Tacrolimus may induce the production of nucleolar anti-nuclear antibody in liver transplant patients.

Abstract

Immunosuppressive drugs have been used to prevent graft rejection in most allo-liver recipients and the immune state of these patients differs greatly before and after transplant operation. This study aims at evaluating the immune state of liver transplant patients treated with tacrolimus by investigating the production of anti-nuclear antibodies (ANA).
A hundred and eighty-eight serum samples from 94 allo-liver recipients treated with tacrolimus and from 94 patients with matched liver diseases were tested for ANA by indirect immunofluorescence assay with HEp-2 cells as substrate.
ANA were detected as positive in 20.2% of the liver transplant patients treated with tacrolimus, and in 12.8% of disease-matched control patients, but this difference was not statistically significant (P=0.17). However, the frequency of nucleolar ANA pattern in ANA-positive cases was significantly higher in the liver transplant patients (63.2%) than in the control group (16.7%) (P=0.01).
Tacrolimus may contribute to producing nucleolar ANA in liver transplant patients. The autoimmune disease susceptibility of allo-liver recipients treated with tacrolimus requires further studying.

Full text

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Received: 12.02.2011 Accepted: 30.06.2011
J Gastrointestin Liver Dis
September 2011 Vol. 20 No 3, 267-270
Address for correspondence:





Lanlan Wang
Department of Laboratory Medicine
West China Hospital of
Sichuan University
Sichuan, China
Email: WangLL87@126.com
Tacrolimus may Induce the Production of Nucleolar Anti-
nuclear Antibody in Liver Transplant Patients
Yongkang Wu, Bei Cai, Jiangtao Tang, Yangjuan Bai, Lanlan Wang
Department of Laboratory Medicine, West China Hospital of Sichuan University, Chengdu, China
Abstract
Background & Aims: Immunosuppressive drugs have
been used to prevent graft rejection in most allo-liver recipients
and the immune state of these patients differs greatly before
and after transplant operation. This study aims at evaluating
the immune state of liver transplant patients treated with
tacrolimus by investigating the production of anti-nuclear
antibodies (ANA). methods: A hundred and eighty-eight
serum samples from 94 allo-liver recipients treated with
tacrolimus and from 94 patients with matched liver diseases
were tested for ANA by indirect immunofluorescence assay
with HEp-2 cells as substrate. Results: ANA were detected
as positive in 20.2% of the liver transplant patients treated
with tacrolimus, and in 12.8% of disease-matched control
patients, but this difference was not statistically significant
(P=0.17). However, the frequency of nucleolar ANA pattern
in ANA-positive cases was significantly higher in the
liver transplant patients (63.2%) than in the control group
(16.7%) (P=0.01). Conclusion: Tacrolimus may contribute
to producing nucleolar ANA in liver transplant patients. The
autoimmune disease susceptibility of allo-liver recipients
treated with tacrolimus requires further studying.
Key words
Tacrolimus – liver transplant – nucleolar ANA.
Introduction
About 8% of the Chinese population, nearly 100 million
patients, suffer from hepatitis B or are carriers of the hepatitis
B virus (HBV) [1]. A definite association of HBV infection
and liver cirrhosis or liver cancer has been demonstrated
[2]. Both liver cirrhosis and cancer could cause liver
dysfunction that severely impairs the quality of life of patients
[3]. Liver transplantation nowadays is a widely accepted
treatment option for liver cirrhosis and cancer [4]. After liver
transplantation, immunosuppressive treatment is necessary to
reduce the risk of subsequent graft rejection and to guarantee
a long-term survival of exogenous liver in recipients.
Although the immunosuppressive regimens for all solid
organ transplants are fairly similar, tacrolimus (also known
as FK506) is one of the most popular immunosuppressants
[5]. As a potent suppressant of T lymphocytes, tacrolimus
is believed to regulate the immune state and balance of
recipients after an operation. So the diseases related to the
immune disorder, such as tumor and infection resulting from
lower immunity, have drawn the attention of clinicians when
applying a tacrolimus-based regime.
Up till now, the relationship between the tacrolimus
treatment and development of autoantibodies or autoimmune
diseases has not yet been deciphered. Since the 90s, several
groups reported the presence of de novo autoimmune
liver diseases as well as serum autoantibodies after liver
transplantation and administration of immunosuppressive
regimen [6-7]. However, the inducing effect of tacrolimus
on autoimmune disease occurrence after liver transplantation
remains to be clarified. Anti-nuclear antibodies (ANAs)
constitute a group of important autoantibodies acting against
the nucleus or cytoplasmic substances of nucleated cells.
The production of these autoantibodies mostly occurs in a
number of autoimmune disorders including systemic lupus
erythematosus and Sjögren’s syndrome [8-9]. The aim of the
current study is to evaluate the immune state of allo-liver
recipients by measuring ANA in the serum in two groups
of liver transplant patients with a long-term tacrolimus
treatment and disease-matched controls.
Patients and methods
This study has been approved by the Ethics Committee
of the West China Hospital of Sichuan University. Written
informed consent was obtained from each participant.

