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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 4 1378-1385
Copyright © 1999 by The Endocrine Society

Original Studies

Effects of Thyroid Hormones on Apoptotic Cell Death of Human Lymphocytes1

Shoji Mihara, Noboru Suzuki, Sueshige Wakisaka, Satoshi Suzuki, Noriaki Sekita, Shoso Yamamoto, Nobuhiko Saito, Takashi Hoshino and Tsuyoshi Sakane

Departments of Immunology and Medicine (S.M., N.S., S.W., T.H., T.S.) and Internal Medicine (S.W., S.S., N.Se., N.Sa.), St. Marianna University School of Medicine, Kawasaki, Kanagawa; and the Department of Dermatology, Hiroshima University School of Medicine (S.M., S.Y.), Hiroshima, Japan

Address all correspondence and requests for reprints to: Dr. Tsuyoshi Sakane, Departments of Immunology and Medicine, St. Marianna University School of Medicine, 2–16-1 Sugao, Miyamae-ku, Kawasaki, Kanagawa 216-8511, Japan.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Apoptosis plays a critical role in the development and homeostasis of tissues, especially those with high cell turnover such as the lymphoid system. We have examined the effects of thyroid hormones, TSH and TRH, on apoptosis of human T lymphocytes. We found that T lymphocytes cultured with T3 and T4, but not TSH nor TRH, in vitro showed enhanced apoptosis, evidenced by DNA ladder formation and characteristic morphological changes. In addition, prolonged cultivation with thyroid hormones of the lymphocytes further enhanced the extent of apoptosis.

We also found that treatment with thyroid hormones of T lymphocytes induced reduction of mitochondrial transmembrane potential ({Delta}{Psi}) and production of reactive oxygen species, both of which are intimately associated with apoptotic cell death. In addition, cellular expression of antiapoptotic Bcl-2 protein was clearly reduced by the treatment of lymphocytes with thyroid hormones in vitro. Thus, T lymphocytes treated with thyroid hormones accompany reduction of Bcl-2 protein expression, production of reactive oxygen species, and reduction of mitochondrial {Delta}{Psi}, resulting in apoptotic lymphocyte death. Moreover, we found that lymphocytes in patients with Graves’ disease showed enhanced apoptosis compared with those in normal individuals. These results suggest that thyroid hormones have the potential to induce apoptotic cell death of human lymphocytes in vivo and in vitro.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
APOPTOSIS plays a critical role in the development and homeostasis of multicellular organisms (1, 2, 3, 4, 5). An increased rate of apoptosis is involved in the pathogenesis of several degenerative diseases (6, 7, 8, 9). Conversely, inhibition of apoptosis has been implicated in autoimmune diseases and carcinogenesis (8, 9, 10). It is now clear that members of the cysteine proteases encoded by the mammalian ICE gene family play a key role in driving apoptosis (11, 12, 13). To date, 10 homologs of human ICE protease, which are now named caspase-1 to -10, have been reported (14). On the other hand, the protooncogene bcl-2 has the ability to inhibit apoptosis of various cell types induced by a variety of stimuli (15, 16).

Glucocorticoid (GC), thyroid hormone, and retinoic acid contribute to vertebrate development and homeostasis by serving as biological signals to control cellular functions, including cell growth and cell death (17, 18, 19). GC receptor, thyroid hormone receptor, and retinoic acid receptor, members of the GC-thyroid hormone-retinoic acid receptor family, share many characteristics for exerting their actions on gene transcription. Upon binding to their respective hormones, intracellular receptors for these hormones act as dimeric transcription factors to activate or repress expression of nuclear target genes by binding to specific DNA sequences termed hormone response elements (20, 21).

It is well known that thyroid hormones contribute to the development and maintenance of homeostasis in multicellular organisms to control cell growth and differentiation (22, 23). Thyroid hormones, for example, influence mammalian central nervous system morphogenesis (24). In addition, cell death during amphibian metamorphosis (involving apoptosis) is under the control of thyroid hormones (25, 26, 27). It has been reported that thyroid hormone induces apoptotic cell death of differentiating erythrocytic progenitor cells (24). On the contrary, thyroid hormone inhibits apoptosis of early differentiating cerebellar granule neurons by inducing enhanced expression of antiapoptotic Bcl-2 protein (28).

