ORIGINAL ARTICLES

Contribution to Antimitochondrial Antibody Production:Cleavage of Pyruvate Dehydrogenase Complex-E2 by

Apoptosis-Related ProteasesShuji Matsumura,1,2 Judy Van De Water,1 Hiroto Kita,1 Ross L. Coppel,3 Takao Tsuji,2 Kazuhide Yamamoto,2

Aftab A. Ansari,4 and M. Eric Gershwin1

Patients with PBC produce a directed, specific response to a single immunodominant auto-epitope of PDC-E2 within the inner lipoyl domain. In contrast, immunized animals react tomultiple epitopes and rarely recognize the inner lipoyl domain. In other autoimmune dis-eases, apoptosis plays a critical role in antigen presentation; the caspases and granzyme B arethe key proteases in the generation of autoepitopes. To determine the specific cleavagepattern of full-length recombinant PDC-E2, we performed in vitro digestion withcaspases-3, -6, -8 and granzyme B. The resulting fragments were immunoblotted and probedwith an extensive panel of monoclonal anti-PDC-E2 antibodies and sera from patients withPBC. Interestingly, on granzyme B digestion, PDC-E2 lost reactivity, suggesting the de-struction of the immunodominant epitope. Because this site contains the major epitope forboth B cells and T cells, it suggests that granzyme B is unlikely to be involved in generationof autoepitopes in primary biliar cirrhosis (PBC). In contrast, following treatment with thecaspase enzymes, immunoreactive fragments were generated. Indeed, by confocal micros-copy, activated caspase-3 is found in the marginal hepatocytes and bile ducts. Moreover,caspase-3 staining was strongest in the small intrahepatic bile ducts, the major site of tissuedestruction in PBC. In conclusion, these data suggest that following apoptosis, the caspasefamily of proteolytic enzymes have the potential to generate immunogenic fragments thatcontribute to the autoantigen reservoir and the production of antimitochondrial antibodies.These findings are also consistent with the generation of an autoimmune response against anintracellular antigen that evades catabolism during apoptosis. (HEPATOLOGY 2002;35:14-22.)

An autoimmune mechanism has been implicated in thepathogenesis of primary biliary cirrhosis (PBC), includingthe presence of a very specific antimitochondrial antibody

(AMA) response to a conserved region of the E2 components of the2-oxo dehydrogenase enzymes, particularly PDC-E2.1-5 AMAs arean early serological marker of PBC and can be detected in theserum of patients before the appearance of elevated serum alkaline

phosphatase and gamma-gluatmyltranspeptidase levels, and longbefore the onset of symptoms.3-5 The immunodominant antigenrecognized by AMA is the E2 component of the pyruvate dehydro-genase complex (PDC-E2). PDC-E2 is composed of an outer andinner lipoyl domain, a binding domain, an inner core domain, andalanine- and proline-rich hinge regions.6 Amongst these domains,the immunodominant epitopes that have reactivity against AMAsare derived primarily from the inner lipoyl domain.7 The relation-ship of the mitochondrial autoantigens to disease and the ontogenyof the autoantibody response remain elusive.

Work in other autoimmune diseases has suggested that apopto-sis contributes to the generation of autoantigenic fragments.8 Bil-iary epithelial cells (BEC) in PBC have been shown to undergoapoptosis. The process of apoptosis is triggered by multiple stimuli,including induction via the TNF receptor family or the perforingranzyme system by cytotoxic T and/or NK cells. For TNF recep-tor induction, binding precedes the initiation of the caspase cas-cades resulting in activated caspases that cleave numerous essentialintracellular proteins. An alternative apoptic pathway involves per-forin release from cytotoxic cells, which generates pores on target-cell membranes, followed by the inflow of granzyme into the cells.Granzyme then induces cells to undergo apoptosis not only by

Abbreviations: PBC, primary biliary cirrhosis; AMA, antimitochondrial antibody;PDC-E2, pyruvate dehydrogenase complex-E2; BEC, biliary epithelial cells; TNF, tu-mor necrosis factor; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.

From the 1Division of Rheumatology, Allergy and Clinical Immunology, University ofCalifornia at Davis, Davis, CA; 2Okayama University Medical School, Okayama,Japan; 3Department of Microbiology, Monash University, Melbourne, Australia; 4De-partment of Pathology, Emory University, Atlanta, GA.

