Dig Dis 1992:10:85-101

Department of Internal Medicine. University of Tubingen, FRG

Antimitochondrial Antibodies in Primary Biliary Cirrhosis and Other Disorders: Definition and Clinical Relevance

Key Words

Abstract

Antimitochondrial antibodies Primary biliary' cirrhosis Syphilis Myocarditis Collagen disorders Drug-induced disorders Naturally occurring mitochondrial antibodies

The nine different antimitochondrial antibody specificities found in nonhepatic and hepatic disorders are described. Anti-Mi and anti-M7 anti­ bodies are associated with infectious disorders such as syphilis or myocar­ ditis. Anti-M3 and anti-M6 have been found in the course of a drug aller­ gic disease due to Vcnocuran and iproniazid, and anti-M5 antibodies seem to occur occasionally in some forms of ANA-positive and ANAnegative collagen disorders. The Ml - and M7-antigens are biochemically defined as cardiolipin and sarcosine dehydrogenase, respectively. AntiM2. anti-M4. anti-M8. anti-M9 are associated with primary biliary cirrho­ sis (PBC). M2 was identified as a-ketoacid-dehydrogenase complex of the inner mitochondrial membrane, anti-M4 as sulfite oxidase, an enzyme of the mitochondrial intermembrane space, and anti-M9 as glycogen phosphorylase. a cytoplasmic enzyme. M8 copurifies with outer mitochondrial membranes derived from pig kidney. Anti-M9 can occur in the absence of anti-M2 while anti-M4 and anti-M8 are always associated with anti-M2. A progressive course of PBC can be predicted with high probability even at early stages of the disease when complement fixing antibodies against M2. M4 and/or M8 are present in patients' sera. In contrast, the presence of anti-M2/M9 antibodies heralds a benign course. The etiopathogenesis of PBC is still unknown. In PBC contact persons a strong stimulation of nat­ urally occurring mitochondrial antibodies (NOMA) has been observed which was in contrast to the lack of this antibody type in PBC patients. Considering the generally accepted role of those antibodies in protecting individuals from infections, the failure of NOMA production may be a predisposing factor to acquire PBC more easily.

Antimitochondrial antibodies (AMA) have been found to be associated with a spec­ trum of different diseases. Thus, they occur in a high frequency in primary biliary cirrhosis (PBC) but can be present also in some forms

of etiologically undefined cardiomyopathies and in collagen disorders. Furthermore, they were detected transiently in sera from pa­ tients with drug-induced systemic and organspecific side effects and also in the course of

Prof. Dr. P. A. Berg Department oflnicrnal Medicine University of Tübingen Otfried-Mül.cr-Strasse D-W-740O Tübingen (FRG)

© I9‘J2 S. Karger AG. Basel 0257-2753/92/ 0 102-008 5S2.75/0

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Peter A. Berg Reinhild Klein

bacterial or viral infections. By fractionation and biochemical analysis of mitochondrial antigens it could be shown that the different clinical entities can be related to distinct AM A types. For the demonstration of these antibodies a spectrum of methods has to be applied in view of the fact that not all subtypes can be detected by the same method. Tables 1 and 2 summarize the principle find­ ings concerning the nine different mitochon­ drial antigen/antibody systems. The clinical relevance as well as the etiopathogenetie role of these antibodies will be discussed in this review focusing mainly on AMA in PBC.

A M A Associated with Infectious Disorders Anti-M 1

In 1970. Wright et al. [1] found a mito­ chondrial antibody in sera from patients with untreated secondary syphilis using the immu­ nofluorescence test (IFL) on cryostat sections (tables 1. 2). It could be absorbed with puri­ fied cardiolipin or Veneral Disease Research Laboratory antigen (a mixture of cardiolipincholesterol-lecithin), and was named 'cardio­ lipin F antibody’. This antibody type - now defined as antiM1 - reacts in the ELISA with inner mito­ chondrial membranes derived from beef heart or rat liver but hardly with purified car-

Table 1. Nine mitochondrial antigen/antibody systems: clinical relevance Clinical relevance

References

AMA in infectious disorders Anti-M l found in patients with syphilis stage II; anti-Ml has to be differentiated from anticardiolipin antibodies of other specificities occurring in collagen disorders (‘antiphospholipid syndrome') Anti-M7 present in sera from patients with acute myocarditis (60%) and dilated cardiomyopathy of unknown etiology (30%) AMA in collagen disorders Anti-M5a ) found in some sera from patients with ANA-positive Anti-M5b f and-negative collagen disorders

8

AMA in PBC Anti-M2 disease-specific marker (occur in 95% of patients with clinical or morphological evidence of PBC) Anti-M9 found in anti-M2-positivc and anti-M2-negative PBC, present in patients with a benign course Anti-M4 I occur exclusively in association with anti-M2; can be taken as a marker Anti-M8 J for a progressive course even when present in patients with stage l/II

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4. 5

6,7

AMA in drug-induced disorders Anti-M3 have been found in sera from patients with Vcnocuran-induced pseudolupus syndrome (drug has been withdrawn from the market) Anti-M6 have been found in sera from patients with iproniazid-induced hepatitis (drug has been withdrawn from the market)

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l-3

9, I0 11

12—16 17-19 20-22 20, 23

Heterogeneity of Antimitochondrial Antibodies

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AMA type

-9 8 9070 -----6 4 61

-5 9

56

1

-4 5

-4 2 37-

H L SMP SMP Antl-M1

* SD L H SD L SMP SMP P Anti-M7

H L SMP SMP Anti-M5b

Fig. 1. Demonstration of the specific antigenic de­ terminants in infectious diseases (Ml, M7). collagen disorders (M5b). and PBC (M2. M9) using different antigen fractions in the Western blot. H = Bovine heart: L = rat liver: P = Pseudomonas aeruginosa; PDH

M2

PDH PDH Y G Ami-M2

Phb M

Phb M9 L Anti-M9

Y = PDH provided by Dr. Yeaman. Newcastle: PDH G = cloned M2a(human PDH) provided by Dr. Gersh­ win. Davis: Phb = glycogen phosphorylase b; M = skel­ etal muscle.