GC and retinoic acid have been shown to induce apoptotic cell death of many cell types, including leukocytes and lymphocytes (29, 30, 31, 32). Regardless of the structural and functional similarities of the GC-thyroid hormone-retinoic acid receptor family, it is not clear whether the thyroid hormone/thyroid hormone receptor complex mediates apoptosis of peripheral blood lymphocytes (PBL) in humans.

In the present study, we have investigated the effects of thyroid hormones on human lymphocyte apoptosis. We show here that thyroid hormones induce apoptotic cell death of a human T cell line and circulating lymphocytes.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients and cells

Ten patients with Graves’ disease (3 males and 7 females) who had goiter with high titer of total T3 (mean ± SD, 5.96 ± 4.40 nmol/L; normal, 1.1–2.9 nmol/L) and total T4 (299 ± 209 nmol/L; normal, 77–170 nmol/L) and low titer of TSH (all <0.1 mU/L; normal, 0.4–4.7 mU/L) were studied. Their mean age ± SD was 31.0 ± 5.3 yr. Lymphocytes from 10 normal volunteer donors (8 males and 2 females; mean age ± SD, 34.7 ± 9.5 yr) were also studied. PBL were isolated by centrifugation of the heparinized blood through Ficoll/Hypaque (Nakarai Tesque, Kyoto, Japan). T lymphocytes were purified using the neuraminidase-treated sheep red blood cell rosette technique (33). CD4+ T lymphocytes and CD8+ T lymphocytes were separated using the magnetic bead method as previously reported (34). Lymphocytes were cultured in RPMI 1640 (Nikken Bio Medical Laboratory, Kyoto, Japan) supplemented with 10% FCS, penicillin (100 U/mL), and streptomycin (100 µg/mL; the latter three from Life Technologies, Gaithersburg, MD), followed by analysis of apoptotic cell death. Original Jurkat cells were weaned from RPMI 1640 containing 10% FCS and were cultured in Nutridoma serum-free medium (Boehringer Mannheim, Mannheim, Germany) to avoid possible influences of various hormones present in FCS. The Jurkat cells, termed Jurkat-NU cells, were cultured in medium with T3, T4, TSH, and TRH (all from Sigma Chemical Co., St. Louis, MO) for the indicated periods. One half-volume of the medium containing the hormones was exchanged every other day.

Western blotting analysis

The cell pellets were lysed in PBS containing 0.1% Nonidet P-40 (Sigma Chemical Co.) and protease inhibitors (35). The samples were spun at 15,000 rpm for 20 min at 4 C. Supernatants were harvested, and equivalent amounts of proteins from the Jurkat-NU cells or the PBL were resolved by 4–20% SDS-PAGE under reducing conditions. The proteins were transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) and were blocked with 2.5% BSA overnight. The blots were probed with the appropriate first antibody described below, followed by biotin-labeled goat antimouse IgG antibody and peroxidase-conjugated streptavidin. Detection was carried out using the enhanced chemiluminescence kit (Amersham International, Aylesbury, UK). The intensity of the detected bands was analyzed with gel-plotting macros in NIH Image 1.55 software. Anti-Bcl-2 (Dako Corp., Glostrup, Denmark) and anticaspase-3 (CPP32) (Transduction Laboratories, Inc., Lexington, KT) were used in this study.

Apoptosis assays

The extent of apoptotic cell death was estimated by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling (TUNEL) method (36). DNA staining with propidium iodide (PI) (37) and DNA ladder formation (38) and microscopic analysis are described below.

To detect DNA strand breaks, which are intimately associated with an apoptotic response, an in situ cell death detection kit (Boehringer Mannheim, Mannheim, Germany), where nicked DNA of fixed cells were labeled by the incorporation of fluorescein-conjugated deoxy-UTP at strand breaks by terminal deoxynucleotidyl transferase, was used (36). The labeled cells were analyzed using a flow cytometer (36, 39).

