Antimitochondrial Antibodies and OtherAntibodies in Primary Biliary Cirrhosis:

Diagnostic and Prognostic Value

Luigi Muratori, MD, PhD*, Alessandro Granito, MD,Paolo Muratori, MD, Georgios Pappas, MD,

Francesco B. Bianchi, MDDepartment of Internal Medicine, Cardioangiology,

Hepatology, Alma Mater Studiorum - University of Bologna,

Policlinico Sant’Orsola-Malpighi, Via Massarenti, 9-40138, Bologna, Italy

Historical overview

Antimitochondrial antibodies (AMA) were described for the first time inpatients who had primary biliary cirrhosis (PBC) more than 40 years ago [1]and continue to be regarded as the most sensitive and specific immunologichallmark of the disease [2,3]. The original indirect immunofluorescence(IFL) technique set-up in the laboratory of Ivan M. Roitt and DeborahDoniach is still the most common assay used to reveal the classical immuno-morphologic pattern of AMA, corresponding to the cytoplasmic staining oftissues rich in mitochondria, such as rat kidney, stomach, and liver [1].

The antigenic target of AMA initially was identified as a non–organ-spe-cific ATPase-associated antigen (M2) present in the inner mitochondrialmembrane [4], consisting of several mitochondrial polypeptides [5]. AMAreactivity tentatively has been classified into nine mitochondrial antigen/antibody systems (M1 to M9), of which only M2, M4, M8, and M9 areconsidered PBC specific [6]. The 70-74–kd mitochondrial polypeptide recog-nized by most AMA-positive sera in immunoblotting [7] was cloned [8] andidentified as the E2 component of the pyruvate dehydrogenase multienzymecomplex (PDC-E2) [9]. In addition to PDC-E2, other members of the of 2-oxoacid dehydrogenase complex (2-OADC) were recognized as additionaltargets of AMA, namely the E2 subunit of branched-chain 2-OADC

Clin Liver Dis 12 (2008) 261–276

* Corresponding author.

E-mail address: luigi.muratori@unibo.it (L. Muratori).

1089-3261/08/$ - see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.cld.2008.02.009 liver.theclinics.com

262 MURATORI et al

(BCOADC-E2), the E2 subunit of oxoglutarate dehydrogenase complex(OGDC-E2), and the E1a subunit and the E3 binding protein of PDC(PDC-E1a and E3BP, respectively). These enzyme complexes play a centralrole in the glycolytic pathway, the tricarboxylic acid cycle, and the pathwayof branched-chain amino acid metabolism. The E2 enzymes share a commonstructure in the N-terminal domain containing lipoic acid moieties. Inmammals, only five proteins contain lipoic acid, and four of them are PBCautoantigens [10]. The dominant epitope recognized by AMA is locatedwithin the lipoyl domain, and the presence of the lipoic acid moiety on theE2 proteins is essential for AMA binding. In addition, anti–lipoic acidantibodies have been detected in the vast majority of PBC sera but not incontrols [11].

Methods of antimitochondrial antibody detection

IFL on cryostatic unfixed sections of rat kidney, stomach, and liver, orig-inally described in 1965 [1], still is the routine assay to detect AMA. Theubiquitous distribution of the mitochondrial antigens determines the typicalgranular, coarse cytoplasmic staining of the renal tubular cells, gastric pari-etal cells, and hepatocytes (Fig. 1). There is another immunofluorescencepattern often misdiagnosed as AMA given by liver/kidney microsomal

Fig. 1. The typical granular ‘‘coarse’’ cytoplasmic reactivity of AMA on different unfixed rat

substrates by IFL: renal tubules are all positive, whereas the glomeruli are not (A); gastric

parietal cells also are positive (B); liver hepatocytes are homogeneously stained (C).

263AMA AND OTHER ANTIBODIES IN PBC

antibody type 1 (LKM1). LKM1 stains the cytoplasm of liver and renaltubules but not the parietal cells of the stomach; in addition, the detectionof alternating positive and weakly positive or even negative renal tubulesis the typical feature of LKM1 renal pattern [12], because its target antigen,the isoform 2D6 of the cytochrome P450, is distributed unevenly along therenal tubule. The misinterpretation of LKM1 as AMA may have importantdiagnostic and therapeutic consequences, as LKM1 is the serologic markerof type 2 autoimmune hepatitis (AIH) [13], usually an aggressive liver dis-ease, often requiring intensive immunosuppression [14]. The technical guide-lines recently issued by the International Autoimmune Hepatitis Group(IAIHG) committee on autoimmune liver serology are a first step towardstandardization of autoantibody detection and interpretation using theIFL technique [15].