Table 2. Nine mitochondrial antigcn/antibody systems: characterization and methods for detection Method for detection

Antigenic determinants (Western blot). kD

AMA in infectious disorders Anti-Mi cardiolipin Anti-M7 sarcosinc dehydrogenase

IFL. CFT, ELISA. WB ELISA, WB

37 90.64. 42

AMA in collagen disorders Anti-M5a not known (IMM) Anti-M5b not known (OMM)

IFL IFL. CFT. ELISA. WB

not detectable 61.57

AMA in drug-induced disorders Anti-M3 not known (OMM) Anti-M6 not known (OMM)

IFL. CFT. ELISA IFL. CFT, ELISA

not detectable not detectable

AMA in PBC Anti-M2 Anti-M9 Anti-M4 Anti-M8

IFL. CFT, ELISA. WB ELISA, WB CFT. ELISA CFT. ELISA

70. 56. 52. 45, 36 98.59 not detectable1 not detectable

AMA types

Corresponding antigen

2-oxoacid-dehydrogenase complex glycogen phosphorylase sulfite oxidase not known (OMM)

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WB= Western blot: IMM = inner mitochondrial membrane: OMM = outer mitochondrial membrane. 1 Sulfite oxidase has a molecular weight of 55-60 kD.

Anti-M 7 Anti-M7 antibodies occur in patients with acute myocarditis or cardiomyopathy of un­ known etiology and can be detected by ELISA using SMP from beef heart and rat liver [4] (tables 1,2; fig. 1). The corresponding antigen was recently identified as an epitope on bacterial and mam­ malian sarcosine dehydrogenase (SD) known to have a molecular weight of 42 kD (bacterial SD) and 90 kD (mammalian SD) [5], Mamma­ lian SD occurs only in liver, and testing antiM7-positive sera against rat liver SMP by Western blotting, the 90-kD epitope could be detected. Interestingly, anti-M7-positive sera revealed a second determinant at 64 kD with liver SMP. and this epitope could be also visualized using SMP from beef heart. By im­ munological studies it could be clearly shown that the SD-related 42- and 90-kD epitopes and the heart-associated 64-kD polypeptide are identical. These findings are suggestive for an organ-specific autoimmune reaction in pa­ tients with anti-M7-positive heart diseases.

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Thus, an acute or latent infection of cardiocytes may lead to the expression of previously hidden (M7-specific?) determinants on the cell membrane, and this antigen may trigger a B cell clone which originally has been sensitized against epitopes present on the evolutionary highly conserved bacterial SD.

AMA-Associated with Collagen Disorders

Anli-M5 Anti-M5 antibodies have been first de­ scribed by IFL in sera from patients with ANA-positive and ANA-negative disorders (tables 1,2). A high association with antiphos­ pholipid antibodies was observed. They rec­ ognize an antigen which copurifies with inner mitochondrial membranes as shown by ab­ sorption studies [6. 7]. We recently defined another AMA type in patients with collagen disorders which could be detected by IFL. ELISA, and Western blot­ ting using outer mitochondrial membranes [8], It recognizes two epitopes at 61 and 57 kD (fig. 1, tables 1,2). In view of the immunological differences of these two AMA types we named them antiM5a and anti-M5b.

AMA-Associated with Drug-Induced Disorders

Anti-M3 Patients with Venocuran-induced pseudo­ lupus syndrome produced an antibody which reacted in the complement fixation test with an outer mitochondrial membrane associated antigen [9. 10] (table 1). High titers were ob­ served during the acute clinical episode, but they decreased rapidly when treatment was stopped.

1Ictcrogcncity of Annmilochondrial Antibodies

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diolipin. It recognizes a determinant at 37 kD when tested in the Western blot against submitochondrial particles(SMP) from heart and liver (fig. 1). The exclusive association ofantiMl with syphilis strongly implies that this antibody was stimulated by cross-reactivity with membranous structures of the agent Tre­ ponema pallidum. Anti-Mi has to be distinguished from another type of anticardiolipin antibodies which gives a strong positive reaction in the ELISA with cardiolipin as well as with throm­ boplastin. a mixture of different phospholip­ ids, but cannot be detected by 1FL. These antibodies were observed preferentially in pa­ tients with collagen disorders who suffered from venous and arterial thrombosis, throm­ bocytopenia. and recurrent fetal loss (‘anti­ phospholipid syndrome’) [2. 3],

M2 determinants

Identification of polypeptides

■

-

a

70 kD

I

E2 subunit of the PDH complex (dihydrolipoamid acetyltransferase)

„

-

b

56 kD

h

protein X of PDH

51 kD IV ll

-

d

45 kD

V

-

e

36 kD

VI

(branched chain acyltransferase) E2 subunit of the 2-oxoglutarate dehydrogenase complex (succinyl transferase) E1 alpha ^ 5 s u b jn it of the PDH complex E l-be ta

Fig. 2. Demonstration of the five major M2 determinants by Western blotting and their identification as the six polypeptides of the 2-oxoacid dehydrogenase complex.

AMA-Associated with Primary Biliary Cirrhosis

Four AM A types. anti-M2, -M4. -M8, -M9, have been detected in sera from patients with PBC using different mitochondrial subfrac­

tions [24] (table 1). Anti-M2 is the most im­ portant marker for the serological diagnosis of PBC: thus. 95% of patients with clinically and histologically defined PBC are positive when tested by ELISA against the purified M2 frac­ tion. while only 85% show the typical AMA pattern in the IFL [ 12, 25]. Anti-M9 is helpful to detect PBC cases which are anti-M2-negative: anti-M4 and anti-M8 occur only in asso­ ciation with anti-M2 and predominate in pa­ tients with advanced PBC. The corresponding antigens copurify either with inner (M2) or outer mitochondrial membranes (M4. M8. M9). The M2 antigen has been cloned by Gershwin’s group [26], M4 and M9 were identified as distinct en­ zymes by conventional methods [19. 22], All three antigens are evolutionary highly con­ served proteins [14-16, 19. 22].

