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The First Department of Internal Medicine (M.T., H.K., T.T., K.A., T.Ki., N.Y., K.E., S.N.), Department of Histology and Cell Biology (T.Ko., P.K.N.), Nagasaki University School of Medicine; and School of Allied Medical Science (T.Y.), Nagasaki University, Nagasaki 852-8501, Japan
Address all correspondence and requests for reprints to: Katsumi Eguchi, M.D., Nagasaki University School of Medicine, First Department of Internal Medicine, 1-7-1, Sakamoto, Nagasaki 852-8501, Japan. E-mail: f1078{at}cc.nagasaki-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Goiter can be experimentally induced by depleting circulating iodine and adding goitrogen. Feeding 6-propyl-2-thiouracil (PTU) as a goitrogen to iodine-deficient rats results in increased mass of the thyroid gland and proliferation of thyrocytes to about ten times the baseline number within 3 months (2). The tissue mass is, however, modulated by not only cell proliferation but also cell death. Examination of thyrocyte proliferation under experimental conditions showed that the actual number of cells found at the end of the experiment is less than expected based on the proliferative activity. This discrepancy has been interpreted as a result of an extensive cell loss within the thyroid gland (3).
At present, the type of eukaryotic cell death is classified into apoptosis and necrosis (4). Apoptosis is of interest because the presence of apoptotic cells in abnormal thyroid glands can be detected by histological examination (5, 6, 7), and the induction of apoptosis of cultured thyroid cells by growth factor deprivation (8) and by cytokines (9) have been reported recently. However, to our knowledge, there are no reports that have serially examined the role of apoptosis in the process of goiter formation and involution in vivo. Apoptosis can be characterized morphologically and biochemically and is usually induced through activation of a set of special genes. Among such genes, activation of a gene for Fas antigen has been well described (10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Fas antigen (Fas), a transmembranous glycoprotein that belongs to the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family, can mediate apoptosis upon forming complexes with Fas ligand (FasL) (10, 11, 12, 13, 14) in a variety of lymphoid and tumor cells, as well as in various normal tissues outside the immune system (15, 16, 17, 18, 19). In the thyroid gland, Fas is expressed in human thyroid tissues in Hashimoto’s thyroiditis and thyroid cancer (6). Studies from our laboratories have also indicated that Fas-mediated apoptosis is induced by cytokines in human thyrocytes in primary cultures (9).
In the present study, rats were fed a low iodine diet (LID) to allow serial examination during the process of goiter formation. Subsequently, rats with fully developed goiter were fed a high iodine diet (HID) to study the phase of involution. This rat model almost equates the clinical condition of endemic goiter or ingestion of goitrogen. To investigate the presence of apoptosis during goiter formation and involution in a rat model of goiter, apoptotic cells were identified by electron microscopy and terminal deoxy-UTP nick end labeling (TUNEL) staining. To assess the involvement of Fas/FasL system in the induction of apoptosis, the spatial and temporal expression of Fas and FasL were localized immunohistochemically and quantified by Western blotting. Furthermore, we examined the proliferating cell nuclear antigen (PCNA) using immunohistochemistry in order to evaluate changes in thyroid gland mass during development of goiter and its involution.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The thyroid glands were divided into three groups; some were fixed with 10% formalin, embedded in paraffin, and sectioned at 4 µm in thickness for histochemical studies. Another group of glands was fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for electron microscopy, while the third group was frozen at -80 C for Western blot analysis.
Biochemical analysis
Serum concentrations of T3, T4, and TSH
were measured by RIA kits (T3, T4; Eiken
Chemical Co., Tokyo, Japan; TSH; rat TSH assay system, Amersham, UK).
Serum iodine concentrations were measured by the Technicon
Auto-Analyzer II system.
