Download expression of cd152 (ctla-4) in children with autoimmune thyroiditis

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2009, 60, Suppl 5, 77-80
www.jpp.krakow.pl
A.M. KUCHARSKA1, E. GORSKA2, M. WASIK2, B. PYRZAK1, U. DEMKOW2
EXPRESSION OF CD152 (CTLA-4) IN CHILDREN WITH AUTOIMMUNE THYROIDITIS
AND +49 A/G POLYMORPHISM OF EXON 1 OF THE CTLA-4 GENE
1
Department of Paediatrics and Endocrinology, Warsaw Medical University, Warsaw, Poland; 2Department of Laboratory
Diagnostics and Clinical Immunology of the Developmental Age, Warsaw Medical University, Warsaw, Poland
CTLA-4 gene is one of the strongest locus of genetic susceptibility to autoimmune thyroid diseases. The aim of the
present study was to investigate surface expression of CTLA-4 on peripheral T cells in homozygotes AA and GG at
position +49 of CTLA-4 gene in children with Hashimoto's thyroiditis and in healthy controls. Blood samples were
obtained from 100 children: 45 with Hashimoto's thyroiditis and 55 controls. CTLA-4 exon 1 polymorphism was defined
by SSCP and RFLP with BbvI enzyme. T cells were analyzed with three color flow cytometry by Coulter EPICS XL. We
found that CTLA-4 expression was significantly lower in the thyroiditis patients than in controls, but CTLA-4 expression
in homozygotes GG and AA was comparable. We therefore conclude that decreased expression of CTLA-4 on T cells in
children with Hashimoto's thyroiditis is not dependent on polymorphic changes at position +49 of CTLA-4 gene.
K e y w o r d s : CTLA-4, Hashimoto's thyroiditis, polymorphism, T cell, homozygotes
INTRODUCTION
CTLA-4 gene is considered to be one of the most important
regions, except HLA, determining the genetic susceptibility to
autoimmune diseases, especially thyroid autoimmune disease
(1). The association between its polymorphisms and
autoimmune disease was found in many populations,
irrespectively of the ethnic origin (2). CTLA-4 gene is located
on 2q33, close to genes of other regulatory molecules: CD28 and
ICOS (3). It consists of 4 exons and 3 introns. The first exon
encodes the leather peptide sequence, the second the
immunoglobulin domain, with a key motive MYPPPY which
binds the ligand, the third encodes the hydrophobic
transmembrane domain, and the fourth the cytoplasmic domain
(4, 5). Polymorphism leading to substitution of alanine by
guanine at position +49 of CTLA-4 gene (+49 A/G) is present in
exon 1 and causes an alteration of the leather peptide sequence
of the CTLA-4, which makes it an attractive candidate for the
functional polymorphism of the CTLA-4 gene.
CTLA-4 (CD152) is a negative regulator of T cell activation
(6). It was first described together with the stimulatory molecule,
CD28 by Brunet et al. (7) in 1987 on T cells in mice. They bind
to the same ligands B7-1 (CD80) and B7-2 (CD86) (6, 8).
CTLA-4 is competitive to CD28 and has much higher affinity
and avidity than CD28 to both ligands B7 (8). CD28 antigen is
constitutively present on naive CD4+T cells and its expression
slightly increases after activation. CTLA-4 antigen is rarely
present on naive CD4+ T cells, but after the activation the
mRNA of CTLA-4 is detectable in T cells as soon as in the first
hour (9, 10). The protein is stored in intracellular follicles and in
that form is detected after 24 hours. The maximal surface
expression of CTLA-4 appears on T cells 48-72 hours after the
activation (10).
The aim of the present study was to evaluate the percentage
of peripheral T cells with the surface CD152 expression in
children with Hashimoto's thyroiditis in relation to the
polymorphic changes at position +49 of CTLA-4 gene.
MATERIAL AND METHODS
The protocol of the study was approved by the Bioethics
Committee of Warsaw Medical University in Warsaw, Poland.
The parents of the patients signed informed consent for the
participation in the study.
One hundred patients were examined: 45 children with
autoimmune thyroiditis of Hashimoto (HT) type and 55 healthy
controls. The mean age in HT children was 15±2 yr, (range: 8.1
to 17.9 yr). The diagnosis of Hashimoto's thyroiditis was based
on the presence of anti-thyroid antibodies (anti-thyroid
peroxidase and/or anti-thyroglobuline antibodies) and a typical
ultrasonographic picture of the thyroid gland. The control group
was age-matched (range: 8.0 to 17.4 yr). Children from the
control group were free of any immunologically mediated
disorder and of the thyroid dysfunction.