Anti-M2 In 1965. Walker et al. [27] described for the first time AMA by IFL on cryostat sec­ tions. It took 25 years until the nature of the corresponding antigen could be identified as the six polypeptides of the 2-oxoacid dehy­ drogenase complex (fig. 1,2), an enzyme com­ plex which is loosely associated with the inner

8‘)

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Anti-M6 A drug-induced mitochondrial antibody was also detected in patients who developed acute hepatitis after the intake of iproniazid [11] (tables 1,2). This anti-M6 antibody could be shown to react preferentially with an anti­ gen associated with outer membranes derived from rat liver mitochondria. Like anti-M3, anti-M6 could be detected by 1FL, comple­ ment fixation test (CFT). and ELISA, but not by Western blotting. The transient rise of anti-M3 and anti-M6 in patients with drug side effects could be due to the loss of tolerance towards an autoan­ tigen induced by the newly formed carrier/ hapten complex. Both drugs have been with­ drawn from the market, and no other antiM3- or anti-M6-specific reactions have since been observed in sera from patients with other drug-induced disorders.

History of the characterization of the M2 antigen inPBC

Year

Authors and Ref. No.

History

1957

Gajdusek, 28

demonstration of complement-fixing antibodies against a cytoplasmic anti­ gen from human tissue in sera from patients with chronic liver diseases

1965

Walker et al.,27

demonstration of a cytoplasmic ‘M’ fluorescence pattern predominantly with sera from PBC patients

1966

Doniach et al.. 29

characterization of the fluorescent antibodies as antimitochondrial anti­ bodies

1967

Berg et a!„ 30

demonstration of complement fixing antibodies against an antigen of the inner mitochondrial membrane

1979

Baum et al., 31

the PBC-specific antigen can be released from inner mitochondrial mem­ branes in association with the ATPase

1981

Sayers et al., 10

ATPase enzyme and antigen activity can be separated

1982

Berget al.. 12

the ATPase-associated antigen (defined as M2) was clinically defined as a marker antigen for the serological diagnosis of PBC

1983

Stephans et al., 33

molecular cloning of the PDH subunit of E. coli (MW 70 kD)

1984/ 1985

Lindenbom-Fotinos et al., 13. 20. 34

demonstration of five antigenic determinants of the M2 antigen by Western blotting (a: 70. b: 56. c: 51. d: 45. e: 36 kD)

1985

de Marucci and Lindsay. 35

definition of protein X as separate component of PDH

1987

Bradford et al., 36

molecular cloning of the lipoate attachment site of E2 ofbovinc heart PDH

1987

Gershwin et al., 26

molecular cloning of the 70-kD determinant

1988

Yeaman et al.. 14

identification of M2a and b as E2 subunit and protein X of the PDH com­ plex

1988

van de Water et al.. 37

identification of the lipoate attachment site of M2 as the PBC-specific epi­ tope of the 70-kD M2 determinant

1988 1989

Fusscy et al.. 38 ) Surh et al., 39 J

identification of band c as E2 subunit of the 2-oxoglutarate and the branched chain 2-oxodehydrogenase complex

1989

Fussey et al.. 40

identification of band ‘d’ and ‘e0’ as El subunit-a and -p of the PDH com­ plex

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Heterogeneity of Antimitochondrial Antibodies

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Table 3.

mitochondrial membrane. The different steps which led to their discovery1 and molecular cloning are shown in table 3. In further studies van de Water et al. [41] and Frcgeau et al. [42] showed that antibodies in PBC sera inhibited the function of pyru­ vate dehydrogenase (PDH) by binding to the lipoyl-lysine residue of PDH-E2 as well as of the 2-oxoglutarate dehydrogenase. Anti-M4 When PBC sera were tested by CFT against purified inner and outer mitochon­ drial membranes, it became evident that these sera contained another complement fixing an­ tibody which reacted with an antigen of the outer mitochondrial membrane (anti-M4) [21], The corresponding antigen was trypsin insensitive in contrast to M2 which was de­ stroyed by trypsin treatment. We now succeeded to show that anti-M4 antibodies recognize epitopes which are as­ sociated with the sulfite oxidase, an enzyme

of the mitochondrial intermembrane space [22]. It is a dimeric enzyme which consists of two subunits each of 55-60 kD and is present also in bacteria. Only PBC sera which were anti-M2 and anti-M4 positive reacted with this enzyme in ELISA and CFT (fig. 3). Incu­ bation of anti-M4 antibodies with sulfite oxi­ dase did, however, not influence the enzyme activity in vitro indicating that they may not bind to the functionally active center. Ab­ sorption of anti-M2/-M4-positive sera with sulfite oxidase abolished anti-M4 but had no effect on the anti-M2 activity (fig. 4) again indicating that M2 and M4 are different anti­ gens. Anti-M4 antibodies could be detected by ELISA and CFT but not by Western blotting, hereby excluding that the trypsin-insensitive 52-kD band observed in the blot using beef heart SMP is an M4-specific epitope as pos­ tulated by Baum [43, 44], Furthermore, we found this 52-kD determinant also in antiM4-negative PBC sera [unpubl. observation].

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Fig. 3. Comparison of anti-M4 with anti-sulfite oxidase (SO) anti­ body titers in 20 anti-M2/M4-positive and 10 anti-M2/M8-positive PBC sera. Only 2 of the 20 antiM4-positive sera did not react with SO. 5 of the 10 anti-M8-positive sera became positive when tested against this enzyme.

