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Functional Fas Ligand Expression in Thyrocytes from Patients with Graves’ Disease

Division of Endocrinology and Metabolism, Department of Medicine (Y.H., M.K., D.Y., K.N.), Department of Immunology (T.H.), and Department of Internal Medicine (S.S., J.H.), Kurume University School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830-0011; and the Department of Immunology, Juntendo University School of Medicine (H.Y., N.K., K.O.), 2–1-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Address all correspondence and requests for reprints to: Yuji Hiromatsu, M.D., Division of Endocrinology and Metabolism, Department of Medicine, Kurume University School of Medicine, 67 Asahimachi, Kurume, Fukuoka 830-0011, Japan. E-mail: yuji{at}med.kurume-u.ac.jp

	Abstract

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Fas/Fas ligand (FasL) interaction has been suggested to play a role in the pathogenesis of Hashimoto’s thyroiditis. This manuscript addressed a role for Fas/FasL interaction in the pathogenesis of Graves’ disease (GD). Apoptosis was detected in 0.5–5.0% of GD thyrocytes, but not in normal thyrocytes from patients with adenoma (N). Fas was constitutively expressed on the basement membrane of both GD and N thyrocytes. Thyrocytes expressed Bcl-2 constitutively in both GD and N thyrocytes. FasL was detected at the messenger ribonucleic acid level in thyroid tissue and cultured thyroid cells by Northern blotting and RT-PCR. FasL protein was detected in the cytoplasm and basolateral surface of thyrocytes from GD, but not in N. Cell surface expression of FasL on cultured thyrocytes disappeared within 48 h after their isolation. However, it was retained by culturing the cells with a matrix metalloproteinase inhibitor. Coculture with thyrocytes induced apoptosis of Fas transfectants, which was blocked by an anti-FasL antibody. Although cultured thyrocytes expressed Fas on the surface, they were not killed by an agonistic anti-Fas antibody. Interferon--induced Fas up-regulation was suppressed by TSH. These results suggest that the increased expression of FasL in GD thyrocytes, the down-regulation of Fas expression by TSH or possibly by TSH receptor autoantibody, and the overexpression of Bcl-2, which could render thyrocytes resistant to FasL-mediated elimination, may thus be involved in the pathogenesis of GD.

	Introduction

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THERE IS considerable evidence that Fas and Fas ligand (FasL) interaction is implicated in the pathogenesis of autoimmune disorders (1, 2). Fas/FasL gene abnormalities are associated with the accumulation of activated lymphocytes in mouse models resembling human systemic lupus erythematosus (3). Fas-based apoptosis is also involved in the T cell-mediated cytotoxicity against target cells, such as virus-infected cells and cancer cells (1, 4, 5). Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) are considered to be autoimmune thyroid disorders. In HT, infiltration of lymphocytes and destruction of thyrocytes are prominent histological features (6). Expression of the Fas antigen on thyrocytes has been reported by several laboratories including ours (7, 8, 9). The presence of apoptosis has been also reported in HT (10). More recently, Giordano et al. (11) reported the constitutive expression of FasL in normal and HT thyrocytes, thereby contributing to the development of clinical hypothyroidism. In contrast, GD is characterized by the hyperplasia of thyrocytes that results from stimulation with anti-TSH receptor autoantibodies. There is little evidence of thyrocyte injury in GD, and lymphocytic infiltrates are less frequent than HT. Rymaszewski et al. (12) reported that FasL was not expressed in GD thyrocytes. However, the role of FasL expression on thyrocytes in GD has not been fully addressed.

In the present study, we explored a possible role of Fas/FasL interaction in the pathogenesis of GD by evaluating the presence of apoptosis and the expression of Fas and FasL in thyroid tissues from patients with GD.

