Serum CD3+,CD4+,CD69+ lymphocytes in Graves' disease patients treated with two different anti-thyroid drugs
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《中华医药杂志》英文版
[Abstract] objective To explore the changes of circulating CD3+,CD4+,CD69+ lymphocyte accompanied with the serum FT3, FT4 and TSH in Graves' disease (GD) patients when using two different anti-thyroid drugs by comparing the difference between the GD patients treated with methimazole and with propylthiouracil and untreated GD patients and control subjects.Methods Fifty-two GD patients were a110 pcated into three groups, one was consisted of untreated GD patients(n=19),the others were consisted of treated patients with two anti-thyroid drugs(methimazole group,n=16;propylthiouracil group,n=17).Serum FT3, FT4 and TSH were measured using radioimmunoassay, and circulating CD3+, CD4+and CD69+T cells by three-color flow cytometry, and compared with those of healthy controls (n=18).Results The percentage of plasma circulating CD4+,CD69+ T cells in untreated GD patients increased as compared with those of patients with the two anti-thyroid drugs groups; the percentage of plasma circulating CD69+ lymphocytes in patients with methimazole decreased more significantly than those with propylthiouracil; the CD69+lymphocyte are significantly inversely correlated with CD4+ T cells in propylthiouracil group(P<0.005).Conclusions Our data showed that the change of plasma circulating CD69+lymphocyte are significant correlated with the plasma circulating CD4+lymphocyte in GD patients with propylthiouracil(P<0.005).This means the immunosuppressive action of propylthiouracil may differ from that of methimazole,which need to further study in the future.
[Key words] Graves' disease; CD69;CD4;CD3；methimazole; propylthiouracil.
INTRODUCTION In Graves' disease (GD) the circulating CD69+ lymphocyte is recognized as the marker of early activation antigens, usually increased in untreated and recurrent GD patients[1].There is no report that the propylthiouracil can reduce the expression of CD69+ lymphocyte in the peripheral blood of patients with GD. This study was carried out to assess the circulating CD69+ T cell in GD patients treated with propylthiouracil or methimazole and its correlation with serum thyroid hormone levels. In addition, we compared the percentages of circulating CD3+, CD4+lymphocyte with the concentration of free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) in those patients treated with one of the two anti-thyroid drugs. In general, thionamide drugs may directly influence the immune response in patients with GD. First, they decreased thyroid antigen expression, and decreased prostaglandin and cytokine releasing from thyroid cells with a subsequent impairment of the autoimmune response. Secondly, they inhibited the generation of oxygen radicals in T cells, B cells and particularly the antigen presenting cells[2].To observe whether propylthiouracil compares favorably or otherwise with methimazole with respect of effects on circulating CD3+, CD4+ and CD69+ lymphocyte as assessed serum FT3,FT4 and TSH by comparing with untreated Graves' disease patients or healthy subjects is our aim.
MATERIALS AND METHODS
Fifty-two patients with GD were enrolled in this clinical comparative observation. They were divided into three groups, the first group (1) was consisted of patients with GD who had been treated with methimazole (n=
Department of Endocrinology,The Affiliated Hospital of North China Coal Medical College, Tangshan 063000,Hebei Province,China
16); the second group(2) was consisted of the untreated GD patients (n=19); the last group(3) was consisted of the GD patients treated with propylthiouracil (n=17). In all, 14 males and 38 females, meanly aged (30±9) y; otherwise, 18 healthy subjects,meanly aged (27±8) y including 9 females and 9 males,were as control group (0). All patients were informed consent and were studied as outpatients during visits to our endocrinology clinic. All patients had diffuse toxic goiter, other signs and symptoms including nervousness, irritability, palpitation, fatigue, heat intolerance, weight loss, etc. We detected the concentrations of circulating CD3+,CD4+, CD69+ lymphocyte in peripheral blood of untreated GD patients when the diagnosis had been made, patients whose thyroid function had been normalized with methimazole or propylthiouracil and the normal controls. FT3, FT4, TSH were measured from fasting plasma samples using immunoradiometric assay kit(Beijing Kewei Clinical Diagnostic Reagents Co.Ltd). CD3,CD4,CD69 were measured in whole blood by three-color flow cytometry(merck-calbiochem kit). Those results were analyzed by SPSS 11.0(T-test, ANOVA)
RESULTS
There was significant reduction in circulating CD69+ T cells in both methimazole group and propylthiouracil group at euthyroidism when compared with untreated GD patients (P<0.014,P<0.016,respectively), but this only reached statistical significance in the methimazole group (1) when it compared with control group (0), whereas the propylthiouracil group(3) whose circulating CD69+ T cells did not changed (Figure 1).