Anti-M9 Anti-M9 antibodies were first detected testing anti-M2-positive sera against trypsintreated liver SMP which were devoid of M2 [17. 18]. The M9-antigen could be further purified by ion exchange chromatography us­ ing a 100,000# supernatant from rat liver SMP. The highest M9 activity was found in the peak eluted by 1 M NaCl. This 'purified' M9 fraction revealed two determinants at molecular weights 98 and 59 kD in the West­ ern blot [ 17] (table 1. fig. 1). Further studies indicated that M9 is an epitope of glycogen phosphorylase. a cytoplas­ mic enzyme which binds easily to particular structures [19], Absorption of patient's sera with this enzyme abolished completely the anli-M9 activity. Glycogen phosphorylase is an enzyme which is involved in intracellular degradation of glycogen and is. therefore, en­ riched in liver and skeletal muscle. Phosphorylasc a (active form) and phosphorylase b (inactive form) consist, respectively, of four and two identical subunits at MW 98 kD. The 59-kD determinant of the M9 antigen was shown to be a degradation product. Anti-M9

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against:

M2 M4 Before absorption

M2 M4 After absorption

Fig. 4. Absorption of an anti-M2/-M4-positive PBC marker serum with sulfite oxidase. Sulfite oxidase absorbed completely the anti-M4 antibodies but had no effect on the anti-M2 activity.

antibodies stimulated in vitro the enzyme ac­ tivity in the presence of A-5-MP which is required for the conversion of inactive phosphorylasc b to active phosphorylase a. This observation indicates that anti-M9 antibodies may have some affinity to the A-5-MP bind­ ing site. The FourAMA Profiles in PBC From follow-up studies in PBC patients it emerged that anti-M2/anti-M9 antibodies were frequently found in patients with the his­ tological stages I/Il while anti-M2-positive pa­ tients with advanced stages predominantly expressed anti-M4 and anti-M8. We. there­ fore. defined four different AMA profiles la­ belled A-D [24. 45. 46]: (A) only anti-M9positive by ELISA, (B) anti-M9- and/or antiM2-positive by ELISA. (C) anti-M2, -M4. and/or -M8-posilivc only by ELISA, and (D) anti-M2. -M4 and/or -M8-positive by ELISA anclC FT.

Helcrogoneiis of Antimilochondrial Antibodies

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Anti-MS Anti-M8 antibodies also recognize an anti­ gen which copurifies with outer mitochon­ drial membranes. It was isolated either from outer membranes or microsomes (known to be contaminated with outer membranes) de­ rived from pig kidney. Subjecting these frac­ tions to Scpharose 6B gel filtration or a dis­ continuous sucrose density gradient M8 was detected in the ascending part of the second peak and the densities between 1.16 and 1.24. respectively [20. 23]. Like M4. the M8 antigen cannot be de­ lected in the immunoblot. One explanation for this finding would be that antibody bind­ ing sites are destroyed by sodium dodecyl sul­ fate used in this method.

Time, years

Table 4. Correlation of the four AMA profiles with the histological stages of PBC evaluated in 327 pa­ tients

AMA profile

Patient number

A B C D

27 135 103 62

PBC stage, % I/II

III/IV

100 94 61 55

0 6 39 45

The validity of the serological classifica­ tion was analyzed in 327 patients correlating liver histology and AMA patterns at the time of first diagnosis (table 4). The AMA profiles C and D were almost exclusively found in PBC patients with stage III or IV, and 41 % of the 165 C/D patients had histological features of chronic active hepatitis [47] (table 4). In another study. 76 patients who were in stage I/II at the beginning of the observation period have been followed for 6-18 years by repeated liver biopsies and AMA screening. 44 of the 76 patients had the profile A/B. 32

Time, years

the profile C/D already in stage I/II. Only 3% of the 44 patients with profile A/B progressed to stage III/IV in contrast to 97% of the 32 patients with profile C/D [45] (fig. 5). Fur­ thermore. survival time was shorter in pa­ tients of group C/D than of group A/B. Pa­ tients who had died of PBC or who had received a liver transplant belonged all to group C/D. AMA profiles remained constant during the whole observation period in all patients except 1 who belonged to profile B and became anti-M4/-M8-positive by ELISA (profile C). Data presented in figures 5 and 6 clearly indicate that anti-M4 and anti-M8 an­ tibodies (profile C/D) can occur already in early stages of PBC and are. therefore, not a marker for advanced but for progressive PBC. A typical clinical course of a patient belonging to group C/D is shown in figure 6. It is our experience from following more than 2,000 PBC patients in the last 10 years that AMA profiles remain unchanged once established. Storage of PBC sera since 1970 allowed this retrospective analysis. As far as the fluctuation of AMA titers is concerned, anti-M2 activity remained rather stable over

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Fig. 5. Predictive value of AMA profiles in relation to the two different courses of PBC. Disease activity is given as mean score 1-4 according to the histological stages. There is a good correlation be­ tween the mean score of patients of group I (patients with a benign course) and 2 (patients with a pro­ gressive course) with that of pa­ tients expressing the AMA profiles A/B versus C/D. 3 patients had re­ ceived OLT. 6 patients had died from hepatic coma or variceal bleeding (f).

Fig. 6. Progressive course of PBC in a patient with AMA profile C. In 1978 transaminases were slightly elevated, and histologically chronic persistent hepatitis (CUP) was diagnosed. In 1982 the alkaline phosphatase also increased, and histologically the typical features of PBC stage I were found. In 1990 the patient received a liver transplant (OLT) because of PBC stage IV.