Received July 3, 2001; accepted September 2, 2001.Supported by a grant from the National Institutes of Health, DK39588.Address reprint requests to: Judy Van de Water, Ph.D., Division of Rheumatology,

Allergy and Clinical Immunology, University of California at Davis School of Medicine,TB 192, One Shields Avenue, Davis, CA 95616. E-mail: javandewater@ucdavis.edu;fax: 530-752-4669.

Copyright © 2002 by the American Association for the Study of Liver Diseases.0270-9139/02/3501-0005$35.00/0doi:10.1053/jhep.2002.30280

14

direct digestion of intracellular proteins, but also by activating thecaspases. Apoptosis-related proteolytic enzymes, such as thecaspases and granzyme B, cleave these autoantigens during apopto-sis.9 We have investigated the effects of proteolytic cleavage on theimmunogenicity of the immunodominant antigen, PDC-E2, fol-lowing in vitro digestion with caspases-3, -6, and -8, and granzymeB. This report provides evidence that, following cleavage bycaspase, but not granzyme, PDC-E2 immunoreactive fragmentsare generated. We suggest that apoptosis of bile duct cells canproduce autoantigenic fragments capable of eliciting or perpetuat-ing an autoimmune response in a genetically susceptible host.

Materials and Methods

Specimens. Sera were obtained from a total of 15 people, in-cluding 10 female patients with PBC, 48.7 6 7.74 years old, aswell as 5 normal volunteers. The PBC patients were all Stage 3 or4 and were known to have AMA in their sera. In addition, forstudies involving liver immunohistochemistry, tissue was obtainedfrom 13 patients with PBC, 7 patients with PSC, and 7 normalcontrols. Multiple serial sections were obtained from these speci-mens and the data interpreted by an unbiased observer. The diag-nosis was based on established disease criteria and confirmed byhistologic review by an independent observer.

Monoclonal Antibodies Against PDC-E2. To help define thereactive fragments following caspase or granzyme digestion, weused an extensive panel of PDC-E2 specific or control monoclonalantibodies (mAb) derived from both human and mouse combina-torial libraries or hybridomas. This panel of mAbs was generated inour laboratory, and the reactive epitopes have been extensivelymapped.10,11 These mAbs have the advantage of recognizingPDC-E2 epitopes at multiple regions of the enzyme and, therefore,are useful in defining digestion products. The specificity and iso-type of these monoclonal antibodies are noted in Table 1. Amongthem, C355.1, 2H4, 4C8, and the human monoclonal antibodyLC2, derived from a patient with PBC, specifically stain the BECcells of patients with PBC and not controls in an apical pat-tern.11,12

Recombinant PDC-E2. The human PDC-E2 gene wascloned into the TOPO TA Cloning Vector (Invitrogen Inc., Carls-bad, CA). We then added the His-tag at the C-terminus of PDC-E2, specific primers by designing the following primers: senseprimer 59-CTCGGATCCCTCGAGCCCATATGAGTCT-

TCCCCCGCATCAG-39; anti-sense 59TAGGATCCTTAGT-GATGGTGATGGTGATGTGAACCACCTCC-CAACAA-CATAGTGAT-39. PCR was performed for 25 cycles of 1 minuteat 94°C, 1.5 minutes at 55°C, and 2 minutes at 72°C. The PCRproduct was recloned into the TA Cloning vector. The fragmentwas then cut from the vector using restriction enzymes, Nde I andBamH I (Gibco BRL Inc., Grand Island, NY) by incubation at37°C for 1 hour and ligated into pET 5a Vector (Promega Inc.,Madison, WI). Escherichia coli strain, BL21Star (DE3) (InvitrogenInc., Carlsbad, CA), was transformed using this construct. There-after the E. coli was harvested after 3.5 hours incubation with 0.5mmol/L ITPG and 0.3 mmol/L thioctic acid to attach lipoic acidto the lipoyl domains of PDC-E2. The attachment was verifiedusing a rabbit antilipoic acid antibody. Finally, the recombinantprotein was purified using a Ni-NTA resin (Qiagen Inc., Valencia,CA) under 6 mol/L guanidine denaturing conditions and dialyzedagainst 25 mmol/L HEPES buffer, pH 7.5 adjusted with KOH.