	Materials and Methods

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Thyroid tissue

Thyroid tissue was obtained from 10 patients with GD, all of whom were women, aged 21–45 yr. At the time of surgery all patients were euthyroid; 7 patients were being treated methimazole (10–30 mg/day), and 3 patients were being treated propylthiouracil (150–200 mg/day). Serum from all of the patients contained anti-TSH receptor antibody, with titers ranging from 18.3–78.0%. Thyroid tissue was also obtained at autopsy from a patient with GD in remission who died accidentally. Normal thyroid tissue adjacent to follicular adenoma was obtained from 5 subjects with follicular adenoma, all women, aged 28–65 yr, as a control.

Cell culture

Thyroid tissue was minced, washed, and digested with 1% trypsin, as previously reported (13). Thyroid cells were cultured in monolayers in DMEM (Sigma Chemical Co., St. Louis, MO) supplemented with 10% heat-inactivated FBS containing 50 µg/mL gentamicin. Thyroid cells were used in the experiments 1–10 days after setting up primary culture. The mouse T lymphoma cell line (WR19L), its human Fas complementary DNA (cDNA) transfectant (hFas/WR19L), and human FasL cDNA transfectants (hFasL/L5178Y and hFasL/BHK) were prepared as previously described (14).

Detection of apoptosis

Apoptotic cells in thyroid tissue were detected by the terminal deoxynucleotidal transferase-mediated deoxyuridine 5'-triphosphate nick end-labeling (TUNEL) method using an Apop Tag in situ apoptosis detection kit and peroxidase (Oncor, Inc., Gaithersburg, MD), as previously described (15).

Immunohistochemistry

Immunohistochemical staining was performed using anti-human Fas mouse monoclonal antibodies (UB2, IgG1, Medical & Biological Laboratories Co., Nagoya, Japan), anti-human Bcl-2 mouse monoclonal antibody (124, IgG1, DAKO Corp., Glostrup, Denmark), anti-human Fas ligand monoclonal antibodies (NOK-1, IgG1 and NOK-2, IgG2a) (14), and mouse IgG1 and IgG2a (DAKO Corp.) as negative controls. Five-micron cryostat sections were stained with antihuman Fas monoclonal antibody (UB2), which was recommended to be used for frozen tissue sections. Paraffin-embedded tissue sections were stained with anti-Bcl-2 monoclonal antibody, anti-FasL monoclonal antibodies (NOK-1 and NOK-2), or control antibodies. Positive reactivity was identified using an avidin-biotin-peroxidase detection system (DAKO Corp. LSAB kit) for the detection of Fas and a catalyzed signal amplification system (DAKO Corp.) for the detection of Bcl-2 and FasL, as previously reported (15, 16).

Confocal laser scanning microscopy

To confirm the expression of FasL in thyrocytes, immunofluorescent cytochemistry for FasL was performed and observed using a confocal laser scanning microscope (LSM-GB200, Olympus Corp., Tokyo, Japan) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm, as previously described (17). Briefly, frozen tissue sections or cultured thyroid cells were fixed with cold acetone for 10 min or with 4% paraformaldehyde-phosphate-buffered saline (PBS) for 1 h at 4 C, respectively. After blocking the nonspecific binding sites by rabbit or goat serum, specimens were incubated with 1:100 diluted antihuman FasL monoclonal antibody (NOK-2) or polyclonal antibody (C-20, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight. After washing with PBS containing 0.05% Tween-20, specimens were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG F(ab')₂ or fluorescein isothiocyanate-conjugated anti-rabbit IgG for 40 min. After further washing with PBS, specimens were mounted using Vectashield (Vector Laboratories, Inc., Burlingame, CA). Human FasL cDNA transfected BHK cells were also stained as a positive control.