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268 Wu et al
The hundred and eighty-eight liver transplant patients and
patients in a control group were recruited from in-patients
and out-patients of West China Hospital from 2007 to 2010.
Patients from the control group were matched by means of
age, gender, etiology, and disease. In the liver transplant
group, 91 (96.8%) cases were detected to be HBV-positive
with preoperative testing, while the control group counted
92 (97.9%) HBV-infected patients. All allo-recipients were
treated with tacrolimus alone as an immunosuppressive
agent. The patients with autoimmune liver disease such as
autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC)
and patients with hepatitis C were excluded from the study
due to a definitely high ANA prevalence.
The blood samples were collected in 4 ml BD Vacutainer
without anticoagulant and allowed to clot up to 1 hour
at room temperature. Within 2 hours after collection the
samples were centrifuged at 1500g for 10 min at 40C to
collect serum. The sera were stored at -800C and thawed at
40C overnight prior to analysis.
ANA detection
ANA were determined by indirect immunofluorescence
assay with commercialized ANA kit (EUROIMMUN Co.,
Germany) with HEp-2 cell lines as substrate at an initial serum
dilution of 1:100 according to the manufacturer’s instructions.
The serum was further diluted as 1:320, 1:1000, 1:3200 and
1:10000 subsequently, when a positive result was obtained with
the lower dilution. ANA fluorescence patterns were defined as
speckled, homogeneous, nucleolar (Fig. 1), centromere, nuclear
dots and cytoplasm staining, etc. ANA fluorescence pattern of
each sample was observed in double blind with a fluorescence
microscope (Model E6000 Nikon Co., Japan).
Statistical analysis
Quantitative data obeying a normal distribution were
described by their mean and standard deviation (SD). When
the distribution of data was skew, the median and range were
used instead. The Mann-Whitney U or Chi-square test was
applied for comparing both patient groups. A probability
value (P) inferior to 0.05 was considered statistically
significant. All data were analyzed with SPSS 16.0.
Results
Age, gender, and disease
The characteristics of the subjects enrolled in the trial
are shown in Table I. There was no significant difference
between liver transplant and control patients in age, gender,
etiology, and disease distribution (P>0.05). This indicates
that the matching based on these 4 variables was adequate.
ANA detection
The incidence of ANA positivity was 20.2% and 12.8%
respectively, in the groups of liver transplant patients treated
with tacrolimus and the control patients. There was no
statistical difference between these two groups (P=0.17).
However, the frequency of nucleolar pattern, which reached
up to 63.2% in the ANA-positive liver transplant patients,
was only 16.7% in the control group, indicating a significant
difference between both groups (P=0.01) (Table II). For
all enrolled transplant patients, the median duration of
tacrolimus treatment was 42 months, and the mean plasma
concentration of the drug was 6.2±2.4 ng/ml.
The transplant patients were divided into a positive
subgroup with 19 cases and a negative subgroup with
75 patients based on the presence of ANAs. The median
durations of treatment with tacrolimus were 36 and 42
months, respectively in the positive and negative subgroups.
No significant difference was found between both subgroups
(P=0.58). The mean plasma concentrations of tacrolimus
in the positive and negative subgroups were 5.8±2.4 and
6.3±2.4 ng/ml, respectively. The difference in mean (0.5 ng/
ml) between both subgroups was not statistically significant
(P=0.40).
Discussion
Because of the risk of rejection after liver transplantation,
immunosuppressive drugs are necessary for sustaining the
graft function and improving the life quality of recipients.
Tacrolimus, an effective calcineurin inhibitor, was approved
for clinical use based on the merit of its immunosuppressive
activity [10]. Its action mechanism can be readily explained.
Tacrolimus binds to 12 kDa FK506-binding immunophilin
protein (FKBP12) of type-3 ryanodine receptor resulting
in a FK506-FKBP12 complex. This complex associates
with the calcineurin with high affinity and inhibits its
serine/threonine phosphatase activity, which results in
the inhibition of the transcription of interleukin 2 and
the blockade of Ca2+ dependent T lymphocyte activation
pathway. The immunosuppressive activity of tacrolimus
determines an impairment of T lymphocyte activation and
proliferation [11], so that cellular and humoral immunity of
liver transplant patients will be imbalanced after long-term
usage of the drug.
Fig 1. Example of nucleolar pattern of ANA detected
by indirect immunofluorescence assay with HEp-2
cells as substrate (40×amplified).