In Vitro Cleavage of Recombinant PDC-E2. RecombinantPDC-E2 (0.3 mg/mL) was subjected to granzyme B and caspase-3,-6, and -8 digestion. Human purified granzyme B was purchasedfrom A.G. Scientific, Inc. (San Diego, CA); caspases-3, -6, and -8were purchased from Becton Dickinson (San Diego, CA) in apreactivated form. Granzyme B was added at a concentration of 6mg/mL to the PDC-E2 solution and incubated at 37°C for 1 hour.Caspases -3, -6, and -8 were added to individual preparations ofPDC-E2 at pH 7.5, in 25 mmol/L HEPES adjusted with KOH,10 mmol/L dithiothreitol (DTT) and 2% CHAPS at a final con-centration of 2.0, 5.0, and 20 mg/mL, respectively, and incubatedat 37°C for 1 hour. The cleavage reactions were stopped throughdenaturation by adding loading buffer and boiling for 5 minutes.The preps were then separated by polyacrylamide gel electrophore-sis (PAGE). As a positive control, PDC-E2 was incubated underthe same experimental conditions as described earlier without theproteases. As a negative control, an irrelevant control antigen (glu-tathione-s-transferase) was digested as described above.

Detection of PDC-E2 Cleavage Fragments. The gels weresilver stained, and the differences between granzyme B orcaspase-3, -6, and -8 treated and untreated samples were com-pared. For verification of the digestion products, the resolved pro-teins were transferred onto a PVDF membrane, and amino acidsequence analysis was determined by Edman degradation sequenc-ing. In addition, the digested proteins were transferred to nitrocel-lulose following SDS-PAGE and blotted using PBC sera or ourmAb panel (Table 2). Staining by mouse anti-His tag antibody wasalso used to track the digested proteins using the His-tag on the Cterminus.

Immunohistochemistry. After collection, liver tissue was fixedin 8% neutral buffered formalin and paraffin imbedded. Six-mi-cron sections were cut and mounted to prevent tissue detachment.The sections were deparafinized by heating overnight at 60°C fol-lowed by four 5-minute changes of xylenes, two 5-minute changesof 100% ethanol, two 5-minute changes of 95% ethanol, and one5-minute change of 70% ethanol, followed by one 5-minutechange in TBS. Sections were blocked with 1.5% goat serum foranticaspase-3 and 1.5% horse serum for antigranzyme B, respec-tively. The individual sections were incubated with rabbit anti-caspase -3, which detects the p17 subunit of caspase-3 with little or

Table 1. Specificity and Isotype of the PDC-E2-SpecificMonoclonal Antibodies

Monoclonal Antibodies Isotype Epitope*

LC2 human IgG ILDC355.1 mouse IgG2b 160-221 (ILD)C150 mouse IgG1 OLD2H4 mouse IgG1k DKA sequence in outer and

inner lipoyl domains4C8 mouse IgG2bk ILD1F1 mouse IgMk ILD

Abbreviations: ILD, inner lipoyl domain; OLD, outer lipoyl domain.*Epitopes of those antibodies were previously determined using recombinant

PDC-E2 fragments (14).

HEPATOLOGY, Vol. 35, No. 1, 2002 MATSUMURA ET AL. 15

no detection of the precursor form, diluted 1: 300 (R & D Systems,Inc.). After incubation, the slides were washed in 2 changes of TBSfollowed by a 30-minute incubation with biotinylated goat anti-rabbit antibody for caspase-3 diluted 1:200. Following incubation,the slides were washed in 2 changes of TBS followed by a 30-minute incubation with avidin conjugated with biotinylated alka-line phosphatase. After incubation, the slides were washed in 2changes of TBS followed by incubation with the Vector Red sub-strate working solution (Vector Laboratory, Inc., Burlingame, CA)until optimal staining was obtained.