The reduced DNA content of apoptotic nuclei appeared as a broad hypodiploid DNA peak in the red fluorescence channels after PI staining. Analysis of hypodiploid DNA was performed as previously described (37). Briefly, the cell pellet was resuspended in hypotonic fluorochrome solution [50 mg/mL PI (Wako, Osaka, Japan) in 0.1% sodium citrate (Wako) plus 0.1% Triton X-100 (Sigma Chemical Co.)] at 4 C in the dark overnight. The PI fluorescence of isolated nuclei was measured, and the percentage of apoptotic nuclei (subdiploid DNA peak in the DNA fluorescence histogram) was calculated using the Consort 30 program (39).

Gel electrophoresis was used to determine nucleosomal DNA fragmentation (39). Jurkat-NU cells were cultured for 2 weeks with or without thyroid hormones. Thereafter, total cellular DNA was recovered using a standard procedure (38). To observe morphological changes, Jurkat-NU cells treated with or without thyroid hormones were cultured for 2 weeks. Cytospin preparations were made and then stained with Diff-Quick Solution (International Reagents Corp., Kobe, Japan).

Flow cytometric analysis of mitochondrial {Delta}{Psi} and ROS generation

Mitochondrial {Delta}{Psi} and superoxide anion (O2-) generation were measured as previously described (40, 41, 42, 43, 44). Briefly, cells (5 x 105) were incubated with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3), 40 nmol/L in PBS; Molecular Probes, Inc., Eugene, OR] and dihydroethidine (HE; 2 µmol/L; Molecular Probes, Inc.) for 15 min at 37 C. In the control experiments, cells were stimulated with a radical-generating agent, menadione (1 mmol/L, 37 C, 30 min; Sigma Chemical Co.), or an uncoupling agent, carbonyl cyanide m-chlorophenylhydrazone (50 µmol/L, 37 C, 30 min; Sigma Chemical Co.). Thereafter, the cells were analyzed on a flow cytometer to measure mitochondrial {Delta}{Psi} and ROS (O2-) generation.

Statistical analysis

Analysis of the percent apoptosis of thyroid hormone-treated Jurkat-NU cells was performed using Dunn’s test. Analysis of the percent apoptosis of thyroid hormone-treated PBL, T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes was performed using Student’s t test. The difference in the extent of lymphocyte apoptosis between patients with hyperthyroidism and normal individuals was compared using Mann-Whitney’s U test. In the statistical analysis, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Effects of thyroid hormones on lymphocyte apoptosis

We first studied the effects of T3, T4, TSH, and TRH on apoptosis of a T cell line, Jurkat cells. To avoid the possible influences of various hormones contained in FCS, we used a serum-free culture medium that is totally free from contaminating hormones. We designated these Jurkat cells as Jurkat-NU cells. Jurkat-NU cells were cultured in the serum-free medium with T3 and T4. The extent of apoptosis was estimated by the TUNEL method, which detects DNA strand breaks of apoptotic cells. We found that Jurkat-NU cells cultured for 2 weeks with T3 and T4 showed enhanced apoptosis compared to the cells without thyroid hormones (T3, P < 0.01 vs. medium; T4, P < 0.01 vs. medium; Fig. 1Go). Thus, it is suggested that T4 as well as T3 are potent inducers of lymphocyte apoptosis. The apoptosis inducing potential of T4 and T3 was confirmed using DNA staining with PI, which is a rapid and reliable method for detecting apoptotic cells with a flow cytometer (Fig. 2AGo) (37). TSH and TRH, on the other hand, did not show any effect on lymphocyte apoptosis in this culture system (Fig. 2BGo). Furthermore, morphological analysis, which showed characteristic apoptotic bodies and nuclear fragmentation (Fig. 3Go) and DNA ladder formation (Fig. 4Go) of Jurkat-NU cells, confirmed the apoptotic nature of thyroid hormone-induced lymphocyte death. These results indicate that thyroid hormones, including T3 and T4, induce apoptotic cell death of a T lymphocyte cell line.