Detection of FasL messenger ribonucleic acid (mRNA) by RT-PCR and Northern blotting

Total RNA was isolated from thyroid tissue from patients with GD and control subjects or from cultured thyroid cells using RNAzol B (Biotech, Houston, TX), as previously reported (18). The cDNA was synthesized from 0.5 µg total RNA, as previously described (18). The following primers were used for PCR of human FasL cDNA (19): forward primer, 5'-GTCCAACCTCTGTGCCCAGAAGGC-3' (nucleotides 180–203); and reverse primer, 5'-ATTCCATAGGTGTCTTCCCATTCCAG-3' (nucleotides 588–563). Paired primers for glyceraldehyde-3-phosphate dehydrogenase were used as controls (18). A thermal cycle was 1 min at 94 C, 1 min at 55 C, and 2 min at 72 C, and the PCR was performed for 35 cycles. The last extension was carried out for 8 min at 72 C. PCR products were applied to a 2% agarose gel and stained with 0.5 µg/mL ethidium bromide.

Northern blot analysis was performed as previously described (18), using antisense RNA probe generated with T3 polymerase from a XhoI-linearized pBluescript SK(+) plasmid carrying a 850-bp fragment of human FasL cDNA (19).

Flow cytometry

Cultured thyroid cell monolayers 1–10 days after setting up the primary culture were trypsinized and then used for the flow cytometric analysis. The surface expression of Fas and FasL proteins and the intracellular expression of FasL and Bcl-2 proteins in cultured thyrocytes were assessed by flow cytometry using anti-Fas (UB2, IgG1 and DX2, IgG1, PharMingen, San Diego, CA) and anti-Bcl-2 (DAKO Corp.) monoclonal antibodies and phycoerythrin- or biotin-labeled monoclonal antibodies against FasL (NOK-1 and NOK-2) and FACScan (Becton Dickinson and Co., Mountain View, CA) as previously reported (20).

Enzyme-linked immunosorbent assay (ELISA)

Soluble FasL in culture supernatant was quantitated by sandwich ELISA using monoclonal antibodies against human FasL (NOK-1 and NOK-3, IgM), as previously described (15).

Statistics

Results were analyzed by paired Student’s t test. P < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detection of apoptosis in thyroid tissue

We investigated whether apoptosis occurred in the thyroid tissues from patients with GD as assessed by the TUNEL method. Positive staining of nuclei was detected in lymphoid follicle area (Fig. 1A) and occasionally in thyrocytes from patients with GD (Fig. 1, B and C). Approximately 0.5–5.0% of thyrocytes were positive in GD thyroids. In normal thyroid glands a few positive nuclei (0–0.5%) were observed (Fig. 1D).

View larger version (150K): [in this window] [in a new window]   Figure 1. In situ detection of apoptosis in thyroid tissues from patients with GD by the TUNEL method. Paraffin sections of thyroid tissues from patients with GD (A–C) and a control subject (D) were stained for apoptotic cells with nuclear DNA fragmentation.

Fas expression was clearly detected on the basal surface of both GD thyrocytes (Fig. 2A) and normal thyrocytes (Fig. 2B). Intense expression of Bcl-2 was observed in the cytoplasm of both GD thyrocytes (Fig. 2C) and normal thyrocytes (Fig. 2D).

View larger version (96K): [in this window] [in a new window]   Figure 2. Fas, Bcl-2, and FasL expression in thyroid tissues. Frozen tissue sections from a patient with GD (A) and a control subject (B) were stained with monoclonal antibodies against human Fas (UB2, IgG1) using the DAKO Corp. LSAB kit. Paraffin sections from GD patients (C and E–G) and a control subject (D and H) were immunohistochemically stained with monoclonal antibodies against human Bcl-2 (IgG1; C and D), human FasL (NOK-2, IgG2a; E, F, and H), and control antibody (IgG2; G) using the DAKO Corp. CSA system kit.

Fas ligand expression in thyroid tissue and cultured thyrocytes from GD patients

To investigate the expression of FasL in thyroid tissues from GD patients, we performed immunohistochemistry using monoclonal antibodies against human FasL. FasL was clearly detected in the thyroid tissues from three of four patients with GD (Fig. 2, E and F). In contrast, FasL was not detected in any of the three normal control thyroid tissues (Fig. 2H).