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Nucleolar ANAs in allo-liver recipients treated with tacrolimus 269
Furthermore the transplanted liver as an exogenous
antigen could stimulate and activate the patients’ immune
responses [12]. Consequently, the patients’ own immune
balance would be re-regulated because of the foreign
organ and tacrolimus usage. In order to achieve a new
immune equilibrium, the immune system including immune
molecules, cells, and organs would be extensively regulated
as compared with that before transplantation [13-15]. Since
the immunological tests, such as the ANA test, were not
performed for most patients before transplantation, it was
impossible to design a self-controlled study. Therefore, it
was decided to choose patients with matched liver diseases
as controls in this trial.
Antinuclear antibodies are one of the effective indicators
to evaluate the disorders of immune system resulting from
the imbalance of immunity against the own cellular antigens
[16]. Normally, a positive result of ANA test may suggest
the emergence of auto-reactivity due to primary autoimmune
or drug-induced autoimmune disorders [17]. Because of the
high prevalence of ANA in autoimmune liver diseases and
hepatitis C, patients with these disorders were excluded from
both groups of liver transplant and control patients in order
to diminish the non-specific influence of these diseases on
the ANA incidence studied in the current trial [18-19]. The
overall incidences and median titers of ANA in the liver
transplant group were comparable to those of the control
group. However, it could not be concluded that the immune
state of the liver transplant patients was similar to that of
the control patients.
Antinuclear antibodies represent a spectrum of
autoantibodies directed against various cellular components
[20]. Different ANA patterns were determined according to
typical fluorescence staining patterns [21]. The frequency
of the nucleolar pattern was significantly higher in liver
transplant patients compared with the control patients in this
study, although nucleolar pattern is reported as a rare pattern
of ANA, especially in autoimmune diseases [22]. Despite the
well known association between nucleolar ANA pattern and
scleroderma [23, 24], no liver transplant patients treated with
tacrolimus had obvious scleroderma-related symptoms.
Since anti-smooth muscle antibody (SMA) and liver
kidney microsomal antibody (LKM) have also been detected
besides ANAs in some post-liver transplantation cases
with immunosuppressive regimens [6-7], a further study
concerning these autoantibodies and the related autoimmune
diseases in allo-liver recipients treated with tacrolimus would
be necessary to clarify the autoimmunity-inducing effect of
tacrolimus.
Conclusions
The current study demonstrates that the long-term
administration of tacrolimus in liver transplant patients
induces the production of nucleolar ANA. As a suggestion,
a set of autoantibodies, including ANA, should be regularly
monitored in liver transplant patients treated with tacrolimus
for monitoring the impact of this drug on disease prognosis
and, if necessary, in time decreasing the dose.
Acknowledgement
This study was supported by the National Natural Science
Foundation of China (Grant No. 30670819, 30900658,
30772051 and 30950010).
Conflicts of interest
None to declare.
References
1. Xu Q, Gu L, Wu ZY. Operative treatment for patients with
cholelithiasis and liver cirrhosis. Hepatobiliary Pancreat Dis Int
2007; 6:479-482.
2. Leung N. HBV and liver cancer. Med J Malaysia 2005; 60 (Suppl
B): 63-66.

Table II. ANA prevalence and titer in liver transplant patients and controls
Group Number of
cases
ANA
prevalence
(%)
Median titer of
ANA [range]
Nucleolar pattern
prevalence of
ANA positive (%)a
Median titer of
nucleolar pattern
[range]
Other pattern
prevalence of
ANA positiveb
Liver transplant
patients
9419 (20.2%) * 100 (100~1000) **12/19 (63.2%) † 100 (100~320) †† 7/19 (36.8%)
Control9412 (12.8%) *100 (100~320) ** 2/12 (16.7%) †210 (100~320) †† 10/12 (83.3%)
Note: a including pure nucleolar pattern and nucleolar pattern mixed with others; b Other pattern including speckled, centromere,
cytoplasm and homogeneous pattern etc; *χ2 = 1.89, P = 0.17; ** Z = -0.26, P = 0.80; †χ2 = 6.42, P= 0.01; †† Z = -0.21, P= 0.83.
Table I. Characteristics of the subjects and disease distribution
Group Number of
cases
Male/
female
Median age
(range)
Etiology of
HBV(%)
Disease distribution (%)
Cirrhosis Liver
cancer
Other liver
diseasesa
Liver transplant patients 9479/1544 (24 ~ 70) 91 (96.8%)52.1%14.9%33.0%
Control 9479/1546 (24 ~ 78)92 (97.9%)55.3%14.9%29.8%
Note: a including severe hepatitis, liver failure, intrahepatic bile duct stone and severe liver rupture etc. excluding autoimmune liver
disease, AIH, PBC and Hepatitis C.