94

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Prognostic Value of A M A Profiles in Comparison to Other Prognostic Models

There are four major models for predicting survival for individual patients with PBC based on different risk variables used for sur­ vival prediction: the Shapiro model (biliru­ bin) [50], the Roll model (age. bilirubin, hepa­ tomegaly. presence of portal fibrosis or cir­ rhosis) [51], the Christensen model (age. bili­ rubin. albumin, presence of cirrhosis or cho­ lestasis, azathioprine treatment) [52], and the Mayo model (age. bilirubin, albumin, pro­ thrombin time, edema) [53]. The latter three groups used the Cox proportional hazard multivariate regression procedure [54], Since these models are based on variables which are indicators for advanced liver disease (biliru­

Heterogeneity of Anlimitochondrial Antibodies

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the years while anti-M4 and anti-M8 titers can fluctuate and are influenced by immuno­ suppressive therapy. In this respect we were unable to confirm a recent report comparing anti-M2 titers with disease progression and histological stages [48] [unpubl. observation]. We also analyzed the AMA profiles in 15 PBC patients who had been selected for or­ thotopic liver tansplantation (OLT) (coopera­ tive study with Prof. Gips, Groningen. The Netherlands) [49], 14 of them had profile C or D. and 1 patient had serological evidence for an overlap between PBC and autoimmune chronic active hepatitis. After OLT. anti-M2 remained positive in 13 of the 15 patients while antibodies .against M4 and M8 grad­ ually decreased within 1-5 years and became negative in all 14 patients.

bin. albumin, edema), the risk scores change during the course of PBC. Therefore, they cannot differentiate at early stages between patients with high or low risk for disease pro­ gression. but they allow a rather accurate pre­ diction of the appropriate timing of liver transplantation in patients prior to death [54]. In contrast, the AMA profiles are not pri­ marily a survival model but can help to dis­ criminate between the two different courses of PBC (benign versus progressive) [51. 55. 56], and to define already at early stages patients with good and bad prognosis.

PBC patients, including husbands, but all 10 patients with acute EBV, cytomegalovirus in­ fection or toxoplasmosis had this antibody type. Since it is known that naturally occur­ ring antibodies can be stimulated in the course of viral disorders, we defined the nonPBC-associated AMA as ‘naturally occurring mitochondrial antibodies' and named them NOMA and the corresponding antigen NOMAg. Interestingly, sera from PBC pa­ tients themselves had this antibody type in only 8 %.

The ‘Naturally Occurring Mitochondrial Antigen/Antibody System'

The presence of autoantibodies in family members of patients with autoimmune disor­ ders has been reported in several studies in­ cluding the demonstration of AMA in PBC families. Using submitochondrial particles from rat liver we could also detect AMA in 57% of 74 relatives including husbands. Sera from technicians who were concerned with the serological diagnosis of PBC were also positive. A PBC-specific reaction with M2, M4. M8. or M9 was definitively ruled out. However, when sera from these contact per­ sons were tested by Western blotting against the purified M2 and M9 fraction, several bands were visualized which could be clearly distinguished from that recognized by antiM2- and anti-M9-positive PBC sera, indicat­ ing that the M2 and M9 antigen fractions con­ tain another still undefined mitochondrial an­ tigen system [57], Interestingly, the same epitopes were ob­ served with sera from patients with acute Epstein-Barr virus (EBV) and cytomegalovi­ rus infections and other infectious disorders. Thus, not only 70% of 81 healthy relatives of

The etiology of PBC remains still obscure. Rough mutants of cnterobacteriaceae have been discussed to be a causative agent based on the observation that sera from rabbits im­ munized with those mutants give a positive reaction with the M2-antigen [58] and that PBC sera can react with Escherichia coli membranes [13, 58], It could be shown, how­ ever, that antibody titers against bovine PHD-E2 were 5-100 times greater than those against E. coli PDH-E2 [59], and these find­ ings indicate that PBC patients produce dis­ tinct antibodies against prokaryotic and euka­ ryotic antigens. We could also show by West­ ern blotting using E. coli membranes as anti­ gen thaï PBC sera recognize the M2a determi­ nant and sera from patients with E. coli infec­ tion another epitope at 65 kD. This indicates that E. coli membranes express PBC- and non PBC-related mitochondrial antigens (NOMAg) (fig. 7) [60]. We, therefore, strongly dispute the concept put forward by Hopf et al. [58] relating the cause of PBC to an infection with enterobacteriaceae. Another strong argu­ ment against the E. coli etiology is our obser­ vation that M2a- and NOMAg-specific epi­ topes are present also on other microorgan-

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A M A and Their Relationship to the Etiopathogenesis of PBC

75 kD -

. -a -6 5 kD

Demonstration of M2- and /:'. (’«//-specific determinants on /:. coli membranes as well as in the M2 fraction testing an anti-M2-positive PBC serum and an anti-/:, («//-positive (but AMA-negative) scrum (derived from a patient with urinary tract infection) in the Western blot. The anti-M2-positive serum recog­ nizes on E. coli membranes PBC-specific determinants at 75 and 51 kD shown to relate to M2a and M2c by elution studies. On the other hand, the anti-/:, coli-pos­ itive scrum reacts with a /:. coli-specific determinant i n the M2 fraction and on E. coli membranes at 65 kD. Purified PBC or the cloned M2a antigen (kindly pro­ vided by Dr. Yeaman and Dr. Gershwin) did not react with anti-/:, (’«//-positive sera (not shown). It is. there­ fore. very unlikely that E. coli infections induce PBCspecific antibodies. Fig. 7.

-4 8 kD

-d

E. coli M2 PBC - serum anti-M2-positive

E. coli M2 Serum from a patient with urinary tract infection

«

Fig. 8.

-a -Epsilon (65 kD)

c-

\

d-

* e-

PDH PDH Y G Serum 1

Berg/Klein

PDH G

PDH M2 Y Serum 2

M2 Serum 3

Dr. Yeaman and Dr. Gershwin) (fig. 8). How­ ever, all these data may indeed be interpreted in favor of a bacterial etiology of PBC. Natural autoantibodies play a major role in the innate immune system providing an effective and instant defense mechanisms

Heterogeneity of Antimiiochondrial Antibodies

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M2

isms. Furthermore, sera from non-PBC pa­ tients preferentially those with EBV or myco­ bacterial infections, can react with the Non­ specific determinant in the immunoblot when tested against the M2 fraction or the purified PDH and the cloned M2a (kindly provided by

96

. .. - c

I 00 rr 1

Demonstration of an M2a-specific reaction testing a se­ rum derived from a patient with acute EBV infection against the M2 fraction as well as purified PDH and the cloned M2a antigen (kindly provided by Dr. Yeaman. Y. and Dr. Gershwin. G). Clini­ cally an association of EBV infec­ tion with PBC could be ruled out. The observation that the EBV-associated anti-M2a antibody disap­ peared after recovery also argues against an association of EBV in­ fection and PBC.