The expression of FasL in thyrocytes was confirmed by confocal laser microscopy. FasL was clearly detected in thyroid cells from three patients with active GD (Fig. 3, A and B), but not in a patient with GD in remission (Fig. 3C) or in two normal controls (Fig. 3D), as determined using both monoclonal (Fig. 3, A–D) and polyclonal (Fig. 3B) anti-FasL antibodies. FasL expression was detectable in cultured thyrocytes only after treatment with a matrix metalloproteinase inhibitor (10 µmol/L KB8301; Kanebo Ltd. Co., Osaka, Japan) (14) that inhibits the shedding of FasL (Fig. 3, E and F). Cell surface expression of FasL on cultured GD thyrocytes was also observed only after the KB8301 treatment as measured by flow cytometric analysis (Fig. 4), but not in normal thyrocytes. The percentage of positive cells in GD thyrocytes was 5.0–21.9% after treatment with 10 µmol/L KB8301 for 24 h and 0.3–3.2% without the treatment.

View larger version (158K): [in this window] [in a new window]   Figure 3. FasL expression in thyrocytes assessed by confocal laser microscopy. Frozen tissue sections from a TSH receptor antibody-positive patient with GD currently being treated with methimazol (A and B), a TSH receptor antibody-negative patient with GD after successful treatment with methimazol (C), a control subject (D), and cultured thyrocytes from a patient with hyperthyroid GD (E and F) were stained with either a monoclonal antibody (NOK-1, IgG1; A and C–G) or a polyclonal antibody (C-20; B) against human FasL. E, Two-day cultured thyrocytes; F, 2-day cultured thyrocytes treated with matrix metalloproteinase inhibitor (10 µmol/L) for 24 h; G, human FasL cDNA-transfected BHK cells stained with NOK-1 as positive controls.

View larger version (14K): [in this window] [in a new window]   Figure 4. Accumulation of FasL on the surface of thyrocytes by matrix metalloproteinase inhibitor (MMPI) treatment. Ten-day cultured thyrocytes were incubated with 10 µmol/L MMPI for 24 h and then assessed for the surface expression of FasL with monoclonal antibody against FasL (NOK-2) by FACS (solid lines). A, Without MMPI; B, with MMPI. Dotted lines indicate background staining with a control monoclonal antibody.

FasL mRNA expression in thyroid tissues from GD patients

To further define the expression of FasL, we investigated the in vivo expression of FasL mRNA by Northern blot analysis and RT-PCR. FasL mRNA was detected only in thyroid tissue from two of five patients with GD, but not in normal thyroids as determined by Northern blot analysis (Fig. 5A). In RT-PCR analysis, FasL cDNA was amplified in thyroid tissues from five of seven patients with GD, but in only one of four normal controls (Fig. 5B).

View larger version (33K): [in this window] [in a new window]   Figure 5. Northern blot analysis (A) and RT-PCR analysis (B) of FasL mRNA expression in thyroid tissues. Total RNA was isolated from frozen tissues from patients with GD (1 and 2, G1–G4) and control subjects (3, N1–N4). Northern blot and RT-PCR analyses were performed. M, One hundred-base pair DNA ladder.

To investigate whether the GD thyrocytes express functional FasL, we set up in vitro coculture of GD or normal thyrocytes with human Fas transfectants (hFas/WR19L). As shown in Fig. 6D, treatment of hFas/WR19L with an anti-Fas IgM antibody (CH-11, Medical & Biological Laboratories; 1 µg/mL) for 24 h induced segmentation of nuclei and cell shrinkage, which are characteristic morphological changes of apoptosis. When hFas/WR19L cells were cocultured with GD thyrocytes, similar apoptotic changes were observed (Fig. 6A). A neutralizing anti-FasL monoclonal antibody (NOK-2; 10 µg/mL) inhibited the apoptotic changes (Fig. 6C). The parental WR19L cells lacking Fas did not show the apoptotic changes (Fig. 6B). In contrast, normal thyrocytes did not induce the apoptotic changes in the hFas/WR19L cells (Fig. 6E). These results indicate that the GD-derived thyrocytes, but not normal thyrocytes, expressed functional FasL.

