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Departments of Endocrinology and Surgery (P.G.), Heinrich Heine University, D-40225 Dusseldorf, Germany; and German Diabetes Research Institute (J.S.), D-40225, Dusseldorf, Germany
Address all correspondence and requests for reprints to: Joachim Feldkamp, M.D., Department of Endocrinology, Heinrich Heine University, Moorenstrasse 5, D-40225 Dusseldorf, Germany. E-mail feldkamj{at}uni-duesseldorf.de
Abstract
In Hashimoto’s thyroiditis, Fas-induced apoptosis is one of the mechanisms leading to cell destruction, whereas thyroid tissue in Graves’ disease is prevented from it. The soluble form of the Fas molecule produced by alternative splicing prevents from apoptosis. We measured soluble Fas in the sera of 112 patients with Graves’ disease, 21 patients with toxic goiter, and 24 patients with subclinical hyperthyroidism due to suppressive therapy with levothyroxine after near-total resection of the thyroid gland for nodular goiter.
Soluble Fas was increased in thyrotoxic patients, toxic goiter, and patients with subclinical hyperthyroidism. Decreased levels of soluble Fas were found in euthyroid patients with Graves’ disease after surgery, whereas soluble Fas was normal in euthyroid patients with Graves’ disease receiving antithyroid drug treatment and in patients in stable remission. There was a good correlation between soluble Fas with free T3 (r = 0.6) and free T4 (r = 0.5). Our results show that soluble Fas is increased in hyperthyroidism independent of the underlying thyroid disease.
APOPTOSIS IS RARELY seen in thyroid tissue from patients with Graves‘ disease, whereas it is common in patients with Hashimoto’s thyroiditis (1, 2, 3). One of the underlying mechanisms of programmed cell death or the prevention of it involves the Fas/Apo 1 molecule (CD95) (4). This member of the TNF/nerve growth factor receptor family induces apoptosis via activation by its physiological ligand or by anti-Fas-specific antibodies (5, 6). The Fas system plays a major role in maintaining homeostasis in the immune system. Deletion of autoreactive T cells is mediated by the Fas/Fas ligand interaction (7, 8, 9, 10, 11). The Fas molecule is expressed by various tissues, including the thyroid gland (12).
Alternative splicing results in a soluble form of the Fas molecule (sFas) that lacks the transmembrane region (13). The killing mechanism of Fas functions via the membrane-bound Fas molecule, whereas sFas protects against apoptosis (14, 15). sFas was found in low concentrations in the sera of healthy subjects and at elevated concentrations in the sera of patients with autoimmune diseases, malignancy, and inflammatory disorders (16, 17, 18, 19, 20, 21). Shimaoka et al. reported elevated levels of sFas in patients with autoimmune thyroid diseases (20). Thyrotoxic patients with Graves’ disease had elevated sFas levels, whereas patients in remission and euthyroid patients with Hashimoto’s thyroiditis had decreased levels. These results were confirmed in one other study from Japan, and elevated levels have been documented in patients with Graves’ opthalmopathy (22, 23).
The aim of our study was to investigate the changes in serum levels of sFas in patients with Graves’ disease in different stages and after definitive treatment and in patients with nonautoimmune thyroid diseases.
Subjects and Methods
Subjects
sFas was measured in 112 patients (mean age, 43.2 ± 11.8
yr) with Graves’ disease recruited in our out-patient clinic (Table 1
). Five subgroups of patients could be
established. Thirty-six thyrotoxic patients (42.9 ± 13.0 yr) were
freshly diagnosed as having Graves’ disease and were untreated. Forty
euthyroid patients were taking stable doses of antithyroid drugs
(methimazole or carbimazole). To determine differences between patients
with regard to duration of therapy, 18 patients (41.9 ± 12.7 yr)
taking antithyroid drug medication for less than 6 months were compared
with 22 patients (42.5 ± 10.6 yr) treated for longer than 6
months (6–30 months). Sixteen patients (45.8 ± 15.9 yr) were in
stable remission without any therapy, and 20 patients (42.9 ±
12.9 yr) had undergone surgery for Graves’ disease at least 6 months
previously and were receiving substitution therapy with levothyroxine.
Graves’ disease was diagnosed by suppressed levels of TSH and elevated
levels of free T3 (fT3) and
free T4 (fT4), increased
99mTc uptake, and positive TSH receptor
antibodies.
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A group of 36 healthy blood donors (mean age, 39.7 ± 11.1 yr) served as controls. All patients without Graves’ disease and all controls were negative for TSH receptor antibodies.
