The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 6 2221-2225
Copyright © 1999 by The Endocrine Society
Immunohistochemical Analysis of Bcl-2, Bax, and Bak Expression in Thyroid Glands from Patients with Subacute Thyroiditis
Mari Koga,
Yuji Hiromatsu,
Atsuo Jimi,
Shuji Toda,
Norimasa Koike and
Kyohei Nonaka
Division of Endocrinology and Metabolism, Department of Medicine
(M.K., Y.H., K.N.), and the Department of Pathology (A.J.), Kurume
University School of Medicine, Kurume 830-0011; the Department of
Pathology, Saga Medical University (S.T.), Saga 849-8501; and Koike
Hospital (N.K.), Saga 840-0862, Japan
Address all correspondence and requests for reprints to: Yuji Hiromatsu, M.D., Ph.D., Division of Endocrinology and Metabolism, Department of Medicine, Kurume University School of Medicine, 67 Asahimachi, Kurume, 830-0011 Fukuoka, Japan.
 |
Abstract
|
|---|
Bcl-2 family proteins are important regulators of apoptosis. To clarify
a role of apoptosis and the expression of Bcl-2 family proteins in the
pathogenesis of subacute thyroiditis (SAT), we evaluated the expression
of Bcl-2, Bax, and Bak by immunohistochemistry and apoptosis by
in situ end labeling of fragmented DNA in thyroid
tissues from 11 patients with SAT. Apoptotic nuclei were found in
granulomas, especially in macrophages/histiocytes and lymphocytes, and
in the regenerating follicular cells, but were rarely found in the area
of fibrosis. The mean (±SD) percentage of apoptotic
follicular cells was significantly greater in SAT than that in controls
(1.4 ± 0.8% vs. 0.4 ± 0.6%). Bcl-2, Bak,
and Bax were strongly expressed in the granulomas and regenerating
thyroid follicular cells from patients with SAT. Bcl-2 and Bak, but not
Bax, were expressed in follicular cells from normal controls. The
percentage of apoptotic cells and the expression of Bax in follicular
cells did not correlate with age or serum levels of thyroid hormones,
C-reactive protein, or thyroglobulin. These data suggest that apoptosis
may be involved in the development of SAT and that Bax expression in
regenerating thyrocytes may be important for the recovery of SAT.
|
Introduction
|
|---|
SUBACUTE thyroiditis (SAT) is a transient
destructive thyroiditis, characterized by painful toxic goiter with
systemic inflammation (1). Most patients with SAT recover within
several months, but about 10–20% of patients treated with
glucocorticoids have recurrences (2). From pathological examination,
various stages of the disease are sometimes found within the same
specimen. Initially, there is extensive follicular cell destruction,
extravasation of colloid, and infiltration of lymphocytes and
histiocytes. The latter tend to congregate around masses of colloid and
coalesce into giant cells. With time, there is a variable degree of
fibrosis, and areas of follicular regeneration are seen. After
recovery, the thyroid appears normal, except for minimal residual
fibrosis. Although it has been reported that SAT may be associated with
viral infection, the etiology of the disease and the mechanisms of the
destruction and regeneration of thyroid follicles have not been fully
elucidated.
Apoptosis is a type of cell death and has been demonstrated to
contribute to cell destruction in a number of diseases. Important
regulators of apoptosis are the Fas/Fas ligand system (3), proteins
encoded by bcl-2 family genes (4), tumor necrosis factor
receptor, and caspases (5). Fas/Fas ligand interaction and apoptosis
have been demonstrated to contribute to the pathogenesis of
Hashimoto’s thyroiditis (6, 7), although Fas ligand expression in
thyrocytes is controversial (8, 9, 10). The ratio of the expression of
death antagonists (Bcl-2, Bcl-XL, and Mcl-1) to death agonists (Bax,
Bak, Bcl-Xs, and Bad) determines the survival or death of cells in
physiological and pathological conditions such as cancer growth (4, 11, 12) and neuronal apoptosis after cerebral artery occlusion (14).
However, there is no report on the expression of Bcl-2 family proteins
in SAT.