In the group(2) the concentrations of CD4+,CD69+ T cells increased as well as those of serum FT3 and FT4. However, once hyperthyroidism was controlled by the two different anti-thyroid drugs(group 1,group 3) CD4+, CD69+ T cells decreased followed in turn by reduction of serum FT3 and FT4 (Table 1).
Figure 1
Table 1 CD4+,CD69+ level after treatment
In group (3) the concentrations of circulating CD69+ T cells were negatively correlated with that of circulating CD4+ T cells when using the Regression Linear Analysis(P<0.05).
DISCUSSION
Data above demonstrated clearly that the hyperthyroid untreated patients with Graves' disease had an increased CD69 expression in peripheral blood which wasrelated in part to elevated thyroid hormone concentration and decreased CD69 expression which was related to a reduction in serum T4 and T3 only by methimazole. They also elucidated that the expression of CD4+ lymphocytes in untreated patients with GD increased when compared with both those of control subjects and patients with GD who had been treated with anti-thyroid drugs. These data were consistent with other previous observations [1,3].Our study demonstrated, for the first time in vivo, that the change of circulating CD69+ lymphocytes were significantly inversely correlated with the circulating CD4+ Tcells in propylthiouracil group when controlling FT3 and FT4 (F=10.707,P=0.005). PTU therapy induced rapid and specific changes within the CD3+ subsets, namely a reduction in the CD4+ subset and an increase in the CD8+ subset, resulting in a marked decrease in the CD4+/ CD8+ ratio[4].These suggested that increased CD69+ T cells and decreased CD4+ T cells in Graves' disease patients treated with propylthiouracil might modulate the clinical course of autoimmune thyroid disease[2].Recent researches found that the imbalance between effector and regulator T cells appeared to be crucial in the pathogenesis of GD[5] and not to be influenced by increased CD69 expression[5,7].And the naive CD4 T cells can differentiate into at least two different types of T helpers, Th1 and Th2 [8],both of the T-helper 2 and the Thelper 1 are involved in the pathogenesis of GD[9，10].Therefore, the decreased CD4+ T cells and the increased CD69+ T cells in GD patients treated with propylthiouracil confirmed that the proprylthiouracil can influence the immune system not as same as the methimazole does. Some researches demonstrated that intrathyroidal lymphocytes[10] from GD patients be accumulated in proximity to thyroid follicles and may change the local immune reaction, thus the increased CD69+ T cells and the decreased CD4+ T cells in intrathyroidal lymphocytes, moreover, may influence recurrence of GD [6，10，11，12].Although we did not detect intrathyroidal lymphocytes and CD8+ T cells, but the changes of peripheral blood CD4+ and CD69+ T cells in GD patients treated with propilthiouracil need to further study.
REFERENCE
1.Corrales JJ, Lopez A, Lissdad J, et al. Methimazole therapy in Graves' disease influences the abnormal expression of CD69( early activation antigen) on T cells. J Endocrinol, 1997,155(3): 491-500.
2. Wilson, Foster, Kronenberg, et al. Williams Textbook of Endocrinology.9th Edition.Saunders:Phildephia,1997,430-450.
3.Gessl A, Waldhausl W. Elevated CD69 expression on naive peripheral blood T-cells in hyperthyroid Graves' disease and autoimmune thyroiditis; discordant effect of methimazole on HLA-DR and CD69. Clin Immunol Immunopathol,1998,87(2):168-75.
4. Walfish PG, Tseng KH. Intrathyroidal activated (Ia+) T-lymphocyte CD+ subsets and B cells in Graves' hyperthyroidism respond rapidly to propylthiouracil therapy: demonstration using fine needle aspirates and two-colour laser flow cytometry. Autoimmunity,1992,13(1):35-41.
5.Saitoh O, Nagayama Y. Regulatin of Graves' Hyperthyroidism with Naturally Occurring CD4+CD25+Regulatory T Cells in a Mouse Model. Endocrinology,2006,147(5):2417-2422.
6.Sieg SF, Harding CV, Lederman MM. HIV-1 infection impairs cell cycle progreessin of CD4+ T cells without affecting early activation responses.J Clin Invest,2001,108(5):757-764.
7. Bohler T, Walcher J, Holzl-Wenig G, et al. Expression of CD69 on T-cells from HIV-1-infected children and adolescents increases with increasing viral load. Eur J Pediatr,1999,158(8):638-644.
8.Zhu J, Yamane H, Cote-Sierra J, et al. GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors. Cell Res,2006,16(1):3-10.
9.Aust G, Sitting D, Steinert M, et al. Graves`disease is associated with an altered CXCR3 and CCR5 expression in thyroid-derived compared to peripheral blood lymphocytes. Clin Exp Immunol,2002,127(3):479-485.