Polymorphism at position +49 of exon 1 of the CTLA-4 gene
Genomic DNA was isolated from blood samples by
Genomic Midi AX (A&A Biotechnology, Gdynia, Poland) and
used for polymerase chain reaction (PCR). A fragment of the
CTLA-4 gene was amplified with the oligonucleotides: forward
78
5'GCTCTACTTCCTGAAGACCT-3' and reverse 5'-AGTCTC
ACTCACCTTTGCAG-3', according to the sequence of human
cDNA of CTLA-4 described by Harper and Balzano (5).
Polymorphism at the position +49 of exon 1 of the CTLA-4 gene
was examined by the method of Single Strand Conformation
Polymorphism (SSCP) followed by confirmation test of
restriction fragment- length polymorphism (RFLP) using Bbv I
enzyme (New England BioLabs, Beverly, MA, USA): 10 µl of a
mixture of 1x Nebuffer 2 and 1.5 µl of a restriction enzyme Bbv
I (New England BioLabs, Beverly, MA, USA) was added to 15
µl of a product of PCR. The samples, after covering with mineral
oil, were incubated for 24 h at 37°C. Then, they were
electrophoresed on 2% agarose (60 min, 500mA, 40W, 120V) at
25°C. Electrophoregrams were visualized with ethidium
bromide. The laboratory evaluation was performed by a
laboratory technician, blinded to the information of the sample
origin (from patient or control).
˜
˜
˜
˜
˜
˜
Homozygotes AA with Hashimoto's disease vs. homozygotes
AA from controls;
Homozygotes GG with Hashimoto's disease vs. homozygotes
GG from controls;
Homozygotes GG with Hashimoto's disease vs. homozygotes
AA from controls;
Homozygotes AA with Hashimoto's disease vs. homozygotes
GG from controls;
Homozygotes AA vs. homozygotes GG in the Hashimoto
disease;
Homozygotes AA vs. homozygotes GG in the control group.
There was no significant difference in the CD152 expression
between AA and GG homozygotes among the Hashimoto
patients (Table 2). In the control group, expression of CTLA-4
on T cells did not differ between AA and GG homozygotes
either. A statistically significant difference in the expression of
CD152 was found only between the children with Hashimoto's
thyroididis and the healthy controls (P<0.05) (Table 2).
Cytometric analysis
Heparinized blood samples from HT children and healthy
controls were diluted three times with saline, and centrifuged for
30 min at 400 x g on Histopaque 1077-1 density gradient (Sigma
Diagnostics, St. Louis, MO). Isolated peripheral blood
mononuclear cells (PBMC) were washed three times with saline
and suspended in PBS. PBMC were then incubated with
monoclonal antibodies for 30 min at 25°C in darkness. An
analysis was performed with the use of a combination of
monoclonal antibodies: CD4- FITC/CD28 -PC5/CD152 -PE and
CD8 -FITC/CD28-PC5/CD152-PE obtained from Immunotech
Beckman Coulter Company (Beckman Coulter S.A. Paris Nord,
France). After incubation, samples were fixed and lysed by the
reagent set Uti-Lyse (Dako Cytomation, Gdynia, Poland). The T
cell phenotype was evaluated using the flow cytometer Beckman
Coulter EPICS XL 4C (EPICS XL/XL-MCL, version 2.0
(Beckman Coulter Company, Paris Nord, France).
The results were statistically analyzed by a t-test, using
STATISTICA XL7.0. Genotype frequency analysis was
performed by a Fisher-Snedecor test.
DISCUSSION
The finding of an association between polymorphic changes
and a disease is one of the first steps to be followed by an
analysis of genetic predisposition to the disease (1, 11). An
increased frequency of the examined in the present study
polymorphism +49 A/G in patients with autoimmune thyroid
diseases is well confirmed in the literature (2) and our results are
consistent with these observations. Nevertheless, the strongest
evidence for association with the disease is the influence of the
polymorphism on the biological function of the gene product,
involved in the disease development (1). One can speculate that
the polymorphic changes of the CTLA-4 gene could impair the
peptide function in several ways. On the one hand, by decreased
production of CTLA-4 and on the other by a decreased surface
expression of CTLA-4 on T cells, lower affinity for the ligand,
or disturbances in extra-/intracellular signal transduction.