51 kD - »» .» :

Fig. 9. Patients with clinically and morphologically defined PBC are serologically classified either as ‘clas­ sical PBC or "PBC syndrome’ depending upon the presence or absence of anti-M2. Patients with ‘PBC syndrome' have antibodies cither against M9. nuclear membranes (200 kD). nuclear dots or keratin. Patients with 'classical PBC' arc always anti-M2-posilivc but may express additionally antibody types found in pa­ tients with the ‘PBC syndrome'.

patients were of the Ta/p and not the T?/g type [67] - the latter being associated mainly with the innate immune system [70] - is a further argument in favor of an antigen-driven im­ mune response leading to the bile duct de­ struction in PBC. Furthermore, granuloma formation frequently observed in patients with early PBC could also be taken as indirect evidence for an immunological reaction against microorganisms. PBC, therefore, seems to be associated with two distinct forms of immunological reactivities: firstly a T-cell specific immune response induced by the agent causing PBC

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against bacterial and viral infections [61.62]. Resistance towards infections probably also depends upon the quality of these protective antibodies. From studying naturally occur­ ring mitochondrial antibodies in sera from PBC patients and their family members it emerged that NOMA were strongly stimu­ lated in PBC contact persons (up to 70%) as compared to PBC patients themselves and controls (8 and 15%) [57], These findings can be interpreted in two ways: firstly, an immu­ nogenic agent derived from PBC patients is responsible for the increased NOMA produc­ tion in PBC families; secondly. PBC patients have an immune defect concerning the pro­ duction of a specific NOMA type in response to a defined antigen as for instance heat shock proteins (see below). Therefore, the increased frequency of E. coli infections reported in PBC patients [63] may be rather a conse­ quence of a lowered resistance towards E. coli and not a clue to the etiology of PBC. It seems, however, most likely that an agent infecting small bile ducts is triggering PBC. Ductular epithelia have been shown to be reservoirs for infectious agents as demon­ strated in patients with Sjogren’s syndrome [64], and cytotoxic T cells attacking epithelial cells of bile ducts as well as immunoglobulins bound to the surface of biliary epithelum have been identified in liver biopsies of PBC pa­ tients [65-67], A hypothesis was put forward that a component of the 2-oxodehydrogenase complex or an as yet undefined protein may be exposed on the biliary epithelial cell sur­ face which becomes then the target for autoreactive T and B cells [68], Krams et al. [69] also examined the role of T cells and autoanti­ bodies by injecting lymphocytes from PBC patients into severe combined immunodeficient mice, and they could demonstrate mor­ phological features which were reminiscent of a graft versus host-like disease. The observa­ tion that T cells in liver biopsies of PBC

Intact B cell reservoir (related to the production of NOMA)

l Intact first line defense protects individuals from becoming infected

l

Defective first line defense allows the agent to intect the target organ (bile ducts)'

Induction of naturally occurring autoanti­ bodies (non-NOMArelated)

V

Pathogenesis

and generating cytotoxic T-cells. and secondly an autoimmune B-cell response probably in­ duced by self epitopes shared by the pathogen [as for instance heat shock proteins. 7 1], The appearance of PBC-specific antibodies and the absence of NOMA would then indicate that due to a somatic point mutation of origi­ nal germline sequences autoantibodies are produced in large quantities in the course of the inflammatory process perpetuated by PBC-agent-specific T-cells and probably also by autoreactive T cell clones.

98

Selective B cell defect (NOMA related)

Berg/Klein

Diagnostic marker

Reflection of quality of the autoimmune B cell response not related to the specific NOMA defect

From this aspect it is also understandable why AMA seem to play no role in the induc­ tion of bile duct lesions. There are several fur­ ther arguments against the pathogenetic role of humoral immunity: firstly, the peculiar manifestation of the histological lesions show­ ing a segmental or focal bile duct destruction: secondly, the fact that anti-M2 antibodies do persist or reappear after liver transplantation in the absence of any biochemical features indicating the recurrence of the disease [72]: thirdly, the observation that in animal models

Heterogeneity of Antimitochondrial Antibodies

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Fig. 10. A simplified view of the ctiopathogenesis of PBC based on the concept that some geneti­ cally determined defect in the pro­ tective natural autoimmune B cell reservoir may be a major predis­ posing factor in acquiring PBC. It is postulated that a population of NOMA is important to protect in­ dividuals from an infection in gen­ eral and from the PBC-related nox­ ious agent in particular (see also text). Self and non-self epitopes of this PBC-spccific agent may be pre­ sented to the immune system ci­ ther via antigen presenting cell (APC) or by the epithelial target cells of bile ducts (not shown). The non-self epitopes induce cytotoxic T cells, which then attack the target cells (segmental or focal inflamma­ tory process). Self epitopes on the other hand may stimulate nonspecifically the natural autoimmune B cell reservoir perhaps via a subpop­ ulation of T cells (Tx). Following the concept of a selective B cell defect the production of protective NOMA in PBC should be weak or absent, and this has. indeed, been observed in PBC patients. Finally, the induction of anti-M2 may be ex­ plained either by a stimulation of an autoreactive B cell clone or by the newly formed epitopes composed of self and non-self determinants.

