23). The distribution of the EZcomponent of PDC was recently examined in the liver and lymph nodes of patients with PBC and other liver diseases using monoclonal or affinity-purified antibodies (23,24). Although the staining of bile ducts in. PBC patients was more intense than in normal or other livers, expression of EZ-components on the cell membranes of bile duct epithelial cells could not
be prove11 (24). In another study, strong expression of the mitochondrial and mycobacterial heat shock protein (hsp) 65 was found in PBC and PSC patients but not in patients with non-cholestatic liver disorders or in healthy controls (25). The increased presentation of intracellular proteins on cell membranes is a common phenomenon in cells exposed to infections or stress in general (26); due to their ability to express major histocompatibility complex (MHC) antigens class II on their surface (27-29), bile duct epithelial cells are known to function as antigenpresenting cells. However, it seems unlikely that these proteins would be major targets of the immune response, First, lymphocytes of PBC patients could not be stimulated in vitro with a hsp-containing mitochondrial antigen fractiocl (personal observation). Second, when sera from PBZ patients with high anti-M2 titers were tested against the cloned hsp 6j by Western blotting (kindly provided by Dr. Maufmann, Ulm), no specific reactions were observed. All these data provide littic evidence that mitochondrial autoantigens play a major role in the pathogenesis of the chronic inflammatory bile duct process. Anti-M2 antibodies do not seem to be involved in the pathogenesis of PBC either. Although immunoglobulins were found on the surface of biliary epithelium in liver biopsies of PBC patients, anti-M2 specificity could not be verified (30). Furthermore, immunization of experimental animals with the EZcomponent of PDC generated mitochondrial antibodies but no bile duct lesions (31). The observation that anti-M2 antibodies persist after orthotopic liver transplantation and that PBC did not reappear in these patients over an observation period of up to 10 years (32) is also intriguing. In recent publications an inhibition of mitochondrial function by antibodies against PDC has been demonstrated in vitro, and its role with respect to the pathogenesis of PBC was discussed (33-36). However, it seems unlikely that these antibodies could interfere with the mitochondrial functions of intact cells in vivo. Also the high and non-fluctuating AMA titers in PBC strongly argue against binding in vivo.
Although great progress has been made in identification of the mitochondrial antigen-antibody systems, the definition of the MZsystem at a molecular level has not really improved our understanding of the disease. This is not surprising since classical PBC can occur in the absence of anti-M2.
P.A. BERG AND R. KLEIN
3
me outstanding immunological phenomenon in PBC is a StrongB-cell activation associated with the production of high-&red antibodies against a spectrum of autoantigens. This hyperreactivity may reflect a genetically determined disposition to respond to self- or non-self antigens in a subset of T-helper cells known to stimulate preferentially B-cells (Tm = T-helper cells, type 2) (37). The agentwhich triggers PBC is probably harboured in the epithelial cells of bile ducts. It is well documented that glandular ducts have a special property as reSeWOirS formicro-organisms (38); due to their ability to express MI-K class I and II molecules in high amounts (27-29) they can also act as antigen-presenting cells and become targets for cytotoxic T-cells. Analysis of portal infiltrates by immunohistochemical methods has revealed that the cellular composition of granuloma is similar to tuberculosis (39), and the histological pattern in mice inoculated with mycoplasma resembles the pattern in PBC (40). Early histological reactions include lymphocytic, eosinophilic and granulocytic infiltrates, morphological phenomena which are more compatible with an infection caused by bacteria or parasites than viruses. The exacerbation of latent intracellular infectious processes of some bile ducts due to insufficient control by the natural immune system (genetically determined specific B-cell defect related to NOMA production?) or other alterations in regulatory immunological mechanisms may be operative in the precipitation of the segmental or focal bile duct lesions. It is still unknown whether T-cell cytotoxicity is directed mainly against self or non-self epitopes. For the moment, there is no strong evidence for MZrelated T-cell cytotoxicity, although T-cells from PBC patients could be stimulated with the PDC-E2 complex to produce interleukin-2 (23). The strong activation of B-cells secreting disease-specific anti-M2 antibodies
could be due to either cross-reacting epitopes which activate T-cells, thereby stimu!ating B-cells to produce AMA, or merely a T-cell-independent activation of B-cells. Since an immune response may be triggered either by THI (responsible for controlling cytotoxic defense mechanisms) or T,,-cells, there is strong evidence for T,,activation in PBC. It is known that T,,-cells preferentially stimulate B-cells which secrete IgGl, IgG3, and IgM immunoglobulins. T,, cells also produce interleukin4, -5 and -6 (37,41), thus stimulating the release of bone marrow cells and especially eosinophils. Interestingly, anti-M2 antibodies have been shown to belong mainly to the IgGl, IgG3 or IgM class but rarely to the IgG2 class. The latter immunoglobulins are preferentially controlled by THL-cells. Furthermore, lymphocytes of PBC patients can spontaneously produce interleukin-6 compared to controls (unpublished observation), and in some patients PBC is accompanied by a rather strong eosinophilic reaction (42). These findings can also be explained by an activation of T,,-cells. The diagnostic exclusivity of antimitochondrial antibodies - so convincingly shown already 27 years ago (2) - makes research in this field fascinating. The statement made by Deborah Doniach at the time that an etiological or pathogenetic role in the perpetuation of liver disease cannot be demonstrated for mitochondrial antibodies or antigens (43) still holds true today.
Acknowledgements R.K. is supported by grants of the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Be 417/19).
References 1 Berg PA, Klein R. Lindenborn-Fotinos J. Antimitochondrial antibodies in primary biiiary cirrhosis. J Hepatol 1986; 2: I2331. 2 Walker JG, Doniach D, Roitt IM, Sherlock S. Serological tests in diagnosis of primary biliary cirrhosis. Lancet 1965; i: 827-31. 3 Berg PA, Doniach D, Roitt IM. Mitochondrial antibodies in primary biliary cirrhosis. I. Localization of the antigen to mitochondrial membranes. J Exp Med 1967; 126: 277-90. 4 Berg PA, Muscztello U, Home RW, Roitt IM, Donia:h D. Mitochondrial antibodies in primary biliary cirrhosis, II. me complement fixing antigen as a component of mitochondrial inner membranes. Br J Exp Pathol 1969; 50: 200-g. 5 Lindenborn-Foiinos J, Baum H, Berg PA, Mitochondrial antibodies in primary biliary cirrhosis. Further characterizationof
the W-antigen by immunoblottingrevealing species and nonspeciesSp‘XifiC determinants. Hepatology 1985; 5: 763-g. 6 Bassendine MF, Fussey SPM. Mutimer DJ, James OFW, Yeaman SJ. Identification and characterization of four M2 mite-
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9
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