Results

Granzyme B Digestion of Recombinant PDC-E2 (rPDC-E2). Material purified from E. Coli-expressing human PDC-E2contains a number of products (Fig. 1, Track 1). The 2 upperbands represent full-length and near-full-length PDC-E2. Thelower of these 2 bands arise as the sequence of human PDC-E2contains a sequence similar to a Shine-Dalgarno region upstreamof 59 methionine. This results in internal initiation and a 64-kdpartial-length recombinant protein (Table 2). The recombinantprotein preparation also contains naturally degraded proteins thatretain reactivity with antimitochondrial monoclonal antibodiesand sera from patients with PBC. Following digestion with gran-zyme B, rPDC- E2 was nearly completely digested into at least 5bands visible at 66, 47.5, 44, 31.4, and 31 kd as detected by silverstaining of the SDS-PAGE gel (Fig. 1). The 66-kd fragment isgenerated by the cleavage of the VETD motif contained in theouter lipoyl domain. This results in preservation of the inner lipoyldomain region and consequent recognition by sera from patientswith PBC and all of the monoclonal antibodies with the exceptionof C150, whose epitope contains the VETD sequence found in theouter lipoyl domain (Table 2) (Fig. 2). The remaining fragmentswere positive only when stained with the His-tag antibody, con-firming that the inner lipoyl domain IETD sequence is abolishedby granzyme B digestion. The identity of these digestion productswas determined based on Edman degradation sequencing. Thisanalysis revealed that the 66 and 47.5 kd bands began at the Lys 46

and Lys 173 in the outer and inner lipoyl domains of PDC-E2,respectively. This shows that PDC-E2 was cleaved after the aspar-tic acid in both the inner and outer lipoyl domains (Fig. 1). The31.4 kd band began at amino acid 296 (glycine).

Caspase-3, -6, and -8 Digestion of Recombinant PDC-E2.Caspase-3 and -8 were less effective in the complete digestion ofrecombinant PDC-E2 compared with granzyme B. However, un-like granzyme B, caspase-3 produced several fragments that wererecognized by PDC-E2-specific mAbs and sera from patients withPBC by immunoblotting (Fig. 3A, Table 3). For example, serafrom patients with PBC recognized up to 12 digestion fragments.Reactivity with C150 revealed a similar complex pattern differingonly in an extra fragment at 31 kd, which also presumably containsthe outer lipoyl domain. Interestingly, the 2 murine mAbs, C355.1

Fig. 1. Silverstained SDS-PAGE gel containing recombinant PDC-E2 di-gested with granzyme B, and caspases -3, -6, and -8. The first lane containsPDC-E2 that was subjected to similar conditions as the granzyme B andcaspase treated samples except for the addition of the proteases. Proteinsequencing revealed that the 66-kd bands (open arrowheads) and the47.5-kd bands (closed arrowheads) correspond to fragments generated bycleavage at the outer lipoyl domain and the inner lipoyl domain, respectively.Note that digestion with caspase-3, while not complete, produced numerousdetectable fragments.

Table 2. Enzyme Cleavage Pattern of PDC-E2 by Granzyme B and Fragment Mapping With PDC-E2-Specific mAbs, anti-His mAb, andSera From Patients With PBC

PDC-E2

Possible Cleavage Site LC2 C355 C150 2H4 4C8 1F1 PBC Sera Hiskd

74 Full length 111 111 111 111 111 111 111 11164 59 M internal start 1 11 2 11 11 11 11 1159 Unknown 2 1 2 1 1 1 1 252 Unknown 2 2 2 11 2 2 2 1

PDC-E2 1

Granzyme B

Possible Cleavage Site LC2 C355 C150 2H4 4C8 1F1 PBC Sera Hiskd

74 Full length 1 1 11 1 1 1 1 266 VETD 1 1 2 1 1 11 1 247.5 IETD 2 2 2 2 2 2 2 1144 Unknown 2 2 2 2 2 2 2 131.4 TGPD 2 2 2 2 2 2 2 131 Unknown 2 2 2 2 2 2 2 1/2