The aim of the present study was to determine the presence of apoptosis
and the expression of Bcl-2, Bax, and Bak at various stages in thyroid
tissues from patients with SAT.
|
Materials and Methods
|
|---|
Thyroid tissue
We obtained thyroid specimens from 11 patients with SAT (all
women; aged 39–61 yr; mean age, 50 ± 8 yr), 2 of whom were
associated with thyroid tumor. The diagnosis of SAT was based on
painful goiter with systemic inflammatory signs and lack of
131I thyroid uptake in the hyperthyroid state. At the time
of biopsy, 8 patients were euthyroid, 2 were hyperthyroid, and the
remaining patient was hypothyroid. Four patients had been treated with
glucocorticoid (Table 1
). Serum C
reactive protein was positive at the time of biopsy in 7 patients.
Normal thyroid tissue was obtained from 6 patients with thyroid cancer
or adenoma, all women, aged 17–62 yr (mean, 44 ± 21 yr), as a
control. The study protocol was approved by the institutional review
board of Koike Hospital, and informed consent was obtained from all
subjects after explanation of the study.
View this table:
[in this window]
[in a new window]
|
Table 1. Bcl-2, Bax, and Bak expression and apoptosis in
regenerating thyroid follicular cells from patients with subacute
thyroiditis and their clinical characteristics
|
|
Detection of apoptosis
Apoptotic cells were detected by in situ end labeling
of fragmented DNA using assay kits (Apop Tag, in situ
apoptosis detection kit, peroxidase, Oncor Inc., Gaithersburg, MD) as
previously described (15). The paraffin-embedded tissue sections were
deparaffinized by xylene and ethanol. Sections were then treated with
proteinase K (20 µg/mL) for digesting protein in tissue.
Digoxigenin-deoxy-UTP was added to the 3'-OH ends of DNA by terminated
deoxynucleotidyl transferase. After incubating with anti-digoxigenin
antibody conjugated with peroxidase, the sections were stained with
diaminobenzidine and counterstained with methyl green. The mean
percentage of positive staining of nuclei was calculated after counting
positively or negatively staining nuclei of 500 thyrocytes in several
fields under light microscope by 2 observers.
Immunohistochemistry
Immunohistochemical staining was performed using mouse
monoclonal antibodies against human Bcl-2 (IgG1
; DAKO Corp. A/S, Glostrup, Denmark) and Bak (IgG2a; Oncogene Science, Inc., Cambridge, MA), rabbit polyclonal antibody
against Bax (IgG; Oncogene Science, Inc.), and mouse IgG1
(DAKO Corp.), mouse IgG2a (DAKO Corp.), and
normal rabbit Ig (DAKO Corp.) as negative controls.
Positive reactivity was identified using a
streptavidin-biotin-peroxidase detection system (DAKO Corp. catalyzed signal amplification system, DAKO Corp.) as previously reported (15). Briefly, the
paraffin-embedded tissue sections were deparaffinized, then put in
Target Retrieval Solution (DAKO Corp.) to amplify the
signal, placed in a water bath (95–97 C) for 20–40 min, and then
cooled for 20 min at room temperature. After blocking endogenous
peroxidase activity with a 3% hydrogen peroxidase solution for 10 min,
intrinsic biotin with an endogenous avidin/biotin blocking kit
(Nichirei Corp., Tokyo Japan), and nonspecific binding by serum-free
protein, the tissue was incubated with prediluted mouse monoclonal
antibodies (10 µg/mL) or rabbit polyclonal antibodies (5 µg/mL) for
15 min, followed by 15-min incubation with a biotinylated rabbit
antimouse IgG+M+A antibody or goat antirabbit IgG antibody and 15-min
incubation with streptavidin-biotin-peroxidase complex, biotinyl
tyramide, hydrogen peroxide, and streptavidin conjugated with
peroxidase. Staining was completed with diaminobenzidine for 5 min, and
the specimens were counterstained with Mayer’s hematoxylin for 2–5
min.
To detect the cell types that undergo apoptosis in the inflamed tissue,
we performed a double immunostaining as follows. After apoptotic signal
was identified as described above, a cell type-specific antibody for
CD45 (DAKO Corp.), CD68 (DAKO Corp.), or
vimentin (DAKO Corp.) was stained using alkaline
phosphatase-conjugated streptavidin (Nichirei Corp.) and visualized
with a fast blue substrate kit (Nichirei Corp.), as described
previously (16).
