Lower concentration of serum soluble CD8 in severe Hashimoto's disease

Abstract

To investigate the roles of soluble CD4 (sCD4) and CD8 (sCD8) in the severity of autoimmune thyroid diseases, we examined serum concentrations of sCD4 and sCD8 in various degrees of severity of Hashimoto's disease (HD) and Graves’ disease (GD) by enzyme immunoassay. The serum concentration of sCD8 was lower in euthyroid patients with HD undergoing treatment for hypothyroidism (severe HD) than in untreated, euthyroid patients with HD (mild HD), but the sCD4 concentration did not differ between patients with severe and mild HD. The serum sCD8 concentration was negatively correlated with the proportion of CD25⁺ cells in CD8⁺ cells in patients with severe HD. Serum sCD4 and sCD8 concentrations did not differ between euthyroid patients with GD in remission and those with intractable GD. These results indicate that serum sCD8 is involved in the severity of HD, possibly by down-regulating the function of cytotoxic T cells.

INTRODUCTION

About 10% of women and 5% of men are positive for antimicrosomal antibodies and are diagnosed with an autoimmune thyroid disease (AITD) such as Hashimoto's disease (HD) or Graves’ disease (GD) [1], both of which are typical organ-specific autoimmune diseases [2,3]. Some patients with HD develop hypothyroidism, but most do not. Some patients with GD treated with antithyroid drugs achieve remission with disappearance of anti-TSH-receptor antibodies, but others do not. The immunological differences between patients with different degrees of disease severity or intractability remain unclear, but we have shown that particular lymphocyte subsets are related to the severity of HD and GD [4–6]. However, there have been few other studies showing immunological differences between these patients.

CD4 and CD8 molecules are members of the immunoglobulin gene superfamily and express as membrane-bound or soluble isoforms [7]. Membrane-bound CD4 and CD8 proteins on CD4⁺ and CD8⁺ cells, both of which proteins are important for immune regulation, are required for interaction with antigen-presenting cells or target cells to induce specific immune responses [7]. The soluble isoforms of these proteins are produced by proteolysis of membrane-bound CD4 and CD8 molecules on T cells or by alternative splicing of the CD4 and CD8 genes [7,8]. Serum concentrations of sCD4 and sCD8 have been measured in the presence of various autoimmune and infectious diseases, and these proteins are believed to participate in immune regulation [9–13]. Interestingly, we have found opposite changes in serum sCD8 in the thyrotoxic stages of HD and GD [14].

In this study, we examined the serum concentrations of sCD4 and sCD8 in euthyroid patients with different severity of HD and GD to clarify the significance of these molecules in relation to disease severity.

MATERIALS AND METHODS

Subjects

The study group comprised 52 patients with HD, 66 patients with GD (Table 1), and 32 healthy volunteers who served as normal controls. HD was diagnosed by antithyroid microsomal antibody (MCPA) and/or antithyroglobulin antibody (TGPA) positivity, a normal TSH-receptor antibody (TRAb) level, and the presence of diffuse goitre. Patients with HD were divided into two groups: 28 euthyroid patients being treated with thyroxine (severe HD) (7 men and 21 women, 55·8 ± 6·8 years of age, mean ± SD) and 24 untreated, euthyroid patients (mild HD) (2 men and 22 women, 54·8 ± 8·6 years of age). GD was diagnosed by TRAb elevation, a history of thyrotoxicosis, and the presence of diffuse goitre. Patients with GD were divided into three groups: 13 untreated, thyrotoxic patients (untreated GD) (3 men and 10 women, 42·5 ± 11·1 years of age), 30 euthyroid patients who had been undergoing antithyroid drug treatment for more than 5 years but were still positive for TRAb (intractable GD) (2 men and 28 women, 44·8 ± 15·0 years of age), and 23 patients in remission who had maintained a euthyroid state and were negative for TRAb for more than 1 years without any treatment (GD in remission) (23 women, 47·2 ± 14·4 years of age). The control group comprised of 32 healthy volunteers (32 women, 45·3 ± 6·9 years of age) who were euthyroid and negative for thyroid autoantibodies. The patients in both HD groups were significantly older than all patients with GD and the control subjects. Venous blood was sampled between 1000h and 1200h. in an EDTA tube for flow cytometry, and in a plain tube for other tests. Informed consent for study participation was obtained from all patients and controls. The study protocol was approved by our local Ethics Committee.

Table 1.