16 MATSUMURA ET AL. HEPATOLOGY, January 2002

and 4C8, that produce an apical staining pattern also had verysimilar caspase-3 digestion profiles with the exception of 2 bands at50 and 47.5 kd. The mAb 1F1, a nonapical PDC-E2 specificantibody, recognized only 3 digestion fragments with a recognitionpattern quite similar to LC2, the human mAb to PDC-E2. ThemAb 2H4, another murine mAb that produces an apical stainingpattern on PBC bile ducts, has an epitope that requires only theamino acids DK and lipoic acid and, therefore, recognizes both theinner and outer lipoyl domains.13 Staining of the caspase-3 digestswith 2H4 produced 15 bands, making this a useful antibody fortracking the inner and outer lipoyl domain containing fragments.These results show that caspase-3 digested PDC-E2 contains nu-merous potentially immunogenic fragments, several of which arerecognized by sera from patients with PBC. Caspase-6 cleavedPDC-E2 more efficiently than caspases-3 and -8 and produced amajor 66-kd band whose molecular weight corresponded to a frag-ment digested at the outer lipoyl domain leaving the inner lipoyldomain intact (Fig. 3B, Table 3). This band was reactive with PBCsera and all of the mAbs except for C150, suggesting that the outerlipoyl domain was cleaved preferentially by this enzyme. Of inter-est, the apically staining mAbs C355.1, 4C8, and 2H4 each recog-nized additional bands. These same bands were faintly stained by

PBC serum. Caspase-8 gave results similar to caspase-6 with theexception of an additional fragment at 47.5 recognized only byPBC sera and mAb 2H4 (Fig. 3C, Table 3). The outer lipoyldomain mAb C150 also recognized a band at 31 kd that was pickedup by 2H4 and the anti-His-Tag antibody as well. This band wasalso found in the caspase-3 digestion products but not withcaspase-6. It is difficult to ascertain identity of this band becausethe recognition pattern suggests that it should contain both theouter lipoyl domain, as recognized by C150, as well as the C-terminal His-Tag. Such a fragment would not be expected to run atthe observed molecular weight. The capacity of caspase-3 to pro-

Fig. 3. Immunoblotting of PDC-E2 fragments digested by caspases-3, -6,and -8 using monoclonal antibodies against PDC-E2 and serum frompatients with PBC. (A) Digestion of full-length PDC-E2 with caspase-3. Notethe numerous digestion products that remain reactive to all of the antibod-ies. Each of the antibodies shows a unique recognition pattern with theexception of LC2 and 1F1. Of interest is the similarity in reactivity betweenthe apically staining mAbs C355.1 and 4C8. (B) While the cleavage ofPDC-E2 was more efficient with caspase-6 than with caspase-3, there weremany fewer bands reactive with the mAbs and PBC serum. Only C355.1, 2H4and 4C8, the apically staining mAbs, recognized additional cleavage prod-ucts. (C) Digestion with caspase-8 produced even fewer reactive cleavageproducts. However, PBC serum recognized 3 additional bands that were alsopositive for 2H4. A band at 31 kd was recognized by C150 and 2H4 as wellas the His-Tag antibody.

Fig. 2. Immunoblotting of PDC-E2 fragments digested by granzyme Busing monoclonal antibodies against PDC-E2 and serum from patients withPBC. (A) Represents full-length PDC-E2 stained by immunoblot with themurine PDC-E2 specific mAbs, the human mAb LC2, PBC serum, and theanti-His-Tag mAb. In addition to the major band at 74 kd, there is a lowerband that is the result of a Shine-Delgarno internal start codon. In addition,some smaller breakdown products are recognized by 2H4, which requiresonly DK and lipoic acid for recognition. In (B) the granzyme B treated sampleshows that the major portion of the full length PDC-E2 was digested leavingone faint band of undigested PDC-E2 (upper arrow) and another fromdigestion of the outer lipoyl domain cut fragment (lower arrow). In the lanestained with the anti-His-Tag antibody, digestion of the inner and outer lipoyldomain, and the TGPD site produced 3 major cleavage products.

HEPATOLOGY, Vol. 35, No. 1, 2002 MATSUMURA ET AL. 17

duce such a large number of immunoreactive fragments led us totest for its presence in PBC liver.

Expression of Activated Caspase-3 in Liver. Liver sectionsfrom patients with PBC, PSC, and healthy controls werestained to determine the location and relative amount of acti-vated caspase-3. In liver sections from 13/13 patients with PBC,small intrahepatic bile ducts exhibited strong, granular stainingwith both positive and negative cells in the same duct (Fig. 4A).This staining was especially apparent in ducts exhibiting thegreatest damage. A similar pattern was also noted in the mar-ginal hepatocytes (Fig. 4B) with intermittent staining of thecentrolobular hepatocytes of these patients. Interestingly, thelarge interhepatic and septal bile ducts in the PBC sectionscontained little if any activated caspase-3 (Fig. 4C). In contrast,

in the healthy controls, the bile ducts and the marginal andparenchymal hepatocytes were all negative for activatedcaspase-3 (Fig. 5A and B). An occasional positive cell in thecentrilobular area was noted. In the PSC liver sections, the bileducts were completely negative, whereas the marginal hepato-cytes showed strong positive caspase-3 staining similar to thatfound in PBC patients (Fig. 5C).