All serum samples were frozen and stored at -20 C until the determination of sFas.
The study was approved by the local ethic committee. All patients and controls gave informed consent.
Determination of sFas
sFas was determined by a nonisotopic sandwich immunoassay (Oncogene Research Products, Cambridge, MA). In brief, a monoclonal mouse antibody, specific for the human Fas protein, is immobilized onto the surface of plastic wells. Serum (100 µl), diluted 1:10, or standards are pipetted into the wells and incubated for 1 h. After washing, a biotinylated detector monoclonal antibody is added and incubated for 1 h. After another washing, this detector antibody, in turn, is bound by horseradish peroxidase-conjugated streptavidin, which catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine from a colorless to a yellow solution after the addition of a stopping reagent. The colored reaction product is quantified by measuring absorbance at dual wavelengths of 450/540 nm. The sFas concentration is determined by interpolation after preparing a standard curve from sFas standards.
fT3, and fT4, and TSH were determined using a commercially available luminescence immunoassay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The normal ranges for fT3 was 2.93–6.16 pmol/liter, and that for fT4 was 1.16–2.7 pmol/liter.
TSH receptor antibodies were measured by RIA (Medipan, Germany), with normal values less than 9 IU/liter. Antibodies against thyroid peroxidase (normal, <100 U/liter) and Tg (normal, <200 U/liter) were determined by RIA (B.R.A.H.M.S., Berlin, Germany). The intra- and interassay coefficients of variation, respectively, were 7.4% and 4.8% for sFas, 5.2% and 6.5% for fT3, 3.9% and 10.3% for fT4, 4.2% and 9.3% for TSH (third generation assay), and 7.8% and 9.7% for TSH receptor antibodies.
Statistics
All variables were analyzed for normal distribution (Kolmogorov-Smirnov test). Data are expressed as the mean ± SE unless otherwise stated. Analyses with more than two variables were analyzed using a standard ANOVA with Scheffé’s test, with post-hoc comparison. Pearson’s correlation was used to assess the strength of association between values with normal distribution. Values not normally distributed were calculated using Spearman’s rank correlation test. P < 0.05 was considered significant. Statistical analyses were performed using StatView (Abacus Concepts, Berkeley, CA).
Results
Compared with controls, thyrotoxic patients with GD, patients with
toxic goiter, and patients with subclinical hyperthyroidism had
significantly higher levels of fT3 and
fT4 (Table 1
), but patients with subclinical
hyperthyroidism had significantly (P < 0.01) lower
levels of fT3 and fT4
compared with thyrotoxic patients.
In normal healthy controls the mean serum level of sFas was 6.35
± 1.6 IU/ml (Fig. 1). No
significant difference was found between men (n = 14) and women
(n = 22). In thyrotoxic patients with Graves’ disease sFas was
significantly increased (9.1 ± 2.2 IU/ml) compared with levels in
controls and patients receiving antithyroid drug treatment
(P < 0.001 for both). In euthyroid patients who had
been treated with antithyroid drugs for less than 6 months sFas was
increased (6.5 ± 1.02 IU/ml) compared with that in patients
receiving long-term treatment (5.41 ± 0.98 IU/ml;
P = 00.2), but was significantly lower
(P < 0.001) than that in newly diagnosed patients. No
significant difference could be seen when all patients receiving
antithyroid drug treatment were compared with controls. Additionally,
no significant difference existed between sFas levels of patients in
remission (5.87 ± 1.08 IU/ml) and controls, but patients who
underwent surgery had significantly decreased (P =
0.02) sFas values (5.35 ± 1.1 IU/ml). sFas was significantly
increased (P < 0.001) in thyrotoxic patients
(10.1 ± 1.76 IU/ml) with toxic goiter compared with all other
groups, with the exception of thyrotoxic patients with Graves’
disease. Patients with subclinical hyperthyroidism had significantly
lower sFas levels (7.7 ± 1.63 IU/ml) than thyrotoxic patients
(Graves’ disease and toxic goiter), but significantly increased values
compared with controls (P < 0.001).
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). A correlation existed also between
fT3 (r = 0.19) and fT4
(r = 0.2), respectively, and TSH receptor antibodies (data not
shown). sFas correlated with fT3 (r = 0.6;
P < 0.01; Fig. 3) and
with fT4 (r = 0.5; P <
0.01). In the subgroups of patients there was also a significant
(P < 0.01) correlation between sFas and free
T3 (r = 0.51 for all patients with Graves’
disease; r = 0.58 for patients with toxic goiter; r = 0.3 for
patients with subclinical hyperthyroidism; r = 0.3 for
controls).