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  		Figure 1. Analysis of apoptotic cell death of Jurkat-NU cells induced by thyroid hormones. Jurkat-NU cells were cultured with T3 and T4 for 2 weeks. Apoptosis of Jurkat-NU cells cultured in the presence of various concentrations of T3 and T4 was estimated using the TUNEL method. T3 (among all concentrations tested) and T4 (from 10-9-10-7 mol/L) induced apoptosis of Jurkat-NU cells in a dose-dependent manner. We obtained similar results in three independent experiments. Data presented are the mean ± SEM of triplicate samples. Dunn’s test was used for statistical comparison. **, P < 0.01 vs. medium.

 


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  		Figure 2. Analysis of apoptotic cell death of Jurkat-NU cells cultured with thyroid hormones, TSH and TRH, using the PI staining method. A, Apoptosis of Jurkat-NU cells cultured in the presence of various concentrations of T3 and T4 was confirmed using the PI staining method. T3 and T4 induced apoptosis of Jurkat-NU cells. We obtained similar results in nine independent experiments. Data presented are the mean ± SEM of triplicate samples. Dunn’s test was used for statistical comparison. **, P < 0.01 vs. medium. B, Apoptosis of Jurkat-NU cells cultured in the presence of various concentrations of TSH and TRH was estimated using the PI staining method. Treatment of cells with TSH and TRH did not induce apoptosis of Jurkat-NU cells. We obtained similar results in three independent experiments. Data presented are the mean ± SEM of triplicate samples.

 


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  		Figure 3. Microscopic examination of apoptotic Jurkat-NU cells cultured with T3 and T4. Jurkat-NU cells were cultured in the presence of T3 (10-8 mol/L) and T4 (10-6 mol/L) for 2 weeks. Cytospin preparations of the samples were stained with Diff-Quick solution. T3- and T4-treated Jurkat-NU cells showed characteristic microscopic features of apoptotic cells, with fragmented nuclei (arrows) and apoptotic body (arrowhead). Magnification, x100.

 


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  		Figure 4. DNA ladder formation in T3- and T4-treated Jurkat-NU cells. Jurkat-NU cells were cultured in the presence of T3 (10-8 mol/L) and T4 (10-7 mol/L) for 2 weeks. Cellular DNA was extracted and subjected to electrophoresis on a 2.0% agarose gel to detect the nucleosomal DNA ladder.

 
To study the effects of thyroid hormones on apoptosis of circulating lymphocytes, PBL, T lymphocytes, and T cell subsets from healthy volunteers were cultured with or without thyroid hormones for 5 days in 10% FCS-RPMI 1640 medium. Cultivation of normal PBL for more than 6 days in this medium induced spontaneous lymphocyte apoptosis. Thus, we used 5 days of culture where spontaneous apoptosis was negligible. As shown in Fig. 5Go, an increase in the percent apoptosis was seen when PBL and T lymphocytes were cultured with T3 hormone. In addition, T3 hormone treatment enhanced apoptosis of both CD4+ T lymphocytes and CD8+ T lymphocytes. The percent increase in apoptotic cells by T3 hormone was not remarkable in this short term culture (for 5 days). The finding was highly reproducible and in the case of T3 hormone, the difference was statistically significant (P < 0.05, by Student’s t test). Treatment with T4 hormone somehow enhanced the apoptosis of normal lymphocytes; however, the enhancement was not statistically significant. These results suggest that thyroid hormones induce apoptotic cell death of normal circulating lymphocytes as well.



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  		Figure 5. Analysis of apoptotic cell death of PBL, T lymphocytes, and T cell subsets induced by thyroid hormones. PBL, T lymphocytes, CD4+ T lymphocytes, and CD8+ T lymphocytes from normal individuals were cultured for 5 days in the presence of T3 (10-8 mol/L). The extent of apoptosis of T3-treated lymphocytes was estimated using the PI staining method. The enhancement by T3 of lymphocyte apoptosis was weak; nevertheless, the results were highly reproducible and significant by Student’s t test. We obtained similar results in five independent experiments. Data presented are the mean ± SEM of triplicate samples. *, P < 0.05 vs. medium. T4 hormone treatment somehow enhanced apoptosis of normal lymphocytes. However, the enhancement was not statistically significant. Thus, the results of such experiments were omitted.