10. Aust G, Kamprad M, Lamesch P, et al.CXCR6 within T-helper and T-cytotoxic tipe1 lymphocytes in Graves`disease. Eur J Endocrinol,2005,152(4):635-643.
11. Nakamara Y, Wetanabe M, Matsuzuka F, et al. Intrathyroidal CD4+T lymphocytes express high levels of Fas and CD4+CD8+ macrophages/dendritic cells express Fas ligand in autoimmune thyroid disease.Thyroid,2004,14(10):819-24.
12. Green S, Pichyangkul S,Vaughn DW, et al. Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever. J Infect Dis,1999,180(5): 1429-1435.
(Editor Emila)(JIA Wei, LI Zhi-guo，YANG )
[Key words] Graves' disease; CD69;CD4;CD3；methimazole; propylthiouracil.
INTRODUCTION In Graves' disease (GD) the circulating CD69+ lymphocyte is recognized as the marker of early activation antigens, usually increased in untreated and recurrent GD patients[1].There is no report that the propylthiouracil can reduce the expression of CD69+ lymphocyte in the peripheral blood of patients with GD. This study was carried out to assess the circulating CD69+ T cell in GD patients treated with propylthiouracil or methimazole and its correlation with serum thyroid hormone levels. In addition, we compared the percentages of circulating CD3+, CD4+lymphocyte with the concentration of free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone (TSH) in those patients treated with one of the two anti-thyroid drugs. In general, thionamide drugs may directly influence the immune response in patients with GD. First, they decreased thyroid antigen expression, and decreased prostaglandin and cytokine releasing from thyroid cells with a subsequent impairment of the autoimmune response. Secondly, they inhibited the generation of oxygen radicals in T cells, B cells and particularly the antigen presenting cells[2].To observe whether propylthiouracil compares favorably or otherwise with methimazole with respect of effects on circulating CD3+, CD4+ and CD69+ lymphocyte as assessed serum FT3,FT4 and TSH by comparing with untreated Graves' disease patients or healthy subjects is our aim.
MATERIALS AND METHODS
Fifty-two patients with GD were enrolled in this clinical comparative observation. They were divided into three groups, the first group (1) was consisted of patients with GD who had been treated with methimazole (n=
Department of Endocrinology,The Affiliated Hospital of North China Coal Medical College, Tangshan 063000,Hebei Province,China
16); the second group(2) was consisted of the untreated GD patients (n=19); the last group(3) was consisted of the GD patients treated with propylthiouracil (n=17). In all, 14 males and 38 females, meanly aged (30±9) y; otherwise, 18 healthy subjects,meanly aged (27±8) y including 9 females and 9 males,were as control group (0). All patients were informed consent and were studied as outpatients during visits to our endocrinology clinic. All patients had diffuse toxic goiter, other signs and symptoms including nervousness, irritability, palpitation, fatigue, heat intolerance, weight loss, etc. We detected the concentrations of circulating CD3+,CD4+, CD69+ lymphocyte in peripheral blood of untreated GD patients when the diagnosis had been made, patients whose thyroid function had been normalized with methimazole or propylthiouracil and the normal controls. FT3, FT4, TSH were measured from fasting plasma samples using immunoradiometric assay kit(Beijing Kewei Clinical Diagnostic Reagents Co.Ltd). CD3,CD4,CD69 were measured in whole blood by three-color flow cytometry(merck-calbiochem kit). Those results were analyzed by SPSS 11.0(T-test, ANOVA)
RESULTS
There was significant reduction in circulating CD69+ T cells in both methimazole group and propylthiouracil group at euthyroidism when compared with untreated GD patients (P<0.014,P<0.016,respectively), but this only reached statistical significance in the methimazole group (1) when it compared with control group (0), whereas the propylthiouracil group(3) whose circulating CD69+ T cells did not changed (Figure 1).
In the group(2) the concentrations of CD4+,CD69+ T cells increased as well as those of serum FT3 and FT4. However, once hyperthyroidism was controlled by the two different anti-thyroid drugs(group 1,group 3) CD4+, CD69+ T cells decreased followed in turn by reduction of serum FT3 and FT4 (Table 1).
Figure 1
Table 1 CD4+,CD69+ level after treatment
In group (3) the concentrations of circulating CD69+ T cells were negatively correlated with that of circulating CD4+ T cells when using the Regression Linear Analysis(P<0.05).