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270 Wu et al
3. Canning C, O’Brien M, Hegarty J, O’Farrelly C. Liver Immunity
and tumour surveillance. Immunol Lett 2006; 107: 83-88.
4. Ng KK, Lo CM. Liver transplantation in Asia: past, present and
future. Ann Acad Med Singapore 2009; 38:322-331.
5. Holt CD, Ingle G, Sievers TM. Inhibitors of calcineurin. J Pharmacy
Practice 2003; 16:414-433.
6. Kerkar N, Hadzic N, Davies ET, et al. De-novo autoimmune hepatitis
after liver transplantation. Lancet 1998; 351:409-413.
7. Evans HM, Kelly DA, McKiernan PJ, Hubscher S. Progressive
histological damage in liver allografts following pediatric liver
transplantation. Hepatology 2006; 43:1109-1117.
8. Aganovic-Musinovic I, Prljaca-Zecevic L, Subasic D. The incidence
of ANA and ETI-dsDNA detected by enzyme immunoassays and
indirect immunofluorescence assay (IFA). Med Arh 2010; 64:68-70.
9. Huo AP, Lin KC, Chou CT. Predictive and prognostic value of
antinuclear antibodies and rheumatoid factor in primary Sjogren’s
syndrome. Int J Rheum Dis 2010; 13:39-47.
10. Sosa I, Reyes O, Kuffler DP. Immunosuppressants: neuroprotection
and promoting neurological recovery following peripheral nerve and
spinal cord lesions. Exp Neurol 2005; 195:7-15.
11. McConnell JL, Wadzinski BE. Targeting protein serine/threonine
phosphatases for drug development. Mol Pharmacol 2009; 75: 1249-
1261.
12 Kuwana M, Okazaki Y, Ikeda Y. Splenic macrophages maintain the
anti-platelet autoimmune response via uptake of opsonized platelets
in patients with immune thrombocytopenic purpura. J Thromb
Haemost 2009; 7:322-329.
13. Di Benedetto F, Di Sandro S, De Ruvo N, et al. Sirolimus monotherapy
in liver transplantation. Transplant Proc 2007; 39:1930-1932.
14. Canadas O, Sáenz A, Orellana G, Casals C. Equilibrium studies of a






fluorescent tacrolimus binding to surfactant protein A. Anal Biochem
2005; 340:57-65.
15. Lilly LB, Grant D. Optimization of cyclosporine for liver
transplantation. Transplant Proc 2004; 36(2 Suppl):267S-270S.
16. Altintas A, Ozel A, Okur N, et al. Prevalence and clinical significance
of elevated anti-nuclear antibody test in children and adult patients
with idiopathic thrombocytopenic purpura. J Thromb Thrombolysis
2007; 24:163-168.
17. Olsen NJ. Drug-induced autoimmunity. Best Pract Res Clin
Rheumatol 2004; 18:677-688.
18. Burak KW, Urbanski SJ, Swain MG. A case of coexisting primary
biliary cirrhosis and primary sclerosing cholangitis a new overlap
of autoimmune liver diseases. Dig Dis Sci 2001; 46: 2043-2047.
19. Zhang FK, Jia JD, Wang BE. Clinical evaluation of serum antibody-
negative primary biliary cirrhosis. Hepatobiliary Pancreat Dis Int
2004; 3:288-291.
20. Hiemann R, Hilger N, Michel J, et al. Automatic analysis of
immunofluorescence patterns of HEp-2 cells. AnnN Y Acad Sci
2007; 1109:358-371.
21. Parker JC, Burlingame RW, Webb TT, Bunn CC. Anti-RNA
polymerase III antibodies in patients with systemic sclerosis detected
by indirect immunofluorescence and ELISA. Rheumatology (Oxford)
2008; 47:976-979.
22. Zabek J, Palacz A, Pyka J, Krzewska I. Antinucleolar antibodies
in diagnostics of antiphospholipid syndrome. Pol Arch Med Wewn
2008; 118:25-30.
23. Błaszczyk M, Jarzabek-Chorzelska M, Jabłońska S, et al.
Autoantibodies to nucleolar antigens in systemic scleroderma:
clinical correlations. Br J Dermatol 1990; 123:421-430.
24. Muro Y. Antinuclear antibodies. Autoimmunity 2005; 38: 3-9.