Kinetics of anti-M2 antibodies after liver transplantation for primary biliary cirrhosis

Journal of Hepatology 1995; 23: 674680 Printed in Denmark AN rights reserved

Journal of Hepatology ISSN 0168-8278

Kinetics of anti-M2 antibodies after liver transplantationfor primary biliary cirrhosis Laurence

Dubel’,

Olivier Farges 2, Hem-i Bismuth2, Mylene Sebagh, Jean-Claude Catherine Johanet’

Hornberg’

and

‘Laboratoire Central d’lmmunologie et d’Ht!matologie, H6pital Saint-Antoine, Paris, and ‘Unit& de Chirurgie Ht;oatobiliaire et de Transplantation Hdpatique, Centre H&patobiliaire, Hdpital P. Browse, Universite Paris Sud, Villejuil; France

Bac~grround/Aims: Orthotopic liver transplantation is currently considered as the treatment of choice for primary biliary cirrhosis in the terminal stage and, as for other autoimmune liver disease, the risk of recurrence of the disease within the graft has been raised. There is, however, some discrepancy about the risk of recurrence based on pathological analysis. In addition, pathological recurrence of primary biliary cirrhosis within the graft is not always associated with a rise in the serological markers of the disease. In order to clarify this situation, we have monitored antimitochondrial antibodies before and after transplantation. Methods: Antimitochondrial antibodies were detected by indirect immunofluorescence (variation in antibody titers) and the antimitochondrial antibodies-2 by western blotting (variation in the number of peptides recognized) in 16 primary biliary cirrhosis patients followed for at least 4 years after transplantation. Results: Antimitochondrial antibody titers had normalized 1 year after transplantation in seven patients, declined in seven others and remained unchanged in two. Over the 4 years of follow up, four patients demonstrated a subsequent increase in antimitochondrial antibody titers. Western blot analysis demonstrated the loss of one or more bands in seven patients during the first operative year after transplantation and in three other patients thereafter; in six patients the

western blotting profile remained identical to that obtained before transplantation. The important changes generally occurred during the first year post-transplantation, without significant changes thereafter, except for three patients who demonstrated a secondary reappearance of the initially lost band. Disappearance of all bands was never observed. There was no concordance between the normalization of antimitochondrial antibody titers (indirect immunofluorescence) and the reduction in the number of peptides recognized (western blotting). Serum bilirubin and alkaline phosphatase levels had normalized by 1 year after transplantation, and remained normal thereafter. Routine liver biopsies performed on a yearly basis did not disclose any pattern suggestive of primary biliary cirrhosis recurrence. Conclusions: Antimitochondrial antibody titers decreased in primary biliary cirrhosis patients after liver transplantation, although antimitochondrial antibodies-2 never disappeared as assessed by western blotting. In the present study these features were not associated with biochemical or histoclogical evidence of primary biliary cirrhosis recurrence.

P

ence of antimitochondrial autoantibodies(anti-M2) found in more than 90% of PBC patients (2,3). By western blotting (WB), these antibodies recognize five autoepitopes (4) that are part of the mitochondrial multienzymatic complex: the pyruvate dehydrogenase complex (PDH) (5-7), the branched chain a-oxoacid dehydrogenase (BCOADH) and the a-oxoglutarate dehydrogenase (OGDC) (8,9). Orthotopic liver transplantation (OLT) is currently

biliary cirrhosis (PBC) is a chronic autoimmune liver disease, characterized by inflammation and destruction of the interlobular and septal bile ducts (1). The major serological feature is the presRIMARY

Received II November 1994; revised I June; accepted 4 July 1995

Correspondence: Laurence Dubel, Hapita Saint-Antoine, Laboratoire Central d’Immunologie du Pr Homberg, 184, Rue du Faubourg Saint-Antoine, 75 012 Paris, France.

674

Key words: Anti-M2; Immunofluorescence; Liver transplantation; Primary biliary cirrhosis; Western blotting. 0 Journal of Hepatology.