Statistical analysis
Differences in the expression of Bcl-2 family proteins and
percentages of apoptotic cells between groups were evaluated using
Mann-Whitney U test or Fisher’s exact probability test. Correlation
was assessed by Spearman’s signed ranks test. P <
0.05 was considered significant.
|
Results
|
|---|
Apoptotic cells in thyroid tissues
Apoptotic nuclei were observed in the area of granuloma (Fig. 1A). Most cell types undergoing apoptosis
in the inflamed tissues were macrophages/histiocytes (Fig. 1B) and
lymphocytes, but not multinucleate giant cells. Apoptotic nuclei were
also found in regenerating follicular cells, which took place as a
group in an insular pattern in the fibrous stroma and showed
hypertropic cytoplasms and enlarged nuclei with prominent nucleoli
(Fig. 1D). Apoptotic nuclei were rarely found in the area of fibrosis,
where many collagen fibers were dominant (Fig. 1C). In normal thyroid
glands, apoptotic nuclei were occasionally found in follicular cells
(Fig. 1E). The mean (±SD) percentage of apoptotic
follicular cells was significantly greater in SAT than that in controls
(1.4 ± 0.8% in SAT and 0.4 ± 0.6% in normal controls; by
Mann-Whitney U test, P < 0.05).
View larger version (117K):
[in this window]
[in a new window]
|
Figure 1. In situ end labeling of
fragmented DNA in thyroid tissues from patients with SAT (A–D) and a
normal control subject (E), stained by the terminal deoxynucleotidyl
transferase-mediated deoxy-UTP nick end labeling method. The
arrows indicate apoptotic nuclei. The dual staining
showed that the apoptotic cells are mainly macrophages/histiocytes
identified with anti-CD68 monoclonal antibody (B). See Materials
and Methods for details. A, Granuloma; B, CD68-positive
macrophage/histiocyte undergoes apoptosis (CD68 and apoptotic signals
are shown in blue and brown,
respectively); C, area of fibrosis; D, regenerating thyroid follicles;
E, normal thyroid follicles.
|
|
Expression of Bcl-2, Bax, and Bak in thyroid tissues
Bcl-2 (Fig. 2, A–C), Bax (Fig. 2, E–G), and Bak (Fig. 2, I–K) were expressed in the area of granuloma
(Fig. 2, A, E, and I) and regenerating thyroid follicular cells (Fig. 2, B, F, and J), but not in the area of fibrosis (Fig. 2, C, G, and K),
from all patients with SAT. Although Bcl-2 and Bak were expressed in
normal follicular cells from the six control tissues examined (Fig. 2, D and M), Bax was not expressed in follicular cells from the six
control tissues examined (Fig. 2H).
View larger version (165K):
[in this window]
[in a new window]
|
Figure 2. Immunohistochemical staining with Bcl-2
(A–D), Bax (E–H), Bak (I–K, M), and control mouse IgG2a (L) in
thyroid tissues from patients with SAT (A, E, and I, granuloma; B, F,
J, and L, regenerating follicles; C, G, and K, fibrosis) and in thyroid
tissue from a control subject (D, H, and M).
|
|
Relation of apoptosis and Bcl-2 family protein expression in
thyroid to the clinical parameters of SAT
Bax expression was weaker in four patients with SAT, and three of
them had been treated with glucocorticoids (Table 1
). The percentages
of apoptotic follicular cells in patients with SAT were not
significantly correlated with the expression of Bcl-2, Bax, or Bak;
age; serum thyroid hormones; C-reative protein; or thyroglobulin
levels.
|
Discussion
|
|---|
We have characterized, for the first time, the presence of
apoptosis and the expression of Bcl-2 family proteins, namely Bcl-2,
Bax, and Bak, in thyroid tissue from patients with SAT. In summary, 1)
apoptosis was observed in the area of granuloma. Apoptotic cells were
mainly macrophages/histiocytes and lymphocytes in the granuloma. 2)
Apoptotic cells were also found in regenerating follicular cells, but
only rarely in the area of fibrosis in SAT. The percentages of
apoptotic nuclei of thyroid follicular cells in SAT were significantly
greater than those in control subjects. 3) Bax, an apoptosis agonist,
was expressed in the area of granuloma and regenerating follicular
cells in all patients with SAT, but was not expressed in normal thyroid
follicular cells from control subjects. 4) Although Bcl-2 and Bak were
also expressed in the area of granuloma and regenerating follicular
cells in SAT, they were constitutively expressed in normal thyroid
follicular cells.