TUNEL
TUNEL was performed using sections of paraffin-embedded tissue
samples according to the method of Gavrieli et al. (21) with
a slight modification as described previously (22) using biotinylated
16-dUTP (Boehringer Mannheim, Germany) as a substrate and horseradish
peroxidase (HRP)-goat antibiotin as the reporting agent. The sites of
HRP were visualized by H2O2 and
3,3'-diaminobenzidine (DAB; Wako Pure Chemicals, Osaka, Japan) together
with nickel and cobalt ions for 5 min (23). Under a light microscope,
more than 10,000 thyrocytes per rat thyroid were counted while we
checked on thyroid epithelial cell with looking at the each serial
section of HE staining and the ratio of apoptotic cells was expressed
as the number of TUNEL-positive thyrocytes per 1,000 thyrocytes.
Control thyroid glands obtained from rats fed normal chow for 21 days
were processed and examined in a similar manner.
Electron microscopy
Thyroid tissue samples were prepared for electron microscopy
using standard procedures and the ultrathin sections were examined and
photographed by a JEOL 1200 EX electron microscope.
Western blot analysis of Fas and FasL
The method of Laemmli (24) was followed by using rabbit anti-Fas
and rabbit anti-FasL as the first antibody and HRP-goat antirabbit IgG
as the second antibody. The rabbit antibodies were prepared from
synthetic oligopeptides, P4 and P5, corresponding to the intracellular
domain (amino acids 292–306) of mouse Fas (18) and intracellular
domain (amino acids 41–55) of rat FasL (10), respectively. The
immunochemical specificities of these antisera have been confirmed in
our previous studies (22, 25). Bands that reacted with antibodies were
visualized with H2O2, DAB, nickel, and cobalt (23). The density of each
band was measured by a Canon CCD camera (Canon Co., Tokyo, Japan)
attached to an Olympus SP-500 Image Analyzer with Image Command program
5098 (Olympus Co., Tokyo, Japan).
Protein measurements
Protein was measured using the Coomassie blue dye method
(Bio-Rad protein assay, Bio-Rad Laboratories, Richmond, CA) with BSA as
standard.
Immunohistochemistry for Fas, FasL and PCNA
Paraffin sections were used for immunohistochemistry. For
localization of Fas and FasL, the polyclonal rabbit anti-Fas and
anti-FasL antibodies used for the Western blot were used as the first
antibody while HRP-goat antirabbit IgG was used as the second antibody.
As a control, normal rabbit serum was used as the first antibody. For
localization of PCNA, monoclonal mouse anti-PCNA (PC10; DAKO, Glostrup,
Denmark) was used as the first antibody and HRP-goat anti-mouse IgG as
the second antibody. As a control, normal mouse IgG was used as the
first antibody. HRP sites were visualized by DAB and H2O2. Under a
light microscope, more than 10,000 thyrocytes were counted and the
ratios of Fas- or PCNA-positive thyrocytes were expressed as the each
number of these cells per 1,000 thyrocytes. Thyroid glands at 21 days
of rats receiving normal chow were used as the control. The PCNA/TUNEL
ratio was obtained by dividing the mean ratio of PCNA-positive cells by
that of TUNEL positive cells.
Statistical analysis
Data were expressed as mean ± SD. In
hormonal data and thyroid weight, differences between groups were
examined for statistical significance using the Student’s t
test. Statistical evaluation of experimental data in
immunohistochemistry and TUNEL among control, goiter formation, the
early stage and the late stage of involution was performed using
analysis of variance (Kruskal-Wallis test). The determination of
significant differences between control and the other stage were
performed by Mann-Whitney’s U test. A P value <0.05 was
selected as the level of significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The serum concentrations of iodine,
T3, and T4 were low in rats fed LID. After
commencement of HID feeding, the concentration of serum iodine level
was markedly higher at 6 h and remained high throughout the
experiment (Fig. 2).
Similarly, serum T3 started to increase at 6 h and
reached the baseline level after 7 days on HID, serum T4
remained low for 3 days and increased after 7 days on HID, although the
level remained below the baseline value after 14 days on HID. Serum TSH
concentration was above 150 ng/ml after 21 days on LID. After
commencement of HID feeding, the high serum TSH level persisted for 3
days but then decreased to the control level after 14 days on HID (Fig. 2). The changes of thyroid hormones and TSH secretion by iodine
depletion and refeeding were similar to those in a previous report
(26).
