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Original Studies |
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.
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Abstract |
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Introduction |
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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.
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Materials and Methods |
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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.
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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.
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Results |
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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).
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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).
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Acknowledgments |
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Received July 6, 1998.
Revised January 4, 1999.
Revised February 17, 1999.
Accepted February 24, 1999.
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References |
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This article has been cited by other articles:
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  H. Tang, K. Chen, Y. Wei, G. C. Sharp, L. McKee, and H. Braley-Mullen Apoptosis of thyrocytes and effector cells during induction and resolution of granulomatous experimental autoimmune thyroiditis Int. Immunol., December 1, 2000; 12(12): 1629 - 1639. [Abstract] [Full Text] [PDF]
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