DiscussionLocal macrophages and dendritic cells internalize apoptotic

cells and present antigens derived from the processing of these cellsto class I–restricted T cells.14,15 BECs experience the normal pro-cesses of cell turnover and as such will undergo apoptosis as part of

Table 3. Enzyme Cleavage Pattern of PDC-E2 by Caspases-3, -6, and -8 and Fragment Mapping With PDC-E2-Specific mAbs,anti-His mAb, and Sera From Patients With PBC

PDC 1 Caspase-3

Possible Cleavage Site LC2 C355 C150 2H4 4C8 1F1 PBC Sera Hiskd

74 Full length 111 111 111 111 111 111 111 11166 VETD 11 11 2 11 11 11 11 1164 59 M internal start 11 11 2 11 11 11 11 1159 Unknown 1 1 1 1 1 1 1 257 Unknown 1 1 6 1 1 1 1 156 HVVD 2 6 1 1 6 2 1 254 SWMD 2 1 1 1 1 2 1 251 SEGD 2 2 2 2 2 2 2 150 SVND 2 6 1 1 2 2 1 247.5 LSID 2 1 1 1 2 2 1 145 Unknown 2 2 6 6 2 2 6 644 VFTD 2 2 1 1 2 2 1 241.2 KDID 2 1 11 11 1 2 11 240 EVTD 2 2 2 2 2 2 2 138 KGID 2 2 1 1 2 2 1 236 EVTD 2 1 11 11 1 2 11 233.5 KGID 2 2 2 2 2 2 2 133 Unknown 2 2 2 1 2 2 2 232.5 KDID 2 2 2 2 2 2 2 131.5 Unknown 2 2 2 1 2 2 2 231.2 VFTD 2 2 2 2 2 2 2 1131 Unknown 2 2 1 1 2 2 2 11

PDC 1 Caspase-6

Possible Cleavage Site LC2 C355 C150 2H4 4C8 1F1 PBC Sera Hiskd

74 Full length 1 11 11 11 11 11 1 166 VETD 11 111 2 111 111 111 11 1164 59 M internal start 1 11 2 11 11 11 1 159 Unknown 2 1 2 1 6 2 6 254 SWMD 2 1 2 1 6 2 6 250 SVND 2 2 2 1 2 2 2 247.5 IETD 2 2 2 2 2 2 2 11

PDC 1 Caspase-8

Possible Cleavage Site LC2 C355 C150 2H4 4C8 1F1 PBC Sera Hiskd

74 Full length 1 11 11 11 111 111 111 1166 VETD 1 11 2 11 111 111 111 1164 59 M internal start 1 11 2 11 11 11 11 156 HVVD 2 1 2 1 1 1 1 254 SWMD 2 6 2 1 2 2 1 250 SVND 2 2 2 1 2 2 2 247.5 LSID 2 2 2 1 2 2 1 1131 Unknown 2 2 1 1 2 2 2 1

18 MATSUMURA ET AL. HEPATOLOGY, January 2002

the ongoing growth and remodeling of small bile ducts. Severalproteases may be involved in this turnover and degradation, par-ticularly the caspases and granzyme B. The roles of these caspasesare to disable critical homeostatic and repair processes, as well as tocleave key structural components resulting in the systematic disas-sembly of the apoptotic cell.16 Caspases are present as inactiveproenzymes that are activated by proteolytic cleavage and caspases8, 9, and 3 are situated at pivotal junctions in the apoptosis path-ways. Caspase 8 initiates disassembly in response to extracellularapoptosis-inducing ligands and is activated in a complex associatedwith the cytoplasmic death domain of many cell-surface receptorsfor the ligands.17 Caspase 9 activates disassembly in response toagents or insults that trigger the release of cytochrome c from