Anti-h42 before and after liver transplantation

considered as the treatment of choice for PBC in the terminal stage (10-12) and, as for other autoimmune liver diseases, the risk of recurrence of the disease within the graft has been raised. The potential for PBC to recur is supported by the possible reappearance of antimitochondrial antibodies (AMA) after an initial postoperative fall (13-15), as well as by the description in some liver grafts of pathological features of PBC (13,15-l 7). These histological features, however, lack specificity as they may also occur as a result of rejection, infection, or bile duct obstruction (17). In addition, histological evidence of recurrence is frequently not associated with a rise in AMA. The long-term outcome of AMA by indirect immunofluorescence (AMA-IIF) is controversial (11,13,14, 18), and very little and incomplete information is available on the qualitative evolution of anti-M2 as assessed by western blot analysis (15,19). The purpose of this study was to monitor the kinetics of anti-M2 by western blot (variation in the number of peptides recognized) as well as by indirect immunofluorescence (variation in AMA titers) in a series of 16 PBC patients followed for at least 4 years after transplantation.

Materials and Methods Patients and sera

We have analyzed the pre- and post-transplant sera of 35 PBC patients treated by OLT at Paul Brousse Hospital since 1986. In order to obtain a homogeneous population, we have excluded patients presenting an

TABLE

Age

stage of PBC and liver function LB

AMA were detected and titrated on a composite block of rat liver, kidney and stomach, with fluorescein conjugated anti-human polyvalent immunoglobulin, according to the method of Roitt & Doniach (21). For screening, sera were diluted I:20 and in case of positive fluorescence, 1:40, 1:80, 1:160, 1:320, 1640 and 1:1280. Samples were considered positive when all renal tubules (and preferentially the distal tubular cells) as well as gastric parietal cells and the cytoplasm of hepatocytes were stained. A separate series of experiments were designed to assess the possible influence of cyclosporine on the detection of AMA by IIF (AMA-IIF). In brief, the serum from a non-transplanted PBC patients (AMA-IIF titers 640) who had not received cyclosporine therapy and the serum from a transplanted PBC patient (with-

58 56 38 48 58 54 50 54 49 30 42 51 54 57 51 49

II/III III IV III IV III III IV IV III IV II III IV III III

tests at the time of transplantation

Before OLT TB

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Indirect immunojluorescence

1

Histological Pt. no.

atypical pattern, called “Wel.“, in IIF (n=5) (Homberg’ JC, unpublished observation), as well as patients whose follow up after transplantation was less than 4 years (n= 14). The mean age of the 16 selected patients was 49.9 years (range, 30-58), and 14 were women. Eight of them had one or more associated autoimmune disorders, including keratoconjunctivitis sicca, scleroderma, Raynaud’s phenomena, sarcoidosis, or CREST syndrome. The immunosuppressive regimen used in all patients was based on an association of steroids, Azathioprine and Cyclosporine, as previously described (20). Sera were sampled at yearly intervals and stored frozen at -80°C until assayed.

6N 25N 20N 27N 35N 14N 13N 10N 5N 4N 5N 20N 3N 52N 4N 20N

AP

OLT+ 1 IIF

WB ab

IIF -

as well as kinetics

of AMA-IIF

OLT+2

and anti-M2

OLT+3

OLT+4

WB

IIF

WB

IIF

WB

b

-

b

_

b a ac acd abed ad abc ab abe acd ab abc abed abed be abed

5N

160

11N 16N IlN 2N 12N 7N 10N

640 640 640 640 80 1280 640

acd ac acd abed abed abed abd

_ 40 80 640 80 80 40

a ac acd abed ad abc ab

160 80 640 80 80 40

a ace acd abed ad abc ab

160 40 80 640 80 160 _

23N 7N 6N 5N 8N 1N 8N 7N

160 1280 640 1280 160 80 80 640

abe acd ab abc abed abed be abed

640 _ 640 160

abe acd ab abc a

80 640 160 640 80

abe acd ab abc abed

: abed

40 640

te abed

80 640 160 160 80 40 20 _

in 16 PBC patients

IIF

_ 80 40 80 640 160 160 160 40 160 80 40 _ -

WB b ac ac ac abc a ac a ab acd ab abc abed abed be abed

LB: Stage of PBC on liver biopsy at the time of OLT, TB: total bilirubin level, AT: serum alkaline phosphatase level, N: TB and AP normal values, OLT+X: X years after OLT, IIF: AMA-titers by indirect immunofluorescence, WB: Anti-M2 profile by western blot, -: negative AMAIIF titers, a (EZ subunit of the pyruvate dehydrogenase complex (PDH)), b (protein X of the PDH), c (E, subunits of the a-oxoacid dehydrogenase and the a-oxoglutarate dehydrogenase complex), d (Eia subunit of PDH), e (El/I subunit of PDH): peptides recognized by anti-M2.