HEp2 cell lines are an additional substrate for AMA detection by IFL.AMA-positive sera give a typical granular cytoplasmic staining on HEp2cells (Fig. 2). In addition, IFL on HEp2 cells allows the immunomorpho-logic definition of antinuclear reactivities, commonly observed in patientswho have PBC [16,17].

Western immunoblotting (W-IB) is considered a second-level test toconfirm the presence of AMA detected by IFL or to identify AMA-specific

Fig. 2. The ‘‘coarse’’ cytoplasmic staining is the typical pattern of AMA by IFL on HEp2 cells,

whereas the nuclei are negative (left panel). W-IB allows further dissection of AMA reactivity,

which is directed against members of the 2-OADCs, a family of polypeptides of different

molecular size. In this representative picture, AMA react with the 74-kd E2 component of

PDC-E2, the 55-kd E3BP, the 52-kd E2 subunit of BCOADC-E2, and the 48-kd E2 subunit

of OGDC-E2, in different combinations (right panel). See Table 2.

264 MURATORI et al

reactivities in patients who have a negative IFL AMA test, given the highersensitivity of W-IB versus IFL [18]. Mitochondrial preparations frombovine or porcine heart are electrophoretically separated on sodium dodecylsulfate-polyacrylamide gels and transferred onto nitrocellulose filters, whichthen are probed with sera from patients who have PBC and controls [19].The single serum sample can react with distinct mitochondrial antigens indifferent associations (see Fig. 2). PDC-E2 and E3BP, with which PDC-E2is cross-reactive, are recognized in the greater part of cases (93%), followedby OGDC-E2 (40%) and BCOADC-E2 (31%) [19]. A synopsis of the hierar-chy of the different AMA reactivities, their frequencies, and associations inthe authors’ PBC cases is in Table 1 [19].

The identification and cloning of the different mitochondrial proteinsrecognized by AMA-positive sera allowed the development of differentELISAs. The standardization of the procedure militates in favor of theELISAs, using purified [20] or recombinant mitochondrial proteins[21,22]. In the authors’ experience, however, comparing different commer-cial and in-house assays, the most efficient approach to detecting AMAwas classical IFL followed by W-IB with beef heart mitochondrial extractor ELISA with recombinant antigens. The W-IB and the ELISA not onlyare helpful confirmatory tests but also may identify AMA in the smallproportion of sera nonreactive with conventional IFL [18].

The authors’ experience comparing sensitivity, specificity, and positivepredictive value of the IFL, W-IB, and in-house and commercial ELISAsfor AMA detection is reported in Table 2.

Notwithstanding continuous and strenuous efforts to maximize the sensi-tivity of the assays to detect AMA in otherwise rigorously AMA-negativesera [22–24], it is commonly accepted that a small number (up to 5%–10%)of patients who have PBC may lack AMA, even after investigation with allthe currently available techniques. From a clinical standpoint, however, theabsence of AMA is almost irrelevant, because patients who have AMA-negative PBC have the same clinical, biochemical, and histologic featuresof classical (ie, AMA-positive) PBC [25,26], the same clinical outcomes,and the same biochemical response to ursodeoxycholic acid treatment [27].

Table 1

Hierarchy of antimitochondrial antibodies reactivities to different mitochondrial antigens in

147 patients who had primary biliary cirrhosis

Mitochondrial antigens Positive patients Prevalence

PDC-E2 þ E3BP 63/147 43%

PDC-E2 þ E3BP þ OGDC-E2 29/147 20%

PDC-E2 þ E3BP þ BCOADC-E2 þ OGDC-E2 17/147 12%

PDC-E2 þ E3BP þ BCOADC-E2 10/147 6%

BCOADC-E2 9/147 6%

None 19/147 13%

The different patterns of reactivities were detected using W-IB with beef heart mitochondria

(see Fig. 2).