mitochondria18,19 and is activated when complexed with apoptoticprotease activating factor 1 (APAF-1) and extramitochondrial cy-tochrome c.20 Caspase-3 appears to amplify caspase-8 andcaspase-9 initiation signals into full-fledged commitment to disas-sembly.16,21 Caspase-8 and caspase-9 activate caspase-3 by proteo-lytic cleavage and caspase-3 then cleaves vital cellular proteins orother caspases.16,21 Granzyme B is a serine protease found in thecytoplasmic granules of CTLs and NK cells that plays a vital role inthe induction of apoptosis in target cells. One of its functions isachieved by the catalysis of caspase cleavage and activation.22-25 Inprevious reports concerning autoimmune disease, it was suggestedthat granzyme B is an important component in the production ofautoantigen. This observation stems from evidence that granzyme

Fig. 4. Liver sections from patients with PBC were stained to determinethe location and relative amount of activated caspase-3. (A) Note the stronggranular staining of the intraheptic duct in the liver from a patient with PBC(arrow). (B) A similar pattern was also noted in the marginal hepatocytes ofthese patients (arrow-H). Of great interest is the lack of staining in the largeinterhepatic bile duct in the PBC (arrow) (C). All sections are 3600, zoomfactor 1.0.

HEPATOLOGY, Vol. 35, No. 1, 2002 MATSUMURA ET AL. 19

B can cleave substrate proteins into novel fragments differentlyfrom the caspase enzymes, and that those granzyme B–generatedfragments are specifically recognized by autoantibodies from pa-tients with SLE and autoimmune myositis.9 Because apoptosis is ageneral phenomenon that participates in maintaining homeostasisin both normal and disease states, it has been speculated that au-toantigens that are cleaved by the caspases contribute to the induc-tion of tolerance in autoreactive T cells rather than to the produc-tion of pathogenic immunogenic fragments.8,9 However, thatstudy did not examine PDC-E2. We find these data of interest,particularly because the protease would not be active during nor-mal apoptosis and may be invoked by a pathogenic process such asPBC. Granzyme B has been found in the periportal region in the

liver of patients with PBC.26 Therefore, we examined the effects ofgranzyme B, caspase -3, -6, and -8 on the digestion of PDC-E2 anddetermined the reactivity of the resulting fragments.

Using a positional scanning synthetic combinatorial peptidelibrary, Thornberry et al. determined the caspase and granzyme Bsensitive amino acid sequences.27,28 They revealed that caspases-6and -8 and granzyme B are potentially capable of cutting after thelysine residue of a similar amino acid motif, such as the inner(IETD) and outer (VETD) lipoyl domain of PDC-E2. In thisreport, we identified that PDC-E2 was almost completely cleavedby granzyme B into fragments that had no immunoreactivity witheither sera from patients with PBC or PDC-E2–specific monoclo-nal antibodies. This loss of antigenicity by cleavage of the major

Fig. 5. Liver sections from patients with PSC and healthy controls werestained to determine the location and relative amount of activatedcaspase-3. (A) A normal bile duct shows no staining for caspase-3 (arrow).Likewise, the marginal and parenchymal hepatocytes are also negative(arrow-H) (B). In the PSC liver (C), the bile ducts are completely negative(arrow-B) whereas the marginal hepatocytes show strong positive caspase-3staining similar to PBC (arrow-H). All sections are 3600, zoom factor 1.0.

20 MATSUMURA ET AL. HEPATOLOGY, January 2002

autoepitopes may also mean a loss of immunogenicity, and sug-gests that granzyme B is not involved in the perpetuation of theautoimmune response. On the other hand, the caspase enzymeswere less efficient in digesting PDC-E2, and fragments retainingAMA reactivity were still present. Caspase-3 cleaved PDC-E2 atseveral aspartic acid residues and produced numerous fragmentswith immunoreactivity against sera from patients with PBC andmonoclonal antibodies against PDC-E2. These findings suggestthat, even though the caspases and granzyme B prefer similaramino acid sequence motifs for cleavage, their efficiency differs asfar as PDC-E2 is concerned, and thus they may play different rolesin processing PDC-E2 during apoptosis. Although these studieswere conducted on recombinant lipoylated PDC-E2, there is noevidence for a difference between the recombinant and nativePDC-E2 with respect to antigenicity and proteolytic digestion.Further, in our hands, recombinant and native PDC-E2 are iden-tical with respect to CD4, CD8, and B-cell recognition (unpub-lished data, 2001). Perhaps the mitochondrial location of PDC-E2explains the differences between this autoantigen and those au-toantigens specific for systemic autoimmune diseases. It is wellknown that during apoptosis, mitochondrial permeability is in-creased, which leads to matrix expansion and the eventual ruptureof the outer membrane. However, little is known about how PDC-E2, which exists on the inner mitochondrial membrane, may beexposed to these enzymes, how it is processed, or its intracellularlocation during apoptosis.29