the production of anti-M2 did not go along with the development of PBC [73]. and fourthly, experiments showing that lympho­ cytes of PBC patients are not stimulated by purified mitochondrial antigen fractions in vitro again indicating that cytotoxic T cells are not directed against mitochondrial epi­ topes [unpubl. data]. Following the concept of the expression of intracellular antigens on the surface of in­ fected cells, as convincingly recently demon­ strated for heat shock proteins, the produc­ tion of so many different antibody types in PBC is not any more surprising. Thus. PBCspecific antibodies to cytoplasmic, nuclear, and cytoskeletal antigens have been found in association with anti-M2 (in about 40%) but also in the absence of anti-M2 [24, 74-78], Since the latter patients expressed clinically and morphologically the typical criteria of

PBC we are inclined to define those AMAnegative but antibody-positive cases as 'PBC syndrome’, and the AMA-positive cases as 'classical PBC’ (fig. 9). In conclusion, concerning the etiopathogencsis of PBC we postulate that the agent triggering the disease expresses strongly im­ munogenic epitopes of mitochondrial nature and that a specific defect in the natural de­ fense system (as defined by the decreased pro­ duction of protecting an individuum against a certain kind of infection) naturally occurring mitochondrial antibodies is a predisposing factor in acquiring PBC (fig. 10). Acknowledgement R.K. is supported by grants of the Deutsche Forschungsgemeinschaft, Bonn Bad-Godesberg (Be 431/19).

References 6 Labro MT. Andricu MC. Weber M. ct al: A new pattern of non-organand non-species-specific anti-organelle antibody detected by immuno­ fluorescence: The mitochondrial an­ tibody number 5. Clin Exp Immu­ nol 1978:31:357-366. 7 Meroni PL. Harris EN. Brucato A. et al: Anti-mitochondrial antibody type 5 and anti-cardiolipin anti­ bodies in autoimmune disorders: Studies on their association and crossreactivity. Clin Exp Immunol 1987:67:484-491. 8 Klein R. Berg PA: Nachweis und Be­ deutung von organunspezifischen antimitochondrialcn Antikörpern (Anti-M5b) bei Patienten mit unkla­ ren Kollagenerkrankungen. Klin Wochenschr I987:65(suppl 9):98. 9 Grob PJ. Müller-Schoop JW. Häckl MA, et al: Drug-induced pseudolu­ pus. Lancet 1975:0:144-148.

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35 De Marucci O. Lindsay JG: Compo­ nent X. An immunologically dis­ tinct polypeptide associated with mammalian pyruvate dehydroge­ nase multi-enzyme complex. Eur J Biochem 1985:149:641-648. 36 Bradford AP. Howell S. Aitken A. ct al: Primary structure around the lipoate-attachment site on the E2 component ofbovine heart pyruvate dehydrogenase. Biochem .1 1987; 245:919-922. 37 Van de Water J. Gershwin E. Leung P. et al: The autoepitope of the 74kD mitochondrial autoanligcn of primary biliary cirrhosis corre­ sponds to the functional site ofdihydrolipoamide acctyltransferase. J Exp Med 1988:167:1791-1799. 38 Fussey SPM. Guest JR. James OFW. ct al: Identification and anal­ ysis of the major M2 autoantigens in primary biliary cirrhosis. Proc Natl Acad Sci USA 1988:85:8654-8658. 39 Surh CD, Danner DJ. Ahmed A. et al: Reactivity of PBC sera with a human fetal liver cDNA clone of branched chain alpha-ketoacid de­ hydrogenase (BCKD) dihydrolipoamide acyltransferasc. the 52kD mitochondrial autoantigen. Hepato­ logy 1989:9:63-68. 40 Fussey SPM. Bassendine MF. Fittes D. et al: The El alpha and beta sub­ units of the pyruvate dehydrogenase complex are M2 'd ‘ and M2 ‘e‘ autoantigens in primary biliary cirrho­ sis. Clin Sci 1989:77:365-368. 41 Van de Water J. Fregeau D, Davis, ct al: Autoantibodies of primary' bil­ iary cirrhosis recognize dihvdrolipoamide acctyltransferase and in­ hibit enzyme function. J Immunol 1988:141:2321-2324. 42 Fregeau DR. Prindiville T. Coppcl Rl.. et al: Inhibition of ketoglutarate dehydrogenase activity by a distinct population of autoantibodics recog­ nizing dihydrolipoamide succinvltransferase in primary biliary cir­ rhosis. Hepatology 1990:11:975981. 43 Ghadimincjad 1. Baum H: Discrimi­ nation between M2 and M4 antimitochondrial antigens in primary bil­ iary cirrhosis. Biosci Rep 1986:6: 1049-1056. 44 Baum H: Nature of mitochondrial antigens of primary biliary cirrhosis and their possible relationship to the etiology of the disease. Semin Liver Dis 1989;9:117-123.

