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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. REFERENCES 1. Tomer Y, Davies TF. Searching for the autoimmune thyroid disease susceptibility genes: from gene mapping to gene function. Endocr Rev 2003; 24: 694-717. 2. Kavvoura FK, Akamizu T, Awata T, et al. CTLA-4 gene polymorphism and autoimmune thyroid disease: a meta analysis. J Clin Endocrinol Metab 2007; 92: 3162-3170. 3. Buonavista N, Balzano C, Pontarotti P, Le Paslier D, Goldstein P. Molecular linkage of the human CTLA-4 and 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. CD28 Ig -superfamily genes in yeast artificial chromosomes. Genomics 1992; 13: 856-861. Dariavach P, Mattei MG, Goldstein P, Lefranc MP. Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains. Eur J Immunol 1988; 18: 1901-1905. Harper K, Balzano C, Rouvier E, Mattei MG, Luciani MF, Goldstein P. CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure and chromosomal location. 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Popko K, Gorska E, Potapinska O, et al. Frequency of distribution of inflammatory cytokines IL-1, IL-6 and TNFα gene polymorphism in patients with obstructive sleep apnea. J Physiol Pharmacol 2008; 59(Suppl 6): 607-614. Wasik M, Gorska E, Popko K, et al. The GLN223ARG polymorphism of the leptin receptor gene and peripheral blood/bone marrow leptin level in leukemic children. J Physiol Pharmacol 2006; 57(Suppl 4): 375-383. Kouki T, Sawai Y, Gardine CA, Fisfalen M-E, Alegre M-L, DeGroot LJ. CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves' disease. J Immun 2000; 165: 6606-6611. 80 15. Maurer M, Loserth S, Kolb-Maurer A, et al. A polymorphism in the human cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene (exon 1 +49) alters T-cell activation. Immunogenetics 2002; 54: 1-8. 16. Ligers A, Teleshova N, Masterman T, Huang WX, Hillert J. CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms. Genes Immunol 2001; 2: 145-152. 17. Kucharska AM, Gorska E, Wasik M, Demkow U. Altered expression of T lymphocyte surface markers in children with chronic autoimmune thyroiditis. J Physiol Pharmacol 2008; 59(Suppl 6): 375-382. 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]