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). There were
significant variations in those positive rates due to the stages of the
thyroids (P < 0.05). Only a few TUNEL-positive cells
were present in the control thyroid gland, but significantly increased
during the development of goiter (at 7–21 days of LID, Table 1). In the early stage of goiter
involution (at 6 h and 3 days of HID), the number of
TUNEL-positive cells further increased but returned to the control
level in the late stage of goiter involution (at 7–14 days of HID,
Table 1). Moreover, the presence of apoptosis was confirmed using an
electron microscopy (Fig. 4).
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). The densities of the 31 and 58
kDa-bands, which were detected in control thyroid glands, increased
during goiter formation and the early stage of involution (Fig. 5B). On
the other hand, FasL was persistently expressed as a single band of
31-kDa in all thyroid gland tissue samples and its density was
virtually constant throughout the experiment (Fig. 6).
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). In the competition study using an excess amount of P4
synthetic peptides, staining of thyrocytes was abolished (Fig. 3C). In
agreement with the results of Western blot analysis, a weak staining
for Fas was observed in control thyrocytes, but the number of such
cells increased significantly during goiter formation (Table 1). In the
early stage of involution, the number of Fas-positive cells
further increased but decreased in the late stage of goiter involution
(Table 1). There were significant variations in those positive rates
due to the stages of the thyroids (P < 0.05). The time
of appearance of Fas-positive cells almost paralleled that of
TUNEL-positive cells. Examination of serial sections of thyroid glands
showed that some Fas-positive cells were also TUNEL-positive (Fig. 3, A-c and B-c).
Compared with immunostaining for Fas, staining for FasL was generally
faint, but significant staining was detected in a number of thyrocytes
(Fig. 7A). In the competition study using
an excess amount of P5 synthetic peptides, no staining of thyrocytes
was detected (Fig. 7B).
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and Fig. 8, goiter formation was associated with a marked increase in the number of
PCNA-positive cells, whereas only a few positive cells were detected in
control tissues. However, in the early stage of goiter involution, the
high number of PCNA-positive cells started to diminish and almost
disappeared in the late stage of goiter involution. The PCNA/TUNEL
ratio was 0.46 in the control thyroid but markedly increased to 12.61
when goiter was established. In the early stage of goiter involution,
the ratio decreased to 6.04 and further decreased in the late stage of
goiter involution to 0.04.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Loss of thyrocytes has been previously established in studies examining cell population kinetics in a rat model of goiter using autoradiography (3). A high level of cell loss, evaluated by the difference between the actual number and expected number of cells based on estimates of proliferative activity, has been demonstrated ranging from 0.30 (in diffuse hyperplasia) to 0.95 (in nodular hyperplasia) in the rat thyroid. On the other hand, several reports have described the presence of apoptosis of thyrocytes in primary cultures (8, 9) and in abnormal thyroid tissues (6, 7). Canine thyrocytes undergo apoptosis after deprivation of FCS, EGF, and TSH (8). Apoptosis has also been identified in thyroid tissues of patients with Hashimoto’s thyroiditis (7). However, no previous studies have actually confirmed the development of apoptosis as a process explaining cell loss in goiter formation. In this study, we confirmed the occurrence of apoptosis and showed increased frequency of apoptotic cells during goiter formation, indicating that apoptosis of thyrocytes is involved in cell loss during goiter formation.
On the other hand, apoptosis of thyrocytes during involution induced by moderate iodine diet in iodine-deficient goiter has only been previously described in an study only using mice (5). The ratio of apoptotic cells in this tissue has, however, not been investigated. In our rat model, we confirmed the occurrence of extensive apoptosis of thyrocytes during the early stage of goiter involution.
What are the mechanisms that induce thyrocyte apoptosis? While several factors may be involved in this process, we focused in this study on the expression of Fas and FasL. Our results showed increased Fas expression during the process of goiter formation and the early stage of involution and decreased in the late stage of involution. More importantly, changes in Fas expression almost paralleled changes in the number of TUNEL-positive cells, and some Fas-positive thyrocytes were also TUNEL-positive. Together with our previous finding of Fas-mediated apoptosis in human cultured thyrocytes (9), the present results suggest that the Fas/FasL system plays an important role in the induction of apoptosis of thyrocytes. Because we found a persistent expression of FasL in all stages of goiter formation and involution, it is likely that Fas expression may serve as a limiting factor for the induction of Fas-mediated apoptosis. These findings are in agreement with a recent report by Giordano et al. (27), in which FasL was constitutively expressed in thyrocytes of patients with nontoxic goiter as well as Hashimoto’s thyroiditis and was found to kill Fas-sensitive subline of human T cell lymphoma, HuT78. These workers concluded that Fas and FasL are simultaneously present in thyrocytes and that their interaction is probably responsible for the induction of apoptosis.