Table 2

Comparison of different techniques for antimitochondrial antibodies detection in patients who

had primary biliary cirrhosis

Sensitivity

(127 cases)

Specificity

(166 þ 100 cases)

Positive

predictive value

IFL on rat tissue 71.6% 97.4% 0.938

IFL on HEp-2 cells 72.4% 93.3% 0.835

W-IB 85% 97.8% 0.947

In-house ELISA 81.1% 97.8% 0.944

Commercial ELISA 78.8% 97.8% 0.943

The sensitivity was evaluated in 127 patients who had PBC and the specificity in 166 patients

whohad type 1 autoimmune hepatitis and in 100 patientswhohadnonalcoholic fatty liver disease.

265AMA AND OTHER ANTIBODIES IN PBC

Clinical significance of antimitochondrial antibodies

The detection of AMA is virtually diagnostic of PBC, even in the absenceof symptoms and with normal alkaline phosphatase. When AMA is detectedin otherwise asymptomatic subjects without cholestasis, PBC already is pres-ent histologically in 40% of cases [28], and in the remaining patients it islikely to develop in succeeding years [29–32].

The detection of AMA is important information in the hands of clini-cians to confirm the diagnosis of PBC [33]; however, the type and strengthof AMA reactivity has no prognostic value. AMA titer may differ by morethan 200-fold among patients who have PBC, but in the single patient itremains stable over the years, and its measurements by IFL or by quantita-tive immunoassays with recombinant mitochondrial proteins are not usefulparameters for predicting disease progression [34], an observation in con-trast with a previous suggestion that quantitation of IgG AMA to purifiedmitochondrial enzymes correlates with advancing of liver disease [20].

The preliminary claims that AMA profiles determined at an early stagemay discriminate between a benign and a progressive course of PBC[35,36] were not validated on a larger series of patients [37]. The differenttype and number of mitochondrial antigens recognized by W-IB at presen-tation is independent of the stage of the liver disease and not associated withpeculiar clinical, biochemical, histologic, and immunologic features or withthe Mayo risk score [19]. The suggestion that IgA class antibodies to2-OADC might be predictive markers of histopathologic progression [38]is not confirmed [39]; however, the serum level of antibodies againstpyruvate dehydrogenase of the IgA class are lowered after treatment withursodeoxycholic acid [40]. Patients who have AMA of the IgG3 subclassmore frequently are cirrhotic, and a positive correlation is reported betweenAMA IgG3 titer and the Mayo risk score [41]. IFL AMA titers correlatewith the number and intensity of W-IB bands but not with reactivity toPDC-E2 [42]. Type and frequency of AMA reactivity are similar in menand women who have PBC [43], despite the fact that men often are diag-nosed later, when the disease has reached a more advanced stage [44].

266 MURATORI et al

Other autoantibodies in primary biliary cirrhosis

The notion that a considerable proportion of patients who have PBC alsohave antinuclear antibodies (ANA) detectable by IFL dates back to the1960s [45], but only 2 decades later ANA patterns were characterized furtheron HEp2 cells [46,47]. ANA are present in nearly half of patients who havePBC irrespective of their AMA status, and overall antinuclear reactivitiesare predominant (up to 85%) in AMA-negative PBC [48].

Using HEp2 cell lines as IFL substrate, the ANA patterns detected in pa-tients who have PBC are heterogeneous and often different patterns coexistin the same serum (Table 3). In patients who have PBC, the most relevantIFL patterns are anticentromere antibodies (ACA), anti–multiple nucleardots (anti-MND), and anti–nuclear envelope antibodies.

The ACA reactivity is directed against discrete granules in interphase cellsand is evident particularly in mitotic cells [49] (Fig. 3). The target antigen ofACA in patients who have PBC is centromere protein B (CENP-B) [50], an80-kd polyprotein that interacts with centromeric heterochromatin in humanchromosomes.