DISCUSSION
Data above demonstrated clearly that the hyperthyroid untreated patients with Graves' disease had an increased CD69 expression in peripheral blood which wasrelated in part to elevated thyroid hormone concentration and decreased CD69 expression which was related to a reduction in serum T4 and T3 only by methimazole. They also elucidated that the expression of CD4+ lymphocytes in untreated patients with GD increased when compared with both those of control subjects and patients with GD who had been treated with anti-thyroid drugs. These data were consistent with other previous observations [1,3].Our study demonstrated, for the first time in vivo, that the change of circulating CD69+ lymphocytes were significantly inversely correlated with the circulating CD4+ Tcells in propylthiouracil group when controlling FT3 and FT4 (F=10.707,P=0.005). PTU therapy induced rapid and specific changes within the CD3+ subsets, namely a reduction in the CD4+ subset and an increase in the CD8+ subset, resulting in a marked decrease in the CD4+/ CD8+ ratio[4].These suggested that increased CD69+ T cells and decreased CD4+ T cells in Graves' disease patients treated with propylthiouracil might modulate the clinical course of autoimmune thyroid disease[2].Recent researches found that the imbalance between effector and regulator T cells appeared to be crucial in the pathogenesis of GD[5] and not to be influenced by increased CD69 expression[5,7].And the naive CD4 T cells can differentiate into at least two different types of T helpers, Th1 and Th2 [8],both of the T-helper 2 and the Thelper 1 are involved in the pathogenesis of GD[9，10].Therefore, the decreased CD4+ T cells and the increased CD69+ T cells in GD patients treated with propylthiouracil confirmed that the proprylthiouracil can influence the immune system not as same as the methimazole does. Some researches demonstrated that intrathyroidal lymphocytes[10] from GD patients be accumulated in proximity to thyroid follicles and may change the local immune reaction, thus the increased CD69+ T cells and the decreased CD4+ T cells in intrathyroidal lymphocytes, moreover, may influence recurrence of GD [6，10，11，12].Although we did not detect intrathyroidal lymphocytes and CD8+ T cells, but the changes of peripheral blood CD4+ and CD69+ T cells in GD patients treated with propilthiouracil need to further study.
REFERENCE
1.Corrales JJ, Lopez A, Lissdad J, et al. Methimazole therapy in Graves' disease influences the abnormal expression of CD69( early activation antigen) on T cells. J Endocrinol, 1997,155(3): 491-500.
2. Wilson, Foster, Kronenberg, et al. Williams Textbook of Endocrinology.9th Edition.Saunders:Phildephia,1997,430-450.
3.Gessl A, Waldhausl W. Elevated CD69 expression on naive peripheral blood T-cells in hyperthyroid Graves' disease and autoimmune thyroiditis; discordant effect of methimazole on HLA-DR and CD69. Clin Immunol Immunopathol,1998,87(2):168-75.
4. Walfish PG, Tseng KH. Intrathyroidal activated (Ia+) T-lymphocyte CD+ subsets and B cells in Graves' hyperthyroidism respond rapidly to propylthiouracil therapy: demonstration using fine needle aspirates and two-colour laser flow cytometry. Autoimmunity,1992,13(1):35-41.
5.Saitoh O, Nagayama Y. Regulatin of Graves' Hyperthyroidism with Naturally Occurring CD4+CD25+Regulatory T Cells in a Mouse Model. Endocrinology,2006,147(5):2417-2422.
6.Sieg SF, Harding CV, Lederman MM. HIV-1 infection impairs cell cycle progreessin of CD4+ T cells without affecting early activation responses.J Clin Invest,2001,108(5):757-764.
7. Bohler T, Walcher J, Holzl-Wenig G, et al. Expression of CD69 on T-cells from HIV-1-infected children and adolescents increases with increasing viral load. Eur J Pediatr,1999,158(8):638-644.
8.Zhu J, Yamane H, Cote-Sierra J, et al. GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors. Cell Res,2006,16(1):3-10.
9.Aust G, Sitting D, Steinert M, et al. Graves`disease is associated with an altered CXCR3 and CCR5 expression in thyroid-derived compared to peripheral blood lymphocytes. Clin Exp Immunol,2002,127(3):479-485.
10. Aust G, Kamprad M, Lamesch P, et al.CXCR6 within T-helper and T-cytotoxic tipe1 lymphocytes in Graves`disease. Eur J Endocrinol,2005,152(4):635-643.
11. Nakamara Y, Wetanabe M, Matsuzuka F, et al. Intrathyroidal CD4+T lymphocytes express high levels of Fas and CD4+CD8+ macrophages/dendritic cells express Fas ligand in autoimmune thyroid disease.Thyroid,2004,14(10):819-24.
12. Green S, Pichyangkul S,Vaughn DW, et al. Early CD69 expression on peripheral blood lymphocytes from children with dengue hemorrhagic fever. J Infect Dis,1999,180(5): 1429-1435.
(Editor Emila)(JIA Wei, LI Zhi-guo，YANG )