It should be noted, however, that not all Fas-positive thyrocytes were TUNEL positive in this study. This may be due to the time lag between the expression of Fas and the internucleosomal cleavage of nuclear DNA. In addition, the effect of suppressing molecules on the apoptotic process should be also considered. For example, Bcl-2, known to suppress several types of apoptotic processes (28, 29, 30, 31), is strongly expressed in human thyrocytes (6, 9) and may prevent thyrocytes to undergo apoptosis. Recently, Baker et al. (32) reported that Fas-mediated apoptosis in thyroid cells was regulated by a labile protein inhibitor. It should also be noted that the induction of apoptosis by anti-Fas antibody requires cytokine stimulation even though about 40% of thyrocytes express Fas antigen in primary cultures (9), indicating that certain cofactor(s) may be required for Fas-bearing thyrocytes to enter the apoptotic cascade.
How is Fas expression regulated on thyrocytes? We have recently
reported that IFN-
and IL-1ß induce Fas expression and
Fas-mediated apoptosis of thyrocytes in primary cultures of human
thyrocytes (9). Moreover, IL-1ß, which is probably released by
monocytes/macrophages or by activated endothelial cells independent of
infiltrating T lymphocytes, actually induces Fas expression as well as
apoptosis in thyrocytes from nontoxic goiter (27). These cytokines may
enhance Fas expression in this rat model of goiter.
Apart from the Fas/FasL system, iodine may also be involved in the proliferation and death of thyrocytes. Excess iodine inhibits proliferation of rat FRTL-5 cells (33) and induces cell toxicity both in vivo and in vitro, probably through excessive production of free radicals (5, 34). The discontinuation of PTU in HID also reinforces the effects of excess iodine during goiter involution because PTU could inhibit iodine oxidation and organification. In addition, recent studies have shown that iodine induces the expression of transforming growth factor (TGF)-ß1 (35, 36, 37), as a potent growth inhibitor, known to induce apoptosis of porcine thyrocytes. Furthermore, Logan et al. (38) showed an enhanced expression of TGF-ß1 during thyroid hyperplasia in iodine-deficient rats, indicating the presence of a close relationship between TGF-ß1 and apoptosis during goiter formation. Thus, in this rat model of goiter, it is possible that iodine may directly or indirectly influence the number of thyrocytes in addition to the Fas/FasL system.
The relation of apoptosis to proliferative activity in the control of thyroid gland mass has not been previously investigated. Our results showed that a change in PCNA expression, which is considered as a parameter of cell proliferation, was similar to the previous data of 3H thymidine prelabelling experiment using similar protocol (20). However it was not always associated with a similar change in the number of apoptotic cells throughout the experiment. During the process of goiter formation, PCNA/TUNEL ratio markedly increased, whereas the ratio tended to decrease particularly in the late stage of goiter involution. These results indicate that both proliferation and apoptosis of thyrocytes are involved in homeostasis of thyroid gland mass; during the stage of goiter formation, the number of thyrocytes increases due to an imbalance in cell proliferation and apoptosis, favoring the former process. In contrast, reduced number of thyrocytes during the early and late stages of involution may be caused by enhanced induction of apoptosis and reduced cell proliferative activity, respectively.
In conclusion, our results indicated that apoptosis of thyrocytes is a main factor of cell loss during goiter formation and involution. Our results also suggested that the Fas/FasL system is involved in the induction of apoptosis of thyrocytes. Moreover, the delicate balance between apoptosis and cell proliferation may play an important role in the control of thyroid gland mass.
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Acknowledgments |
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Footnotes |
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Received December 29, 1997.
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