The antigenic targets of anti-MND are discrete nuclear dots, differentfrom nucleoli and from the ACA granules by being larger, fewer in number,and not seen in mitotic cells or on metaphase chromosome spreads (seeFig. 3). The molecular target of anti-MND is a 100-kd soluble protein calledsp100 [51,52] and, less often, a transformation and cell growth suppressing

Table 3

Antinuclear antibodies in 195 patients who had primary biliary cirrhosis

All patients

(195)

AMA-positive

patients (173)

AMA-negative

patients (22)

ANA patterns 97 (50%) 82 (47%) 15 (68%)

� Speckled 56 (30%) 47 (24%) 9 (41%)

� MND 41 (21%) 30 (17%) 11 (50%)

� Rim-like/membranous 35 (18%) 24 (14%) 11 (50%)

� Centromere 37 (18%) 34 (20%) 3 (14%)

� Homogeneous 6 (3%) 4 (2%) 2 (10%)

ANA specificities 108 (55%) 92 (53%) 16 (73%)

� Anti-sp100 65 (33%) 53 (31%) 12 (54%)

� Anti-gp210 42 (21%) 32 (18%) 10 (45%)

� Anti-LBR 11 (6%) 10 (6%) 1 (5%)

� Anti-CENP-B 41 (21%) 38 (22%) 3 (14%)

� SSA-Ro52 54 (28%) 46 (27%) 8 (36%)

� Anti-dsDNA 10/125 (8%) 10/118 (8.4%) 0/7

ANA patterns were evaluated on HEp2 cell lines by IFL. All ANA specificities (except anti-

dsDNA) were evaluated with ELISA or immunoblot assay with recombinant or purified

proteins. Anti-dsDNA antibodies were evaluated by IFL on Crithidia luciliae in 125 patients.

The sum of the different ANA patterns and specificities appears greater then the cumulative

number of positive patients because multiple reactivities were commonly observed in the same

serum.

Abbreviation: LBR, lamin B receptor.

Fig. 3. ANA patterns by IFL on HEp2 cells. ACA react with discrete granules in interphase

cells and typically in the mitotic cells (A). Anti-MND react with 5–20 nuclear dots distinct

from nucleoli and from the ACA targets. Punctate staining of chromosomes in mitosis clearly

distinguishes ACA from anti-MND (B). Anti-MND are isolated (B), whereas there is the

concomitant AMA cytoplasmic staining (C). A triple reactivity is showed (D): in addition to

cytoplasmic AMA and the nuclear anti-MND pattern, the rim-like/membranous reactivity is

evident as a thin ring around the nuclear envelope.

267AMA AND OTHER ANTIBODIES IN PBC

protein named PML, aberrantly expressed in promyelocytic leukemia cells[53,54].

The IFL pattern of the anti–nuclear envelope antibodies is called rim-like/membranous, giving the appearance of a thin ring confined to thenuclear membrane (see Fig. 3) [55]. The molecular targets of this rim-like/membranous IFL pattern are identified as structural components of thenuclear pore complex, such as gp210 [56] and nucleoporin p62 [57], andof the nuclear membrane, such as lamin B receptor [58,59].

With conventional IFL, the concomitant presence of AMA technicallymay hamper the detection of the rim-like/membranous pattern. To facilitatethe detection of ANA patterns in AMA-positive patients, the use of specificantisera to each of the four IgG isotypes as secondary antibody recently hasbeen suggested [60]. Another expedient is the pretreatment of cultured HEp2or HeLa cells with 1% formaldehyde: the fixative reduces antigenicity and,therefore, intensity of the cytoplasmic AMA signal, without altering the an-tigenic epitopes on the nuclear envelope, so that the concomitant rim-like/membranous ANA pattern is revealed more efficiently [61].

268 MURATORI et al

Diagnostic and prognostic value of antinuclear antibodies

in primary biliary cirrhosis

At variance with AMA, the fine dissection and classification of the ANAspecificities may offer not only diagnostic support but also prognostic infor-mation on the disease.

ANA reactivities, such as the anti-MND and the rim-like/membranouspatterns, which are relatively rare if not absent in normal and pathologiccontrols, are strongly associated to PBC [62–64], and now there is enoughevidence to consider these ANA IFL patterns as surrogate positive markersof the disease in AMA-negative patients [48,65,66]. Furthermore, severalANA specificities, in particular anti-sp100, anti-gp210, and antilamin Breceptor, seem predominant in patients who have PBC [16,48,54,64].