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The clinical significance of soluble forms of the human Fas molecule is poorly understood. Various conditions may lead to increased levels of sFas in human serum (16, 17, 18, 19, 20, 21, 22, 23). The intact Fas molecule consists of an intracellular, a transmembrane, and an extracellular domain. A region called the death domain, which shows homology to TNF receptors, is required to propagate its apoptotic signal (4). Activation of the Fas molecule either by the Fas ligand or by specific antibodies leads to cell destruction, which is mediated by a family of cysteine proteases (caspases) (24).
In human thyroid tissue the Fas antigen is functionally expressed on the surface of thyrocytes (25, 26, 27, 28, 29). The role of the Fas system in different autoimmune thyroid diseases may be either in promoting apoptosis or protecting the follicle cell from programmed cell death. In Hashimoto’s thyroiditis Fas-mediated apoptosis has been observed and is regulated by various cytokines (26, 28, 30, 31, 32, 33, 34, 35, 36).
In thyroid tissues from patients with Graves’ disease apoptosis is a rare event, probably due to increased expression of the Bcl-2 protein, which exerts a high antiapoptotic activity (1). As the soluble form of the Fas molecule has been shown to prevent apoptosis, it could be expected that sFas might be increased in serum of patients with Graves’ disease (14). Recently, it has been demonstrated that sFas was increased in thyrotoxic patients and decreased in patients in remission (20).
In our own study all patients with elevated levels of fT3 and fT4 and patients with suppressed TSH had increased sFas values. This was true for patients with autoimmune Graves’ disease as well as for patients with nonautoimmune thyroid disease (toxic goiter). Patients with subclinical hyperthyroidism due to suppressive doses of levothyroxine had increased sFAs levels, but did not attain the levels in patients with frank hyperthyroidism. This suggests that increases in sFas levels are much more a consequence of hyperthyroidism itself than a sequel of autoimmune thyroid diseases. This was confirmed by a good correlation of sFas with fT3 and fT4 in the overall group of patients and in all subgroups. Shimaoka et al. reported a high correlation of sFas with TSH receptor antibodies and speculated that sFas levels might be dependent on activation of the autoimmune process (20). If sera with negative TSH receptor antibodies were removed in their study, there was no longer a correlation between TSH receptor antibodies and sFas. In our own cohort of patients with Graves’ disease, TSH receptor antibodies correlated much weaker with sFas (r = 0.25).
In conclusion, the elevated sFas levels in hyperthyroidism seem to be dependent on the extent of hyperthyroidism. It cannot be ruled out that minor changes in sFas levels might be attributed to autoimmune thyroid diseases. Shimaoka (20) reported decreased sFas levels in euthyroid patients with Hashimoto’s thyroiditis, whereas they were normal in hypothyroid patients with the same disease, but a total of only 32 patients were investigated. We did not have the opportunity to determine sFas levels in hypothyroid subjects due to the very small number of cases observed during the study period.
One of the main functions of the Fas/Fas ligand system is the deletion
of activated B and T cells. It can be speculated from the results of
our study that thyroid hormones exert direct effects on the immune
system via activation of the Fas/Fas ligand system. Whether this leads
in consequence to a reduced rate of Fas-induced apoptosis of target
cells via protection by sFas or to an increase in apoptotic events by
up-regulation of the membrane-bound Fas molecule is speculative. Other
cofactors, such as cytokines (IL-1, interferon-
, and others), may
modulate this system, and further investigations are necessary to
elucidate the role of thyroid hormones in apoptotic processes. The
source of sFas in patients with hyperthyroidism is lymphocytes rather
than thyrocytes, as patients with only very small thyroid remnants and
subclinical hyperthyroidism after surgery also had elevated sFas
values. Our results are in concordance with other observations showing
an influence of hyperthyroidism on the immune system (37, 38). Similar results have been shown for serum levels of the
soluble IL-2 receptor and adhesion molecules such as intracellular
adhesion molecule-1, indicating the interaction between thyroid
hormones and the immune system (39, 40).
Acknowledgments
We thank T. B. West for critically reading the manuscript.
Footnotes
Abbreviations: fT3, Free T3; fT4, free T4; sFas, soluble Fas.
Received October 11, 2000.
Accepted May 8, 2001.
References
chain. Endocr J 46:437–442[Medline]
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