According to this consideration in our laboratory were evaluated
physiological consequences of gene polymorphisms in obesity
or leukemia (12, 13). The polymorphism at the position +49 of
exon 1 of the CTLA-4 gene changes the sequence of leather
peptide of CTLA-4 and, theoretically, can change only the
CTLA-4 expression, but not the affinity to ligands or signaling
pathway. Polymorphic homozygotes GG (Ala/Ala) should
potentially have decreased CTLA-4 expression and impaired
inhibitory function of CTLA-4 compared with wild
homozygotes AA (Thr/Thr). Such hypothesis has been
confirmed by Kouki et al. (14). The authors stated that G allele
at the position +49 of the CTLA-4 gene is associated with
impaired control of T cell proliferation. Maurer et al. (15) also
have suggested that the surface expression of the CTLA-4 and its
intracellular distribution correlates with the genotype at position
+49. Homozygotes GG present with decreased surface
expression of the CTLA-4, whereas activated T cells in
RESULTS
In the group of 45 children with Hashimoto's thyroiditis,
13 (29%) patients were homozygous for A allele and 14 (31%)
patients were for G allele. In the control group, 12 (22%)
subjects were homozygous for A allele and 8 (15%) subjects
were for G allele. The statistically significant difference in
Fisher-Snedecor test showed the increased frequency of GG
homozygotes in patients with Hashimoto's thyroiditis (P<0.04,
odds ratio, OR=2.65; confidence interval, CI: 0.99-7.06)
(Table 1).
The surface expression of the CTLA-4 (CD152) on T cells
was compared between AA and GG homozygotes in following
subgroups:
Table 1. Frequency of particular alleles at position +49 of the CTLA-4 gene.
Homozygotes AA Homozygotes GG Heterozygotes AG
(%)
(%)
(%)
Children with Hashimoto’s disease
(n=45)
29
31*
40
Healthy children
(n=55)
22
15*
64
*Significant difference (P<0.05)
79
Table 2. T cell antigen expression in patients compared with controls in relation to the frequency of alleles at position +49 of CTLA-4 gene.
GROUPS FOR COMPARISON
CD4+CD152+ CD8+CD152+
Homozygotes AA with Hashimoto’s disease
vs. homozygotes AA from controls
P=0.004
NS
Homozygotes GG with Hashimoto’s disease vs.
homozygotes GG from controls
P=0.02
P=0.02
Homozygotes GG with Hashimoto’s disease vs.
homozygotes AA from controls
P=0.01
NS
Homozygotes AA with Hashimoto’s disease vs.
homozygotes GG from controls
P=0.01
P=0.005
Hashimoto’s homozygotes AA vs. homozygotes GG
NS
NS
Control homozygotes AA vs. homozygotes GG
NS
NS
Groups compared statistically with a t-test; NS- no statistical difference.
homozygotes AA show another pattern of intracellular
distribution (14). The association between polymorphism at
position +49 and CTLA-4 expression has also been confirmed
by Ligers et al. (16). These authors showed that homozygotes
AA present with a higher surface expression CTLA-4 after
activation and with a higher level of mRNA for CTLA-4 in
unstimulated T cells. In the present study, the baseline
percentage of peripheral T lymphocytes with the surface
expression of CTLA-4 was evaluated in children with
Hashimoto's thyroiditis and compared with healthy controls. In
Hashimoto patients, the percentage of T cells with the surface
expression of CTLA-4 was significantly decreased. This
difference was not dependent on the presence of the
polymorphism at position +49 of exon 1 of the CTLA-4 gene, as
in both examined groups, the expression of CTLA-4 did not
differ between AA and GG homozyotes. The finding suggests
that a lower expression of CTLA-4 in children with Hashimoto's
thyroididis is associated with the presence of disease, but it is not
related to the examined polymorphism. In our previous paper,
we reported that patients with Hashimoto's thyroiditis have a
decreased expression of CTLA-4 on CD4+ and CD8+ T cells
(17). Accordingly, one can conclude that this observation is not
a simple consequence of the presence of +49 A/G
polymorphism. The association of the frequency of the
polymorphic G allele with Hashimoto's disease and lack of its
influence on CTLA-4 expression may suggest that the +49 A/G
polymorphism is probably in linkage disequilibrium with other
unknown genetic changes responsible for this effect. Thus,
decreased surface expression of CTLA-4 observed in patient
with Hashimoto's thyroiditis is not dependent on the genotype at
position +49 of exon 1 of the CTLA-4 gene.
Conflict of interests: None declared.
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R e c e i v e d : July 27, 2009
A c c e p t e d : October 15, 2009
Author’s address: Dr. Anna M. Kucharska, 26 J. Sibeliusa
St., 02-641 Warszawa, Poland; Phone: +48 22 600815554; Fax:
+48 22 6214155; E-mail: [email protected]