Sicca syndrome [67,68], systemic sclerosis [69], and CREST (calcinosis,Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangi-ectasia) syndrome [70] are connective tissue disorders significantly associ-ated with PBC; therefore, the corresponding ‘‘rheumatologic’’ ANAreactivities (anti-SSA/Ro, anti-Scl70, and ACA, respectively) are not unex-pected in these patients. In the setting of autoimmune liver disease, however,irrespective of concomitant rheumatologic manifestations, anti-SS-A/Ro-52kD and ACA/anti-CENP-B may attain diagnostic relevance asadditional AMA-independent specific serologic markers of PBC [71].

Some ANA specificities, such as anti-gp210 and possibly anti-p62, alsooffer prognostic indication, being significantly associated to aggressive dis-ease with poor prognosis [48,63,72,73]. In addition, in Japanese patientswho had PBC, not only anti-gp210 is a significant risk factor for diseaseprogression toward hepatic failure but also ACA positivity offers prognosticinformation, being significantly associated to the development of portalhypertension [74].

An overview of the IFL ANA patterns and ANA specificities detected inthe authors’ patients who had PBC is summarized in Table 3.

Other autoantibodies in primary biliary cirrhosis

Small ubiquitin-related modifiers (SUMOs) are linked covalently to sp100and PML, and autoantibodies against SUMOs were exclusively detected ina proportion of anti-MND–positive patients who had PBC [75]. Autoanti-bodies directed to other components of the nuclear envelope, such as nuclearlamins and translocated promoter region (Tpr), were observed in some pa-tients who had PBC and also in patients who had other systemic autoimmunedisorders [76]. Antibodies against SOX13, a transcriptional regulatory pro-tein with DNA-binding capacity, were detected in 18% of patients who hadPBC [77] and also are present in AIH, autoimmune cholangitis, and type 1diabetes mellitus. Anti–lamina-associated polypeptide 2 (anti-LAP2) were de-tected in 6% of patients who had PBC [73] but are not disease-specific [78].

269AMA AND OTHER ANTIBODIES IN PBC

Antimitochondrial antibodies and liver transplantation

In the first months after liver transplantation for end-stage PBC, AMA ti-ter declines slightly and then rapidly returns to pretransplantation values; theantibody subclass/isotype remains unchanged and AMA fluctuations offerno insight on the possible recurrence of the disease [79]. A similar behaviorafter liver transplantation is observed for PBC-specific ANA: anti-gp210and anti-sp100 antibodies levels remain stable irrespective of the clinicaloutcome and do not reflect recurrent disease activity in the graft [79–81].

Antimitochondrial antibodies, autoimmune hepatitis, and overlap syndrome

The erroneous interpretation of LKM1 as AMA in patients sufferingfrom AIH may delay the diagnosis and the correct immunosuppressivetreatment. Patients who have genuine AMA and definite diagnosis ofAIH exist, however, and AMA presence does not preclude a satisfactory re-sponse to corticosteroids [82].

In recent years, an increasing number of studies have reported patientspresenting, or developing over time, mixed clinical and laboratory featuresconsistent with the hepatitic (ie, AIH) and the cholestatic (ie, PBC orprimary sclerosing cholangitis) forms of autoimmune liver disease [83–87].

In the authors’ experience, a retrospective analysis of 125 patients who hadPBC identified six cases with IAIHG score [88] consistent with AIH,a ‘‘hepatitic’’ biochemical picture, and liver histology with aspects of interfacehepatitis, all features suggesting a diagnosis of ‘‘overlap AIH-PBC syn-drome.’’ The distinctive serologic feature of these six patients who had over-lap AIH-PBC, in comparison with the remaining 119 patients who had‘‘pure’’ PBC, was the positivity of high titers (R1:80) anti–double-strandedDNA (anti-dsDNA) antibodies by IFL on Crithidia luciliae (Fig. 4)(G. Pappas, personal communication). Anti-dsDNA were already observedin nearly one third of patients who had advanced stage AIH [89]. If confirmedin larger series, high-titer anti-dsDNA antibodies could acquire the role ofserologic marker of the overlap AIH-PBC syndrome.