 
Disruption of the mitochondrial {Delta}{Psi} and generation of O2- in the thyroid hormone-induced apoptotic lymphocytes

It has been shown that cells induced to undergo apoptosis accompany an early reduction in the incorporation of mitochondrial {Delta}{Psi}-sensitive dye, DiOC6(3), indicating that a fall in the mitochondrial {Delta}{Psi} is associated with apoptotic cell death (45, 46). This mitochondrial {Delta}{Psi} disruption can be detected in many different cell types regardless of the apoptosis-inducing stimulus. Indeed, GC- and Fas-induced apoptotic lymphocytes showed reduction of mitochondrial {Delta}{Psi} (29, 40, 41, 42, 43, 44). It has been reported that reduction of mitochondrial {Delta}{Psi} is an early irreversible step of apoptotic response (46). Concomitantly, mitochondrial ROS generation was detected during apoptosis (40, 41, 42, 43, 44, 46, 47, 48). We next examined the reduction of mitochondrial {Delta}{Psi} and the generation of O2-, one of ROS, in lymphocytes treated with thyroid hormones. To this end, Jurkat-NU cells and normal PBL were cultured for 2 weeks and 5 days, respectively, followed by incubation with DiOC6(3) and HE. T3 and T4 clearly reduced mitochondrial {Delta}{Psi} in Jurkat-NU cells (Fig. 6AGo) and normal PBL (Fig. 6BGo). We also found generation of O2- in Jurkat-NU cells (Fig. 7AGo) and normal PBL (Fig. 7BGo) treated with thyroid hormones. Taken together, these data indicate that thyroid hormones reduce the mitochondrial {Delta}{Psi} and induce O2- production, both of which lead to apoptotic cell death of lymphocytes.



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  		Figure 6. Analysis of mitochondrial {Delta}{Psi} of Jurkat-NU cells and normal PBL cultured with thyroid hormones. Jurkat-NU cells and normal PBL were cultured for various periods to analyze mitochondrial {Delta}{Psi} in the presence of various concentrations of T3 and T4. The cells were recovered and stained with DiOC6(3 ), as described in Subjects and Methods. A reduction of mitochondrial {Delta}{Psi} was evident in Jurkat-NU cells cultured for 2 weeks (A) and normal PBL cultured for 5 days (B) with the hormones. Values indicate the percentage of cells with reduced mitochondrial {Delta}{Psi} potential in the total cells. We obtained similar results in four independent experiments. The uncoupling agent, carbonyl cyanide m-chlorophenylhydrazone, was used to reduce mitochondrial {Delta}{Psi} as a positive control.

 


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  		Figure 7. Analysis of O2- production by Jurkat-NU cells and normal PBL cultured with thyroid hormones. Jurkat-NU cells and normal PBL were cultured for various periods to analyze ROS production in the presence of various concentrations of T3 and T4. The cells were recovered and stained with HE. Production of O2- is evident in Jurkat-NU cells cultured for 2 weeks (A) and in normal PBL cultured for 24 h (B) with thyroid hormones. Menadione was used as an O2--inducing agent as a positive control. We obtained similar results in three independent experiments. Values indicate the mean fluorescence intensity of the samples.

 
Apoptosis-associated protein expression of Jurkat-NU cells cultured with thyroid hormones

We next examined the expression of apoptosis-associated proteins of Jurkat-NU cells cultured with T3 and T4 by the Western blotting method. We were interested in the expression of a representative antiapoptotic protein, Bcl-2, that has been reported to have antioxidant activity (49, 50). We found that Bcl-2 expression was clearly reduced in Jurkat-NU cells treated with T3 (Fig. 8Go, upper panel; the band image analyzed by NIH Image software was as follows: medium, 100%; 10-9 mol/L T3, 93%; 10-8 mol/L T3, 59%; 10-7 mol/L T3, 43%; and 10-7 mol/L T4, 68%). The reduction of Bcl-2 protein expression was depend on the dose of thyroid hormones. On the other hand, there was no significant change in apoptosis-inducing proteases such as caspase-3 (CPP32; Fig. 8Go, lower panel; medium, 100%; 10-9 mol/L T3, 105%; 10-8 mol/L T3, 91%; 10-7 mol/L T3, 93%; and 10-7 mol/L T4, 95%).