It has been shown by morphological studies at the light micro-scopic and ultrastructural level that apoptosis of biliary epithelialcells occurs in the liver of patients with PBC.26 It is also apparentthat mononuclear cell infiltrates surround the injured bile ducts.Using immunohistochemical analysis, the epithelial cells of theinjured bile ducts of PBC patients strongly expressed Fas on the cellmembrane, and were in close proximity to FasL-expressing mono-nuclear cells. In contrast, granzyme B and perforin staining wasweaker, suggesting that these 2 proteins play a minor role in thebile duct destruction of PBC compared with Fas-FasL system. Ofgreat interest is the fact that the caspases can be activated throughcytochrome P450–generated reactive metabolites. Recently, Ha-ouzi et al. showed that the reactive metabolites generated duringthe processing of skullcap diterpenoids by CYP3A in rat hepato-cytes activate Ca21-dependent transglutaminase and open the mi-tochondrial permeability transition pore.30 This lead to the rup-ture of the outer mitochondrial membrane, the release ofcytochrome c, followed by caspase induction.30 The role of theliver in the processing of xenobiotics through cytochrome P450provides a sound argument in favor of a nongranzyme B pathwayfor caspase activation and supports our current findings that sug-gest the importance of caspase digestion of PDC-E2 during apo-ptosis in the pathogenesis of PBC. In other words, the effectorcaspases are responsible not only for the destruction of BECs, butalso the production of autoantigenic fragments of PDC-E2. This isfurther substantiated by our findings that activated caspase-3 isfound only in the small bile ducts and ductules and not the largeducts in PBC liver. Whether or not such fragments are responsiblefor the generation of autoreactivity, or merely provide a continu-ous source of autoantigen for the ongoing stimulation of high titerAMA is still unknown. Finally, it should be noted that the gener-

ation of novel autoantigenic fragments, or the inhibition of epitopedigestion need not occur in the target cells to effect a perpetuationof the immune response.

The digestion of PDC-E2 by the proteolytic enzymes associatedwith apoptosis would also have a potential effect on the T-cellresponse in PBC. In a report by Shimoda et al.,31 a significantpopulation of CD41 T cells, which recognize the 14 amino acidinner lipoyl domain sequence, GDLLAEIETDKATI, exist in theperipheral blood of HLA DRB40101-positive patients withPBC.31 The study also indicated that the sequence ExDK is essen-tial for T-cell recognition. This motif corresponds to the granzymeB cleavage site, and thus if PDC-E2 were to be digested by gran-zyme B, the immunogenic epitope would no longer be available forpresentation by APCs. As a result, the ability to stimulate ExDKpeptide-specific CD41 T cells would be lost. It can also be hypoth-esized that the inability of APC to present the granzyme B–derivedPDC-E2 peptide fragments could allow autoreactive T cells, whichmay be elicited by molecular mimicry, to escape tolerance andcontribute to the destruction of target BECs.32

The studies described herein generate a number of additionalquestions, such as: How would the proteolytic cleavage of PDC-E2cause a disease-specific AMA response corresponding to what isseen in PBC? Perhaps proteolytic cleavage by the caspases does notgenerate the initial breakdown in tolerance but merely serves toperpetuate the antibody or T-cell response. This would also ex-plain why only antibodies to PDC-E2 and not other mitochon-drial enzymes are produced as there are numerous more abundantproteins in the cells that undergo similar proteolytic cleavage. Inconclusion, the data obtained in this study suggest a potential rolefor the proteolytic enzymes involved in the digestion of intracellu-lar proteins in the generation of immunogenic fragments. More-over, it would be of great interest to determine in future studies ifthe localization of activated caspase-3 in liver sections correspondsto cells undergoing apoptosis.

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