675

L. Dubel et al.

out AMA) under cyclosporine therapy (1.3 mlx2 a day) were titrated, with the serum of a blood donor used as a control. The first serum was incubated with the second (dilution l/2) and with the control serum (dilution l/2) and titrated over again.

Statistical

analysis

The evolution after OLT of each parameter (AMA(IIF), anti-M2(WB), TB, AP) was performed using Spearman’s test.

Results Western blotting

Findings before OLT

To obtain liver mitochondrial antigens for western blotting, livers collected from Wistar rats were homogenized with a Potter instrument in 9 times their weight of a 0.25 M saccharose, 2 mM TRIS buffer, pH 7.4. Subcellular fractions were prepared by fractional centrifugation as described by De Duve et al. (22). The rat liver mitochondrial antigens so recovered contain 20 g protein per liter (23). Samples were denatured by heating at 100°C for 3 min in sample buffer and were applied to a 13% SDSPAGE gel according to the method of Laemmli (24). The mitochondrial proteins were separated at 14 v/cm for about 3 h, and they were electrophoretically transferred to nitrocellulose at 1 mA/cm* for 3 h (trans-blot, Biorad, Brussels, Belgium) (25). After incubation in a solution containing 5% (W/V) non-fat dry milk in PBS-Tween to block nonspecific protein binding sites, nitrocellulose strips carrying the separated mitochondrial proteins were incubated for 1 h at 22°C with 4 ml of a 1: 100 dilution of the patient sera in non-fat dry milk. After washing in PBS-Tween, the strips were incubated for 45 min in peroxidase-conjugated goat antihuman IgG, IgA, IgM (Pasteur Institut Production, Paris, France) at a dilution of 1:200. Peptide bands to which anti-M2 had bound were visualized with 4-chloro I-naphthol substrate. These bands have previously been described as: a) E2 subunit (dihydrolipoamide acetyl transferase) of the pyruvate dehydrogenase complex (PDH); b) protein X of PDH; c) E2 subunits of the branched chain a-oxoacid dehydrogenase and the a-oxoglutarate dehydrogenase; d) Eros subunit of PDH; e) E&I subunit of PDH (4).

The clinical, biological and histological status of the 16 patients at the time of transplantation are summarized in Table 1. Of these, 14 had advanced disease (histologic stages III and IV). Total bilirubin and serum alkaline phosphatase levels were raised in all patients

Liver function

p co.05

Pre OLT

OLT+4

AMA median values

Number of peptides 5 p co.05

tests

Total bilirubin (TB) and serum alkaline phosphatase (AP) were measured at least monthly. Data are expressed as N times the normal upper range. Liver histology

Histologic examinations were performed to assess the histologic stage of PBC at the time of transplantation (using the Scheuer classification (26)) and to search for histological evidence of either rejection or PBC recurrence on liver biopsies performed at yearly intervals after transplantation. All sections were stained with hematoxylin and eosin. Selected sections from each case were stained with trichrome. 676

Pre OLT

OLT+4

Number of peptides recognized by anti-M2 (Mean values) Fig. 1. AMA titers (by indirect immunofluorescence) and number of bands recognized by the anti-M2 antibodies (by western blot) before and4 years after OLT (OLT+4). Data are expressed as means for the 16 PBCpatients studied.

Anti-M2

(mean TB: 16 times normal value, mean AP: 8 times normal value). AMA-IIF were present in all patients. By western blot, the sera of all patients recognized at least two bands, but none of them simultaneously presented the five antigenic bands. The most frequently represented were the a (93%) and b (75%) bands. Findings after OLT TB and AP levels normalized rapidly after transplantation. The kinetics of antimitochondrial antibodies as assessed by indirect immunofluorescence and western blot are summarized in Table 1. One year post-transplant (OLT+l), AMA-titers had normalized in seven patients, declined without returning to the previous negative value in seven other patients, whereas they

before and after liver transplantation

had remained identical to pretransplant titers in two patients. A subsequent decrease in IIF titers over the 4 years of follow up was observed in four patients (who had declined without return to previous negative value). Conversely, five other patients experienced a secondary rise in IIF titers including three patients who had normalized 1 year after OLT, one patient who had a decrease without return to previous negative value 1 year after OLT and one patient without previous modification. Mean values of AMA titers, before and 4 years after OLT are shown in Fig. 1. Overall, by western blot, loss of 1 or more bands was observed in ten patients. Of these, seven occurred during the first year and three after the first year. In the former, a subsequent disappearance of a band was observed 4 years after OLT, in three patients (nos.