Although the close association with human leukocyte antigen Bw35
supports the role of cytotoxic T cell immunity in SAT (2), the precise
mechanism of the thyroid cell destruction is unclear. The present
demonstration of apoptotic nuclei in the area of granuloma suggests the
involvement of apoptosis in the development of SAT. Apoptosis was
rarely seen in sites of fibrosis, which might be a healing process. The
increased levels of apoptosis in the regenerating follicular cells may
be related to shorter turnover of regenerating follicular cells
compared with that of well differentiated normal thyroid follicular
cells. This may represent a physiological feature of regenerating
follicular cells.
It is now known that the bcl-2 gene family regulates
apoptosis (4). The present study confirmed that normal thyroid
follicular cells expressed Bcl-2 and Bak, but not Bax (12, 17, 18),
suggesting that Bcl-2 and Bak expression in differentiated normal
thyroid follicular cells might regulate apoptosis in the physiological
state. In contrast, in the area of granulomatous changes in SAT, Bax
expression was detected in follicular cells and inflammatory cells.
These data suggest that the increased expression of Bax may contribute
to the apoptosis in the lesion.
Furthermore, we demonstrated the expression of Bax in the regenerating
follicular cells for the first time. Basile et al. (19)
reported the up-regulated expression of Bax in regenerating proximal
tubules after ischemic injury. The up-regulation of Bax gene expression
and Bax expression have also been reported in rat (20) and mice (21)
liver regeneration after partial hepatectomy and in muscle regeneration
in muscular dystrophies (22). Those reports support that Bax expression
in regenerating thyroid follicular cells may play an important role in
the recovery of SAT.
In conclusion, results from the present study suggest the involvement
of apoptosis in the development of SAT in its various stages.
Furthermore, Bax expression in the regenerating follicular cells may
play a role in the recovery of SAT. As the number of patients studied
was small, two patients were associated with thyroid cancer, and four
patients had been treated with glucocorticoids, which might
down-regulate Bax expression (23), further studies are indicated to
clarify the precise role of Bcl-2 family proteins in thyroid cell
damage and recovery in SAT and their implications with respect to its
therapy.
|
Acknowledgments
|
|---|
We thank Jack R. Wall, M.D., Ph.D. (Division of Endocrinology
and Metabolism, Queen Elizabeth II Health Science Center, Halifax,
Canada), for the valuable suggestions and contributions to this
study.
Received July 6, 1998.
Revised January 4, 1999.
Revised February 17, 1999.
Accepted February 24, 1999.
|
References
|
|---|
-
Nikolai TF. 1991 Silent thyroiditis and
subacute thyroiditis. In: Braverman LE, Utiger RD, eds. Werner and
Ingbar’s the thyroid: a fundamental and clinical text, 6th ed.
Philadelphia: Lippincott; 710–727.
-
Volpé R. 1995 Subacute and sclerosing
thyroiditis. In: DeGroot LJ, ed. Endocrinolgy, 3rd ed. Philadelphia:
Saunders; 742–751.
-
Nagata S, Golstein P. 1995 The Fas death factor.
Science 267:1449–1456.
-
Kroemer G. 1997 The proto-oncogene
bcl-2 and its role in regulating apoptosis. Nat Med. 3:614–620.[CrossRef][Medline]
-
Ashkenazi A, Dixit VM. 1998 Death receptors:
signaling and modulation. Science 281:1305–1308.
-
Giordano C, Stassi G, Maria RD, et al. 1997 Potential involvement of Fas and its ligand in the pathogenesis of
Hashimoto’s thyroiditis. Science 275:960–963.
-
Kotani T, Aratake Y, Hirai Y, Fukazawa Y, Sato H,
Ohtaki S. 1995 Apoptosis in thyroid tissue from patients with
Hashimoto’s thyroiditis. Autoimmunity 20:231–236.