Antimitochondrial antibodies and chronic graft-versus-host disease

Chronic graft-versus-host disease (GVHD) and PBC share many clinicaland laboratory features, the most intriguing of all being portal lymphocyticinfiltration and destruction of small bile ducts. Several studies reported theoccurrence of AMA in a significant proportion of patients who had chronicGVHD, suggesting a potential common pathogenic background [90]. Fur-ther investigations, however, using not only IFL but also immunoblottingwith beef heart mitochondria and ELISA with recombinant polypeptidesof human PDC-E2, bovine BCOADC-E2, and rat OGDC-E2 failed to de-tect anti–PDC-E2 in bone marrow transplant recipients who had chronic

Fig. 4. Anti-dsDNA antibodies are directed against the kinetoplast (arrows) of Crithidia

luciliae.

270 MURATORI et al

GVHD [91], whereas a variety of other non–organ-specific autoantibodieswere observed, in particular, ANA, the presence of which is significantlyassociated with the development of chronic GVHD [92].

One possible exception is a subgroup of patients who had multiple mye-loma who developed high-titer AMA specifically targeting PDC-E2 afterallogeneic hematopoietic stem cell transplantation and donor lymphocyteinfusions. The epitope specificity of these anti–PDC-E2 antibodies wasnot located, however, within the lipoyl domain of PDC-E2, which is thetypical antigenic target of PBC sera [93].

Antimitochondrial antibodies and fulminant hepatic failure

AMA reactivity can be detected transiently in a proportion of patientssuffering from acute liver failure, possibly as a consequence of the oxidativestress-induced liver damage: 28 (40.6%) of 69 male and female patients whohad fulminant hepatitis resulting from different causes (paracetamol poison-ing, drug-induced liver injury, and hepatitis A, hepatitis B) developed AMA;the AMA specificity using immunoblot with recombinant antigens was thesame as that observed in patients who had PBC, albeit at lower titer;AMA reactivity remained rather constant during the first week but disap-peared rapidly within 1 year [94]. A concomitant study, however, identifiedonly one subject (1.3%) as AMA positive by IFL among 72 patients present-ing with acute liver failure [95]. The expected lower sensitivity of the IFLtechnique might explain such a discrepancy.

Concluding remarks

AMA remain the serologic cornerstone in the diagnosis of PBC, even ifthey are not detectable in a proportion of patients, notwithstanding the

271AMA AND OTHER ANTIBODIES IN PBC

most sensitive and sophisticated technologies used. To fill in the serologic gapin AMA-negative PBC, there now is sound evidence to consider ANA pat-terns, such as anti-MND and anti-rim-like/membranous, as PBC-specificsurrogate hallmarks of the disease, and their detection can be consideredvirtually diagnostic. Furthermore, particular ANA specificities, such asanti-gp210, anti-p62, ACA, and anti-dsDNA, may provide additional diag-nostic and prognostic information.

Summary

AMA are the most sensitive and specific immunologic markers of PBC;the E2 component of the pyruvate dehydrogenase complex and other mem-bers of the 2-OADC are their molecular targets. Even if AMA titers or pat-terns have no prognostic significance, their presence is diagnostic of PBCand predictive of disease occurrence even in asymptomatic patients whodo not have cholestasis. A small proportion of patients believed to havePBC (up to 5%–10%) test AMA negative, even with the most sophisticatedtechniques available; however, AMA-positive and AMA-negative PBCshare the same clinical and prognostic features.

ANA are found in nearly 50% of patients who have PBC, irrespective oftheir AMA status, the most informative immunofluorescence patterns beinganti-MND, anti–rim-like/membranous, and anticentromere. The most im-portant ANA specificities are nuclear pore complex proteins (gp210 andnucleoporin p62), a nuclear membrane protein (lamin B receptor), nuclearbodies (sp100 and PML), CENP-B, and double-stranded DNA.

Immunofluorescence ANA patterns, such as anti-MND and anti–rim-like/membranous, are extremely PBC-specific and can be considered posi-tive hallmarks of the disease, with the same diagnostic value of AMA.

Several ANA specificities have additional diagnostic or prognostic value:anti-gp210 is associated with rapidly evolving liver disease, anticentromereenhances the risk for developing portal hypertension, and anti-dsDNA oftenis found in patients who have AIH-PBC overlap syndrome.

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