Fig. 2. Representative examples of the kinetics of anti-A42 as assessed by western blotting. a: loss of 2 bands after OLT (patient no. 8), lane 1: pretransplant sera, lane 2: 1 year post-transplant. lane 3: 2 years post-transplant, lane 4: 3 years post-transplant, lane 5: 4 years post-transplant. b: no mod$cation in western blot profile (patient no. 12). lanes 1 and 2: before OLT lanes 3-7: l-4 years after OLT A: E2 subunit of the pyruvate dehydrogenase complex (PDH), B: protein X of the PDH, C: Ez subunits of the a-oxoacid dehydrogenase and the a-oxoglutarate dehydrogenase complex, D: E,a subunit of PDH.

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L. Dubel et al.

6,7,8). Conversely, bands lost within the first year in three patients had recurred at 4 years (nos. 2,13,14). In seven patients, the western blot profiles have remained unchanged over the 4 years of follow up. Mean values of the number of peptides recognized by these antibodies before and 4 years after OLT are shown in Fig. 1 and representative examples are shown in Fig. 2a and b. There was no concordance between the post-transplant kinetics of AMA-IIF and anti-M2. In order to explain the discrepancy between the persistence of antibodies as assessed by WB and their disappearance by IIF (observed for five patients 4 years after transplantation), we have studied the influence of Cyclosporine on the detection of AMA titers as described in the Methods section. Cyclosporine did not inhibit the detection of AMA by indirect immunofluorescence (data not shown).

TABLE

Histologic examination after OLT During the first postoperative month two patients developed an acute episode of rejection that resolved with two bolus steroids in one and a course of OKT3 in addition to steroids in the other, without subsequent recurrence of rejection. During the course of follow up, six patients (nos. 3,5,6,7,13,14) developed chronic active hepatitis which occurred l-4 years posttransplant and was related to the hepatitis C virus in five patients and to the hepatitis B virus in one patient. All patients had negative HBV and HCV serologies at the time of transplantation, and there was no correlation between the occurrence of the acquired hepatitis and the kinetics of antibodies assessed by IIF or WB. One other patient developed mild steatosis 2 years after transplantation. The remaining nine patients had either normal routine liver biopsies or a mild and usually transi-

2

Evolution

of antimitochondrial No. of patients studied

Reference

antibodies

and histological

No. of uatients with a follow up of at least 4 years after OLT

analysis

Methods

after orthotopic

liver transplantation

from different

groups

Results after OLT Kinetics

of AMA titers

Histologic

findings

on liver biopsy

3

1

IIF

Undetectable levels after OLT and subsequent rise with titer identical to or even greater than before OLT.

Histological evidence of PBC recurrence in the 3 cases.

Polson et al. (13)

23

4

IIF

Initial fall in the first few months after OLT, with subsequent rise.

Histological features with PBC recurrence 10 patients.

Haagsma

et al. (14)

13

3

IIF CFT RIA

Fall or even loss, 1 month after OLT, with unchanged data during the following years. A rise was observed in 2 patients after 4 and 5 years.

No histological evidence PBC recurrence.

Esquivel et al. (18) Demetris et al. (27)

52

7

IIF

Decline in most patients (43/45). Thereafter the titers remained at the same level or decreased slightly, except for a subsequent increase in 3 of the 7 patients after >3 years.

No histological evidence of PBC recurrence.

Balan et al. (17)

60

22

ELISA

Significant decrease after OLT in all patients. In 3 of the 5 patients in whom recurrent PBC was diagnosed (26 years after OLT) anti-M2 titers were undetectable.

Histological evidence of PBC recurrence (5 patients).

Gouw

19

9

IIF RIAWB

Overall decrease in titer, with steepest fall during the first year.

Histological evidence of PBC recurrence.

Klein et al. (19)

28

15

IIF ELISA CFTWB

Decrease of anti-M2 titers by ELISA. By WB, the same MZ-determinant as before OLT was visualized.

No histological PBC recurrence for any of the followed patients.