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57 Klein R. Berg PA: Demonstration of 'naturally occurring mitochondrial antibodies' in family members of patients with primary biliary cirrho­ sis. Hepatology 1990; 12:335-341. 58 Stemerowicz R, Hopf U. Möller B: Are mitochondrial antibodies in primarv biliary cirrhosis induced by r(rough) mutants of enterobacteriaceae? Lancet I988:ii:l 166-1170. 59 Fussey SPM. Yeaman SJ. James OFW. et al: Reactivity of antimitochondrial antibodies in PBC: Little support for a bacterial etiology. Hepatology 1990:12. ’ 60 Berg PA. Klein R: Mitochondrial M2 autoantigens and primary bil­ iary cirrhosis. Lancet I989:i:447. 61 Naparstek Y. Andres-Schwartz .1. Manser T. et al: A single germline VH gene segment of normal A/J mice encodes autoantibodies char­ acteristic of systemic lupus erythe­ matosus. J Exp Med 1986:164:614— 626. 62 Avrameas S. Dighiero G, Lymberi P. et al: Studies on natural anti­ bodies and autoantibodics. Ann Im­ munol 1983; 134D: 10 3 -113. 63 Burroughs AK, Rosenstein IJ. Ep­ stein O. et al: Bacteriuria and pri­ mary biliary cirrhosis. Gut 1984:25: 133-137. 64 Green JE. Hinrichs SH. Vogel J. et al: Exocrinopathy resembling Sjo­ gren's syndrome in 4TLV-I tax transgenic mice. Nature 1989:341: 72-74. 65 PapeGR. Rieber EP. Eisenburg J. et al: Involvement of the cytotoxic/ suppressor T-cell subset in liver tis­ sue injury' of patients with acute and chronic liver diseases. Gastrocnlerology 1983:85:657-662. 66 Hoffman RM. Pape GR. Spengler U, et al: Clonal analysis of liverderived T-cells of patients with pri­ mary biliary cirrhosis. Clin Exp Im­ munol 1989:76:210-215. 67 Krams SM. van de Water J, Coppel RL. et al: Analysis of hepatic T lym­ phocyte and immunoglobulin deposits in patients with primary bil­ iary'cirrhosis. Hepatology 1990:12: 306-313. 68 Mackay 1R, Gershwin ME: Molecu­ lar basis of mitochondrial autoreactivity in primary biliary' cirrhosis. Immunol Today 1989;10:315-318.

69 KramsSM, Dorshkind K, Gershwin M E: Generation of biliary lesions af­ ter transfer of human lymphocy tes into severe combined immunodeficienl (SO D ) mice. J Exp Med 1990; 170:1919-1930. 70 Modlin R. Pirnicz C. Hofmann FM. et al: Lymphocytes bearing antigenspecific T-cell receptors accumulate in human infectious disease lesions. Nature 1989:339:544-547. 71 Kaufmann SHE: Heat shock pro­ teins and the immune system. Im­ munol Today 1990:11:129-136. 72 Haagsma EII. Manns M. Klein R, et al: Subtypes of antimitochondrial antibodies in primary biliary cirrho­ sis before and after orthotopic liver transplantation. Hepatology I987;7: 129-133. 73 Krams SM. Surh CD, Coppel RL, et al: Immunization of experimental animals with dihydrolipoamide acetvltransfcrase. as a purified recombi­ nant polypeptide, generates mito­ chondrial autoantibodics but not primary biliary cirrhosis. Hepatol­ ogy 1989:9:411-416. 74 Penner E. Kindas-Miigge I. Hitehmann E. et al: Nuclear antigens rec­ ognized by antibodies present in liver disease sera. Clin Exp Immu­ nol 1986:63:428-433. 75 Pisi E. Bianchi FB. Ballardini G. et al: New immunopathologic features in primary biliary cirrhosis. Front Gastroenterol Res 1986:9:132—142. 76 Lozano F. Raris A. Borche L. cl al: Autoantibodics against nuclear en­ velope-associated proteins in pri­ mary biliary cirrhosis. Hepatology 1988:8:930-933. 77 Lassoued K. Brenard R. Degos F. et al: Antinuclear antibodies directed to a 200-kilodalton polypeptide of the nuclear envelope in primary bil­ iary cirrhosis. A clinical and immu­ nological study of a series of 150 patients with primary biliary cirrho­ sis. Gastroenterology 1990:99:181 — 186. 78 Klein R. Gebel M. Schollhom V. et al: Definition of antimitochondrial antibody (AMA) negative ‘primary biliary cirrhosis' by the presence of antibodies against laminin and kera­ tin. J Hepatol 1987;5/S I :S 152.

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45 Klein R, Klöppel G. Garbe W. et al: Anlimitochondrial antibody pro­ files determined at early stages of primary biliary cirrhosis differen­ tiate between a benign and a pro­ gressive course o f the disease: A ret­ rospective analysis of 76 patients over 6-18 years..I Hepatol 1991:12: 21-27. 46 Berg PA. Klein R: Heterogeneity of antimitochondrial antibodies. Sem­ in Liver Dis 1989:9:103-116. 47 Klein R. Berg PA: Prognostische Be­ deutung der antimitochondrialen Antikörperprofile A-D bei Patien­ ten mit primär-biliärer Zirrhose. Dtsch Med Wochenschr 1988:113: 1549-1553. 48 Hescltine L. Turner IB. Fussey SPM, et al: Primary biliary cirrho­ sis. Quantitation of autoantibodics to purified mitochondrial enzymes and correlation with disease pro­ gression. Gastroenterology 1990:98: 1786-1792. 49 Klein R. Klöppel G. Gips CH. et al: Die primär-biliäre Zirrhose vor und nach Transplantation - Antikörper­ verlauf. Z Gastroenterol 1989:24: 288. 50 Shapiro JM. Smith H. Schaffner F: Serum bilirubin: A prognostic factor in primarv biliarv cirrhosis. Gut 1979:20:137-140.' 51 Roll J. Boyer JL. Barry D. et al: The prognostic importance of clinical and histologic features in asymp­ tomatic and symptomatic primary biliary cirrhosis. N Engl J Med 1983:308:1-7. 52 Christensen E. Neuberger .1. Crowe J. et al: Beneficial effect of azathioprine and prediction of prognosis in primary biliary cirrhosis. Final re­ sults of an international trial. Gas­ troenterology 1985:89:1084-1091. 53 Dickson ER. Grambsch PM, Flem­ ing TR. et al: Prognosis in primary biliary cirrhosis: Model for decision making. Hepatology 1989:10:1-7. 54 Cox DR: Regression models and life-tables (with discussion). J R Stat Soc(B) 1972:34:187-202. 55 JctTrey GP. Hoffman NE, Reed WD: Validation of prognostic mod­ els in primary biliary cirrhosis. Aust N Z JM cd 1990:20: ¡0 7 -110. 56 Beswick DR. Klatskin G. Boyer JL: Asymptomatic primary biliary cir­ rhosis. A progress report on long­ term follow up and natural history. Gastroenterology 1985:89:267-271.