View larger version (151K): [in this window] [in a new window]   Figure 6. FasL-mediated cytotoxic activity of thyrocytes against Fas transfectants. Ten-day cultured GD thyrocytes were cocultured with hFas/WR19L (A and C) or control WR19L (B) cells in the presence (C) or absence (A and B) of NOK-2 for 24 h. D, Culture of hFas/WR19L with anti-Fas monoclonal antibody (CH-11, IgM; 1 µg/mL). E, Culture of hFas/WR19L with 10-day cultured normal thyrocytes for 24 h. F, Ten-day cultured GD thyrocytes were cultured with anti-Fas monoclonal antibody (CH-11; 1 µg/mL) for 24 h.

Although both GD and normal thyrocytes expressed Fas on the surface, they were resistant to both an agonistic anti-Fas monoclonal antibody (CH-11; 1 µg/mL; Fig. 6F) and soluble FasL (50 ng/mL; data not shown). The pretreatment of thyrocytes for 2 days with interferon- (IFN; 50–1000 U/mL), interleukin-1 (10–500 U/mL), or tumor necrosis factor- (TNF; 50–1000 U/mL) did not increase the susceptibility to anti-Fas monoclonal antibody (CH-11)-induced apoptosis (data not shown).

TSH suppresses IFN-induced up-regulation of Fas expression on thyrocytes

To investigate the mechanisms for thyrocyte resistance to the Fas-mediated apoptosis, we studied the effect of TSH (human recombinant TSH, Sigma Chemical Co.) and 8-bromo-cAMP (Sigma Chemical Co.) on Fas expression on GD thyrocytes. IFN induced Fas expression on cultured thyrocytes (Fig. 7). Both TSH and 8-bromo-cAMP suppressed the induction of Fas expression on IFN--stimulated thyrocytes in a dose-dependent manner (Fig. 7).

View larger version (50K): [in this window] [in a new window]   Figure 7. Effects of TSH and 8-bromo-cAMP on IFN-induced Fas expression on thyrocytes. Two-day cultured thyrocytes were incubated with IFN (200 U/mL) and various concentrations of human TSH or 8-bromo-cAMP for 2 days. Fas expression on thyrocytes was analyzed by FACS using monoclonal antibody against human Fas (UB2). *, P < 0.05; **, P < 0.01

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

FasL is a type II membrane protein that belongs to the TNF family. FasL was initially reported to be expressed in activated T cells and NK cells (1). Recent studies have revealed that FasL is also expressed in the immune-privileged sites, such as retina (21) and testis (22). More recently, it has been reported that some tumor cells, including those of epithelial origin, expressed FasL (4, 5). In thyroid diseases, Giordano et al. (11) reported the constitutive expression of FasL on thyrocytes using polyclonal antibody C-20 and monoclonal antibody clone 33, and they postulated that the Fas/FasL system might cause thyrocyte damage in HT. Mitsiades et al. (23) also reported the increased expression of FasL in thyrocytes in HT using the same polyclonal antibody. However, Fiedler et al. (24) brought into question the specificity of these antibodies by flow cytometry, immunofluorescence, or immunohistochemistry because of nonspecific staining unrelated to FasL. Furthermore, Stokes et al. (25) claimed the expression of FasL on the surface of thyrocytes with the use of RT-PCR, ribonuclear protection techniques, immunohistochemical staining, and protein immunoassays. They failed to show the expression of mRNA for FasL in primary cultured thyrocytes from normal and thyroiditis tissue samples. Rymaszewski et al. (12) also failed to show FasL mRNA expression in GD thyroid tissue by immunohistochemistry. In our study, we employed monoclonal antibodies against human FasL, NOK-1 and NOK-2, which are specific for human FasL in flow cytometry and immunofluorescence (14, 24). FasL expression was only observed in GD thyroids, not in the normal thyroids. The discrepancies in FasL mRNA expression in thyroid tissue among them may be due to the differences in methods, or patient characteristics, including disease activity, preoperative treatment, and degree of lymphocytic infiltration, may explain the apparent discrepancies. The localization of FasL in GD thyrocytes was mainly in the cytoplasm. Cell surface expression of FasL on GD thyrocytes was detectable by flow cytometry only after treatment with a matrix metalloproteinase inhibitor that inhibits the shedding of FasL.