Present study

16

16

IIF WB

Significant decrease in anti-M2 titers in most patients (14/16); became negative in S/14 cases. Loss of bands for 8 patients by WB without becoming negative.

No histological evidence PBC recurrence.

Neuberger

et al.

(16)

et al. (15)

IIF: indirect bodies

678

immunofluorescence,

CFT: complement

fixation

test, RIA: radio

immunoassay,

WB: western

blot, AMA:

compatible in 9 of of

of

antimitochondrial

anti-

Anti-M2 before and after liver transplantation

ent portal infiltrate. In no patient was a granulomatous destruction of the bile duct, the so-called “florid duct lesion” ever observed.

Discussion PBC is a disease associated with numerous abnormalities of the immune system, including the presence of anti-M2. It is now clearly established that these antibodies represent the hallmark of this disease, although, they do not appear to be closely related to the pathological changes occurring within the liver. Liver transplantation is the treatment of choice for PBC, which is one of the indications associated with the longest survival and the best quality of life (11,27-29). Some information is available on the long-term serological and histological outcome after liver transplantation for PBC, including several controversial points: the longterm outcome of AMA-IIF, the recurrence of the disease within the graft (13,15-l&27,30,31) and the fact that those patients with histological signs of recurrence lacked, in certain cases, detectable, anti-M2 antibodies (15,17). In fact, post-transplant monitoring of AMA has, in the past (13-19,27), been assessed by indirect immunofluorescence (IIF), radio-immunoassay (RIA) complement fixation test (CFT), ELISA and recently by western blot, over variable follow-up periods after transplantation and, as demonstrated in Table 2, they have yielded conflicting results. In an attempt to clarify this issue, we have used the qualitative contribution of the western blot method for the analysis of the anti-M2 after OLT, and have compared it with indirect immunofluorescence, in a series of 16 PBC patients. Although this study population is small, we believe it is representative as its epidemiologic, clinical, biological, histological and immunological characteristics, and furthermore the indications for transplantation were comparable to that in other studies of transplanted PBC patients (32-35). Our study confirms that AMA-IIF titers decrease in most patients (14/16) after OLT and, in addition, demonstrate that this decrease is usually long-lasting (12/ 14). This evolution is, however, not paralleled by the kinetics of anti-M2 that persist in the sera, as demonstrated by western blot. In fact, disappearance of all antigenic peptides recognized by these anti-M2 was never observed after transplantation, as assessed by WB. The reason for this discrepancy is unclear. We do not believe that the difference in the results yielded by IIF and WB is merely related to a difference in the sensitivity or specificity of these two techniques. A recent study from our laboratory on 142 PBC patients has revealed that IIF and WB have comparable sensitivities (0.85 for IIF vs 0.91 for WB) and equal speci-

ficities (1 for both) and that the level of discordance is low (8%) (36). It is also unlikely that the initial fall in AMA titers was related to blood transfusions; there was no correlation between the amount of blood products transfused and the kinetics of anti-M2 antibodies (not shown). In addition, transfusions were, almost exclusively, performed intraoperatively and any dilution effect 1 month post-transplant is unlikely to have persisted. Finally, immunosuppressive agents used after transplantation hardly have any influence on the humoral arm of the immune response. This discrepancy between the IIF and the WB assessment of antimitochondrial antibodies is not unprecedented. Fusconi et al. (37) have previously described patients with a positive IIF and a negative WB profile related to a denaturation of the M2 autoepitopes by the western blot method. However, in our study, the difference between the two tests was “reverse” (i.e. positive WB and negative IIF) and was only observed after transplantation (i.e. not in the pretransplant sera). We have ruled out a putative interference of cyclosporine on the detection of the AMA-IIE Our data indicate that antimitochondrial antibodies directed against mitochondrial antigenic peptides (the whole antigen being denatured during the heating step of the western blot) persist in the serum after OLT, whereas antimitochondrial antibodies directed against undenatured antigen (as assessed by IIF) seem to disappear. This observation may explain why some authors have observed histological evidence of PBC recurrence within liver allografts, while AMA-titer (by IIF) was markedly decreased or had even disappeared (17). The significance of the failure of anti-M2 antibodies to disappear, as well as their influence on the long-term risk of PBC recurrence after OLT, warrants further investigation.

Acknowledgements This study was supported by a grant from the Conseil Scientifique de la Faculte de Medecine Saint-Antoine, Universite Pierre et Marie Curie, Paris VI, France.

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