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  		Figure 8. Analysis of apoptosis-associated protein expression of Jurkat-NU cells cultured with thyroid hormones. Cytoplasmic proteins were recovered from Jurkat-NU cells cultured with various concentrations (10-9-10-7 mol/L) of thyroid hormones for 2 weeks. Immunoblotting was carried out using anti-Bcl-2 and anticaspase-3 (CPP32) monoclonal antibodies. It was evident that Bcl-2 protein expression was reduced from 100% (medium) to 43% (10-7 mol/L T3) by NIH Image analysis when cultured with thyroid hormones. Similar results were obtained in three independent experiments.

 
Enhanced apoptosis of PBL in patients with Graves’ disease

Our in vitro study clearly revealed that thyroid hormones induce apoptosis in a Jurkat-NU cell line and normal lymphocytes. Because circulating thyroid hormone levels are high in patients with hyperthyroidism, lymphocyte apoptosis should be enhanced in patients with Graves’ disease, which is the most common cause of hyperthyroidism. Reports that there is a significant decrease in peripheral blood T lymphocytes in patients with Graves’ disease (51, 52, 53) may relate to the accelerated apoptosis by thyroid hormones in the patients. Indeed, when we analyzed the extent of apoptosis of PBL from the patients, 5–25% of PBL from the patients spontaneously underwent apoptosis, whereas PBL from almost all normal individuals did not die via apoptosis spontaneously (Fig. 9Go). Thus, thyroid hormones induce accelerated lymphocyte apoptosis in vivo and in vitro.



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  		Figure 9. Analysis of apoptotic cell death of normal PBL in patients with hyperthyroidism. PBL from 10 patients with hyperthyroidism and 10 normal volunteers were recovered and cultured for 24 h in the culture medium consisting of 10% FCS-RPMI 1640. The extent of apoptosis was estimated by DNA staining with PI. Lymphocyte apoptosis was enhanced significantly in patients with hyperthyroidism compared with that in normal controls (P < 0.05, by Mann-Whitney U test).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study, we found that thyroid hormones reduce Bcl-2 expression and induce intracellular oxidation, resulting in the induction of lymphocyte apoptosis. In addition, prolonged cultivation with thyroid hormones of the lymphocytes further enhanced the extent of apoptosis (data not shown). We also found that dehydroepiandrosterone did not enhance apoptotic cell death of Jurkat-NU cells (data not shown), confirming the specificity of the action for thyroid hormones. It has been reported that thyroid hormones exert positive and negative influences on cellular apoptosis; thyroid hormone induces apoptotic cell death of differentiating erythrocytic progenitor cells (23); T3 potentiates all-trans-retinoic acid-induced apoptosis during differentiation of the promyeloleukemic cells HL-60 (54). In contrast, thyroid hormone inhibits apoptosis of early differentiating cerebellar granule neurons through an increase in the amounts of Bcl-2 protein (28). Retinoic acid, which is also one of the nuclear receptor gene superfamily, induces apoptosis in leukemic HL-60 cells, peripheral T cell lymphoma, and embryonal stem cells, P19EC (16, 31, 32, 55). Retinoic acid reduces Bcl-2 expression, resulting in the induction of apoptotic cell death of leukocytes (55). Overexpression of Bcl-2 inhibits retinoic acid-induced apoptosis in HL-60 and P19EC cells (16, 55). It is well known that GC induce apoptosis of lymphocytes (30). Thus, it is now clear that members of the nuclear receptor gene superfamily, including thyroid hormone receptor, modulate apoptotic cell death of leukocytes/lymphocytes. We found that thyroid hormones have an apoptosis-inducing effect on PBL. However, the effect seems to be rather weak. This may be due to the fact that the thyroid hormones have a weak apoptosis-inducing effect on PBL. Or, alternatively, fresh PBL may be apoptosis resistant. It is known that anti-Fas antibody, which has a potent apoptosis-inducing effect, induces weak apoptotic cell death of fresh PBL (56). GC hormones, which are well known as having an apoptosis-inducing effect on lymphocytes, show instead a weak effect on fresh PBL, and higher concentrations of GC hormones are required for inducing apoptosis of PBL (57). To clarify this issue, further studies are needed to identify the critical apoptosis-inducing molecule(s) whose expression/function is modulated by the thyroid hormone.