Clinical characteristics of patients with Hashimoto's disease and Graves’ disease

		Hashimoto's disease (HD)		Graves’ disease (GD)
	Euthyroid		Euthyroid		Thyrotoxic
	Severe n = 28	Mild n = 24	Intractable n = 30	In remission n = 23	Untreated n = 13
Age (years)	55·6 ± 6·8	54·8 ± 8·6	44·8 ± 15·0	47·2 ± 14·4	42·5 ± 11·1
FT3 (pg/ml)	3·18 ± 0·42	3·03 ± 0·32	3·38 ± 1·13	3·11 ± 0·38	13·25 ± 8·05*
TSH (µU/ml)	1·21 ± 1·15§	2·70 ± 1·55	0·96 ± 1·30	1·48 ± 0·72	N.D.
TRAb (%)			33·1 ± 26·9‡	1·6 ± 4·2	47·4 ± 22·1

Data are mean ± SD. FT3, free triodothyronine; TSH, thyroid-stimulating hormone; TRAb, anti-TSH receptor antibody; N.D., not detected.

^*P < 0·05 versus euthyroid GD;

^‡P < 0·01 versus GD in remission;

^§P < 0·05 versus mild HD.

Serum concentrations of sCD4 and sCD8

Serum sCD4 and sCD8 concentrations were measured with available ELISA kits (Cellfree™ sCD4 test kit and Cellfree™ sCD8 kit, Endogen, Inc., Woburn, MA, USA) according to the manufacturer's instructions. Both assays were sandwich ELISAs that used two monoclonal antibodies, the second being peroxidase-conjugated. Briefly, 96-well microplates were coated with a murine monoclonal antibody to either CD4 or CD8. Sera were applied to the plates directly (for sCD4) or at a final dilution of 1 : 10 (for sCD8) and incubated. The plates were washed and incubated again with horseradish peroxidase (HRP)-conjugated murine monoclonal antibodies directed against different epitopes of human CD4 or CD8. After unbound HRP-conjugate antibodies were removed by a washing, o-phenylenediamine (for sCD4) or tetramethylbenzidine (for sCD8) was added to each well, and the plates were again incubated. The reaction was stopped by sulphuric acid stop-solution, and absorbance was measured at 490 nm (for sCD4) or 450 nm (for sCD8). The results, expressed as U/ml, were determined from standard curves plotted with data from six standard sCD4 samples (ranging from 0 to 230 U/ml) and five standard sCD8 samples (ranging from 0 to 866 U/ml).

Monoclonal antibodies

Fluorescein-isothiocyanate (FITC)-conjugated anti-CD8 (Becton Dickinson, Mountain View, CA, USA), phycoerythrin (PE)-conjugated anti-CD25 (Becton Dickinson), and Peridinin Chlorophyll Protein (PerCP)-conjugated anti-CD4 monoclonal antibodies (Becton Dickinson) were used.

Lymphocyte subsets

Samples of 100 µl of EDTA-treated whole blood were incubated for 30 min at 4°C with 20 µl of each antibody. The samples were then haemolysed and fixed with lysing reagent (FACS™ Lysing Solution; Becton Dickinson). They were then washed once and subjected to three-colour flow cytometry in FACSCalibur™ (Becton Dickinson) to determine the percentage of lymphocyte subsets.

Thyroid function test and thyroid autoantibodies

Serum concentration of free T₃ (FT₃) (normal, 2·4–4·6 pg/ml; 3·8–7·2 pmol/l) and TSH (normal, 0·6–5·4 µU/ml) were measured with a radioimmunoassay kit (Japan Kodak Diagnostic Co., Ltd, Tokyo; Daiichi Radioisotope Laboratories, Tokyo, respectively). MCPA and TGPA were measured with a particle agglutination kit (Fuji Rebio, Tokyo, Japan). A reciprocal titre of 1 : 100 or more was considered positive. Serum TRAb was measured by radioreceptor assay with a commercial kit (Cosmic Corporation, Tokyo, Japan) and expressed as percent inhibition of labelled TSH binding. The normal value is less than 10%.

Statistical analysis

We used Student's t-test to analyse differences between two groups and one-way analysis of variance followed by Scheffe's test to analyse differences between three or more groups. Correlation was established with Pearson's correlation coefficient. Probability values ≤0.05 were considered significant.

RESULTS

Serum concentrations of sCD4 and sCD8

Serum sCD4 concentrations also did not differ significantly between euthyroid patients with severe HD and those with mild HD (Fig. 1a), whereas serum sCD8 concentrations were significantly lower in severe HD patients than in mild HD patients (P < 0·01)(Fig. 1b). Serum sCD8 concentrations (203·5 ± 49·7 U/ml) in age-matched healthy control subjects (11 women, age; 51·3 ± 2·2 years old) were not significantly different from those in either group of HD patients (Fig. 1b). Serum concentrations of sCD4 and sCD8 did not differ significantly between patients with intractable GD and those with GD in remission (Figs 1a,b). Serum sCD4 concentration (10·6 ± 3·4 U/ml) and sCD8 concentration (203·5 ± 49·7 U/ml) in age-matched healthy control subjects were not significantly different from these in either group of GD patients (Fig. 1a,b). Serum concentrations of both sCD4 (24·1 ± 17·3 U/ml) and sCD8 (302·5 ± 88·3 U/ml) were significantly higher in thyrotoxic GD patients than in other euthyroid GD patients and in age-matched normal control subjects.