-
Xerri L, Devilard E, Hassoun J, Mawas C, Birg F. 1997 Fas ligand is not only expressed in immune privileged human organs
but is also coexpressed with Fas in various epithelial tissues. J
Clin Pathol Mol Pathol. 50:87–91.[Abstract/Free Full Text]
-
Stokes TA, Rymaszewski M, Arscott PL, et al. 1998 Constitutive expression of FasL and thyrocytes. Science 279:2015
(www.sciencemag.org/cgi/content/ full/279/5359/2015a).
-
Mitsiades N, Poulaki V, Kotoula V, et al. 1998 Fas/Fas ligand up-regulation and Bcl-2 down-regulation may be
significant in the pathogenesis of Hashimoto’s thyroiditis. J
Clin Endocrinol Metab. 83:2199–2203.[Abstract/Free Full Text]
-
Krajewska M, Krajewski S, Epstein JI, et al. 1996 Immuno-histochemical analysis of Bcl-2, Bax, Bcl-X, and mcl-1
expression in prostate cancers. Am J Pathol. 148:1567–1576.[Abstract]
-
Branet F, Brousset P, Krajewski S, et al. 1996 Expression of the cell death-inducing gene bax in carcinoma
developed from the follicular cells of the thyroid gland. J Clin
Endocrinol Metab. 81:2726–2730.[Abstract]
-
Manetto V, Lorenzini R, Cordon-Cardo C, et al. 1997 Bcl-2 and Bax expression in thyroid tumours. An immunohistochemical and
western blot analysis. Virchows Arch. 430:125–130.[CrossRef][Medline]
-
Gillardon F, Lenz C, Waschke KF, et al. 1996 Altered expression of Bcl-2, Bcl-X, Bax, and c-Fos colocalizes with DNA
fragmentation and ischemic cell damage following middle cerebral artery
occlusion in rats. Brain Res Mol Brain Res. 40:254–260.[Medline]
-
Koga M, Hiromatsu Y, Jimi A, Inoue Y, Nonaka K. 1998 Possible involvement of Fas-mediated apoptosis in eye muscle
tissue from patients with thyroid-associated ophthalmopathy. Thyroid4
:311–318.
-
Toda S, Matsumura S, Fujitani N, Nishimura T, Yonemitsu
N, Sugihara H. 1997 Transforming growth factor-ß1 induces a
mesenchyme-like cell shape without epithelial polarization in
thyrocytes and inhibits thyroid folliculogenesis in collagen gel
culture. Endocrinology 138:5561–5575.
-
Brocker M, De-Buhr I, Papageorgiou G, Schatz H, Derwahl
M. 1996 Expression of apoptosis-related proteins in thyroid tumors
and thyroid carcinoma cell lines. Exp Clin Endocrinol Diabetes.
104(Suppl 4):20–23.
-
Kiefer MC, Brauer MJ, Powers VC, et al. 1995 Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak.
Nature 374:736–739.
-
Basile DP, Lipis H, Hammerman MR. 1997 Expression of bcl-2 and bax in regenerating rat
renal tubules following ischemic injury. Am J Physiol.
272:F640–F647.
-
Kren BT, Trembley HH, Krajewski S, Behrens TW, Reed JC,
Steer CJ. 1996 Modulation of apoptosis-associated genes
bcl-2, bcl-x, and bax during rat liver
regeneration. Cell Growth Differ. 7:1633–1642.[Abstract]
-
Tzung SP, Fausto N, Hockenbery DM. 1997 Expression
of bcl-2 family during liver regeneration and identification
of bcl-x as a delayed early response gene. Am J Pathol. 150:1985–1995.[Abstract]
-
Tews DS, Goebel HH. 1997 DNA-fragmentation and
expression of apoptosis-related proteins in muscular dystrophies. Neuropathol Appl Neurobiol. 23:331–338.[CrossRef][Medline]
-
Lotem J, Sachs L. 1995 Regulation of Bcl-2, bcl-XL
and Bax in the control of apoptosis by hematopoietic cytokines and
dexamethasone. Cell Growth Differ. 6:647–653.[Abstract]
Top
Abstract
Introduction