It has been reported that the addition of ROS or the depletion of endogenous antioxidants can induce apoptotic cell death, and that apoptosis can be inhibited by endogenous or exogenous antioxidants (47, 49, 58). Moreover, apoptosis is sometimes associated with increases in intracellular ROS levels (47, 49, 58). It has been reported that Bcl-2 protein exerts antioxidant effects in certain in vitro systems (49, 50). We observed that intracellular oxidation was increased in thyroid hormone-treated lymphocytes. Thus, enhanced apoptosis of thyroid hormone-treated lymphocytes may be due to the enhanced ROS production and/or the reduction of antioxidant effects by decreasing Bcl-2 protein expression.

Several models of Bcl-2 function have been proposed. Bcl-2 acts in an antioxidant pathway to decrease the generation of ROS and free radical, which induces apoptotic cell death (49, 50). Bcl-2 prevents activation of the caspase cascade (59). Overexpression of Bcl-2 prevents both mitochondrial {Delta}{Psi} disruption and apoptosis induced by DNA damage, GC, and ceramide (46). Disruption of the mitochondrial {Delta}{Psi}, which results from the asymmetric distribution of protons and other ions on both sides of the inner mitochondrial membrane, can be detected in many different cell types regardless of the apoptosis-inducing stimulus, such as dexamethasone, Fas cross-linking, and irradiation (46). This mitochondrial {Delta}{Psi} disruption occurs before nuclear DNA fragmentation and constitutes an early irreversible step of apoptosis. Our study clearly showed that thyroid hormones induce mitochondrial {Delta}{Psi} disruption and reduce Bcl-2 expression, further confirming the apoptosis-inducing potential of thyroid hormones.

We found that thyroid hormone receptor is expressed constitutively on both normal PBL and Jurkat-NU cells by RT-PCR (data not shown). The results suggest that thyroid hormone receptor (and thyroid hormone complex) may be involved in the apoptosis-inducing effect of thyroid hormones, even though we cannot neglect the possibility that thyroid hormones directly exert the proapoptotic effect without participation of the receptor.

Graves’ disease, the most common cause of hyperthyroidism, is caused by an agonistic anti-TSH receptor antibody, resulting in overproduction of thyroid hormones (60). It has been shown that there is a significant decrease in peripheral blood T lymphocytes in Graves’ disease (51, 52, 53). In fact, we found that PBL from patients with Graves’ disease with a high titer of circulating T3 and T4 showed enhanced apoptosis compared with those from normal donors. Thus, it is possible that thyroid hormones induce apoptotic cell death of lymphocytes, resulting in lymphocytopenia of circulating blood in patients with Graves’ disease. We have also studied Bcl-2 protein expression in patients with Graves’ disease (data not shown). We found reduction of antiapoptotic Bcl-2 protein expression in most, but not all, patients with Graves’ disease. These data indicate that the mechanism of lymphocyte apoptosis in patients with Graves’ disease is at least in part shared with that elucidated by our present in vitro study. Autoimmune responses, including production of anti-TSH receptor autoantibody, are associated with the pathogenesis of Graves’ disease (60). It is of interest to study whether enhanced lymphocyte apoptosis may contribute to the autoimmune responses that are associated with the pathogenesis in patients with Graves’ disease.

In summary, our results indicate that thyroid hormones induce lymphocyte apoptosis in vitro and in patients with Graves’ disease.


   Footnotes
 
1 This work was supported in part by the 1996 Naito Memorial Foundation (Tokyo, Japan) and the 1996–1997 SRF Foundation Grant for Biomedical Research (Tokyo, Japan). Back

Received March 20, 1998.

Revised October 27, 1998.

Accepted January 5, 1999.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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