Fig. 1.

Serum concentrations of (a) sCD4 and (b) sCD8 in patients with Hashimoto's disease and Graves’ disease, and in age-matched healthy control subjects.

Correlations between serum sCD4 (sCD8) concentrations and proportion of CD25⁺ cells in CD4⁺ (CD8⁺) lymphocyte subsets

In the CD4⁺ cells, the proportions of CD25⁺ cells were not significant different between each subject. In the CD8⁺ cells, the proportions of CD25⁺ cells were significantly increased in patients with severe HD (n = 28, 11·2 ± 7·8%, P < 0·05) than those of mild HD (n = 24, 7·5 ± 5·0%), as well as our previous report [4]. There was no significant correlation between the serum sCD4 concentration and the proportion of CD25⁺ cells in CD4⁺ cells. However, there was a significant negative correlation between serum sCD8 concentration and the proportion of CD25⁺ cells in CD8⁺ cells in patients with severe HD (n = 28, r = −0·43, P < 0·02) (Fig. 2).

Fig. 2.

Correlation between the serum concentration of soluble CD8 and the proportion of CD25⁺ cells in peripheral CD8⁺ lymphocytes in patients with severe Hashimoto's disease.

Correlation among serum concentrations of sCD4, sCD8, and thyroid hormones

There was no significant correlation between the serum concentrations of sCD4 and sCD8 in any GD or HD patient group. In all GD patients (n = 66), serum sCD4 and sCD8 concentrations were significantly correlated with serum FT₃ concentrations (r = 0·53, P < 0·001, and r = 0·59, P < 0·001, respectively), and serum sCD8 was significantly correlated with serum TRAb concentration (r= 0·29, P < 0·05). In thyrotoxic GD patients (n = 13), serum sCD4 and sCD8 concentrations correlated significantly with serum FT₃ concentrations (r= 0·71, P < 0·01, and r = 0·62, P < 0·05, respectively).

DISCUSSION

In this study, we found the serum sCD8 concentration to be lower in patients with severe HD than in patients with mild HD. Furthermore, we found a significant negative correlation between the serum concentration of sCD8 and the proportion of activated cytotoxic T cells (CD25⁺CD8⁺ cells) in patients with severe HD. These findings suggest that sCD8 down-regulates the function of CD8⁺ cytotoxic T cells and prevent the progress of hypothyroidism in HD patients, and that the decrease of sCD8 may increase activated cytotoxic T cells and aggravate thyroid destruction in HD patients. These possibilities are consistent with the previous reports indicating that sCD8 antagonizes CD8⁺ T cell activation and down-regulates the function of cytotoxic T cells in vitro [15], that cytotoxic T cells are involved in thyrocyte destruction in HD patients [16], and that the proportions of activated cytotoxic T cells (CD25⁺CD8⁺ cells) were higher in patients with severe HD than in those with mild HD [4].

Serum concentrations of sCD4 and sCD8 do not differ significantly between patients with intractable GD and those with GD in remission. This indicates that neither sCD4 nor sCD8 is related to the severity or intractability of GD. On the other hand, in untreated and thyrotoxic patients with GD, the serum concentrations of sCD4 and sCD8 were significantly increased and significantly correlated with the concentrations of thyroid hormones. However, thyrotoxicosis itself dose not always cause an increase of serum sCD8 [14,17], Furthermore, sCD8 down-regulates the function of cytotoxic T cells [15] which produce inflammatory cytokines such as interferon-γ [18,19]. Therefore, an increase of sCD8 may suppress inflammatory cytokines which inhibit the function of thyrocytes [20,21], provide the circumstances in which thyrocytes are easily stimulated by TRAb, and create a vicious circle of thyrotoxicosis in GD. This possibility may also well explain the previous reports that serum sCD8 increases well before the relapse of thyrotoxicosis in cases of GD [17] and that a high level of sCD8 predicts failure to respond to antithyroid drug therapy in GD patients [22]. It is also interesting that the amount of sCD8 produced by thyroid-infiltrating lymphocytes is greater than that produced by peripheral lymphocytes in patients with GD [23].

Serum concentrations of sCD4 decrease dramatically during pregnancy [24] and increase in patients with infectious mononucleosis and some autoimmune diseases [10,11,25–28], as well as in thyrotoxic GD patients. Some autoimmune diseases ameliorate during pregnancy [29], but, the significance of serum sCD4 concentration in AITD was not clarified in this study.