Furthermore, cultured thyrocytes from GD, but not those from normal, thyroids exhibited consistent FasL-mediated cytotoxic activity against Fas-bearing target cells, indicating that the GD-derived, but not normal, thyrocytes express functional FasL. A substantial amount of soluble FasL was always found in the supernatant of cultured GD thyrocytes. These results suggest that the expression of FasL in thyrocytes might be induced by some factor associated with the pathogenesis of GD. In our preliminary experiments, we examined the effects of IFN, TNF, interleukin-1, and/or TSH, but none of these factors could induce FasL expression in cultured normal thyrocytes (data not shown). It has been reported that some anticancer drugs, such as daunorubicin and bleomycin, could induce FasL expression in some tumor cells (7). In our preliminary experiment, we found that bleomycin could induce FasL expression in thyrocytes, suggesting that DNA damage might be a trigger for FasL induction (unpublished observations). Further studies will be required to elucidate the mechanisms responsible for the FasL induction in GD thyrocytes.

Although the thyrocytes from GD patients expressed both Fas and FasL, apoptosis was only occasionally found in the GD thyroids. Indeed, cultured thyrocytes were resistant to Fas-mediated apoptosis induced by anti-Fas antibody or soluble FasL (data not shown). The increased expression of Bcl-2, which is an inhibitor of programmed cell death (26), in GD thyrocytes may inhibit the process of Fas-mediated apoptosis, although it is controversial whether Bcl-2 inhibits Fas-mediated apoptosis (26, 27, 28, 29). Kawakami et al. (8, 29) reported that TSH and GD Igs inhibited the Fas-mediated apoptosis of normal thyrocytes. We also demonstrated that TSH and 8-bromo-cAMP suppressed the IFN-induced Fas up-regulation on cultured GD thyrocytes. This may explain why GD thyrocytes rarely undergo apoptosis. Alternatively, TSH stimulation may elicit some protective mechanism against the Fas-mediated apoptosis in thyrocytes. It is noteworthy that the pathogenesis of GD is associated with serum autoantibodies to TSH receptor. The anti-TSH receptor autoantibodies may act like TSH to prevent the Fas/FasL-mediated apoptosis of thyrocytes, thus leading to thyroid hyperplasia, although the explanation provided by Arscott et al. (30), indicating that the Fas death pathway is normally blocked by a protein inhibitor in thyrocytes, appears more reasonable.

In conclusion, the increased expression of FasL in thyrocytes of patients with GD may help to maintain homeostasis in the thyroid by eliminating infiltrating lymphocytes and hyperplastic thyrocytes by Fas-mediated apoptosis. Overexpression of Bcl-2, aberrant stimulation with anti-TSH receptor autoantibodies, and a labile protein inhibitor may render thyrocytes resistant to the Fas-mediated apoptosis and thus lead to the thyroid hyperplasia that characterizes GD. Further studies are now under way to clarify the correlation between FasL expression in the thyroid and the clinical course of GD.

	Acknowledgments

 
We thank Dr. Howard A. Young (Laboratory of Experimental Immunology, DBS, NCI-Frederick Cancer Research and Development Center, Frederick, MD) for scientific discussion.

Received July 9, 1998.

Revised January 4, 1998.

Accepted February 8, 1999.

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