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Departments of Medicine (P.L.A., J.K., M.R., J.L.B., J.D.B., J.R.B.) and Surgery (N.W.T.), University of Michigan Medical School, Ann Arbor, Michigan 48109-0666
Address all correspondence and requests for reprints to: James R. Baker, Jr., University of Michigan Medical School, 1520 MSRB I, Ann Arbor, Michigan 48109-0666.
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or
interleukin-1ß to the anti-Fas-treated cell cultures also did not
promote apoptotic signaling through this pathway. In contrast, the
concomitant administration of cycloheximide allowed the induction of
apoptosis through the activation of Fas in thyrocytes. These results
suggest that Fas is constitutively expressed in thyrocytes, but that
the induction of apoptosis through the Fas pathway is blocked by a
labile protein inhibitor. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Currently, the mechanisms by which thyroid cells undergo apoptosis in
thyroiditis are not well defined. One potential pathway involves
activation of Fas antigen on thyroid cells by Fas ligand present on
lymphocytes. Fas antigen is a type I membrane protein and a member of
the nerve growth factor/tumor necrosis factor receptor family (6, 7).
It is found constitutively expressed on lymphocytes and has been
detected on some cells of nonhematopoeitic origin (7, 8). Activation of
Fas antigen by Fas ligand initiates intracellular signals that result
in the death of the cell through activation of caspases (7, 9).
Regulation or modulation of this pathway can occur at multiple levels
throughout the pathway including changes in the level of the expression
of Fas antigen or its ligand (7, 8, 10, 11), the regulation of
components of intracellular signaling (12, 13, 14, 15), and the expression of
proteins that promote cell survival such as members of the Bcl-2 gene
family (2, 14, 15, 16). It is possible that this system plays a role in the
pathogenesis of thyroiditis, because CD8 lymphocytes are abundantly
present in the thyroid in areas where apoptosis is observed, and the
Fas pathway is a major mechanism of CD8-mediated cytotoxicity (7, 17).
However, two recent reports have presented differing conclusions as to
the expression and regulation of Fas and the induction of apoptosis on
thyroid cells. One report indicated that Fas antigen is constitutively
expressed on thyroid cells and is down-regulated by the addition of TSH
(18). These studies also suggested that thyroid Fas antigen does not
function to induce apoptosis unless the cells are exposed to
interferon- (IFN-). However, this report only used a single
technique to examine the cell surface expression of Fas protein (flow
cytometry) and did not examine the expression of Fas messenger RNA
(mRNA). Another recent publication suggested that Fas antigen was not
constitutively expressed on thyroid cells, but that its expression was
induced by interleukin-1ß (IL-1ß) leading to autologous apoptosis
by thyrocyte expressed Fas ligand (19). However, these investigators
did not use a cell death assay specific for apoptosis (rather than
necrosis) and looked only at Fas protein expression. In addition, the
acute and massive apoptosis that would be expected from autologous Fas
activation is not observed in thyroiditis.
To determine whether thyroid cell apoptosis can be related to
activation of the Fas pathway, we examined the expression and function
of Fas antigen on thyroid follicular cells. We used two different
techniques (immunohistochemical staining and western blotting) to
examine Fas antigen protein expression, and two techniques
(ribonuclease protection and RT-PCR) to examine and quantify mRNA for
Fas antigen. We also employed techniques specific for apoptotic cell
death (detection of fragmented DNA) and quantitative cell viability
[3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
conversion] to determine thyroid cell susceptibility to apoptosis from
Fas activation, and the role of the inflammatory cytokines IL-1ß and
IFN- in modulating this process.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thyroid cell culture
Thyroid tissue was digested overnight with 40 mg collagenase, 4
mg hyaluronidase, and 4 mg DNase I (all from Sigma Chemical Co., St.
Louis, MO) in 40 ml RPMI-1640 (GIBCO-BRL). Red blood cells were lysed
with ammonium chloride lysis buffer (0.15 M
NH4Cl, 10 mM KPO4, and 1
mM EDTA, pH 7.3), and cells were cultured in RPMI-1640 with
20% NuSerum IV (Collaborative Biomedical Products, Bedford, MA) at 37
C in 5% CO2. After 24 h, nonadherent cells were
removed by washing with RPMI. Adherent cells were weaned over 5 days
from 20% NuSerum/RPMI to Cellgro media (Mediatech, Herndon, VA) either
with or without supplementation of 10 mIU/ml bovine TSH every 1–2 days
(Sigma Chemical). To assure that lymphocytes were not present in
samples analyzed for RNA, cell cultures were washed daily with removal
of the nonadherent cells and supernatant.
RT-PCR
RNA was isolated from cells using TRI Reagent (Molecular
Research Center, Cincinnati, OH). RT was performed on 2–4 µg total
RNA by first annealing with 100 ng oligo(dT)18 for 10 min
at 70 C. The reaction was then brought to a final volume of 20 µl
with 0.5 mM deoxynucleotide triphosphate, 32 U RNase
inhibitor, 10 mM dithiothreitol, and 200 U SuperScript II
reverse transcriptase in 1x RT buffer (all from Boehringer Mannheim
Corp., Indianapolis, IN). A parallel reaction without reverse
transcriptase was included as a negative control. Incubation at 42 C
for 1 h was followed by 10 min at 94 C, after which the samples
were kept at 4 C. PCR was carried out using 1/20 (1 µl) of the RT
reaction as a template cDNA with sequence-specific primers for Fas,
thyroglobulin (Tg), thyroid peroxidase (TPO), or ß-actin. Each
20-µl reaction contained 0.2 mM deoxynucleotide
triphosphate, 1x AmpliTaq PCR buffer (Perkin Elmer Corp.,
Foster City, CA) containing 1.5 mM MgCl2, 40 ng
each specific forward and reverse primer (listed below), and 0.5 U
AmpliTaq DNA polymerase (Perkin Elmer). PCR was performed on
a GeneAmp 2400 (Perkin Elmer) for 30 cycles of 94 C for 30 sec, 60 C
for 30 sec, and 72 C for 45 sec, followed by a single incubation at 72
C for 7 min then 4 C. One half of each PCR reaction was evaluated for
size and relative quantity of product by loading onto a 1.5% agarose
gel. Amplified products were visualized by staining with ethidium
bromide.
Primers (complementary nucleotides):
Fas (710–1019) forward-TAACTTGGGGTGGCTTTGTCT reverse-AACTTTCTGTTCTGCTGTGTCTTG
Tg (104–866) forward-CTTCGAGTACCAGGTTGATGCC reverse-GGTGGTTTCAGTGAAGGTGGAA
TPO (1512–2105) forward-TGTGTCCAACGTGTTCTCCACAG reverse-AAGACGTGGCTGTTCTCCCAC
ß-actin (258–657) forward-CACGGCATTGTAACCAACTG reverse-TCTCAGCTGTGGTGGTGAAG
Ribonuclease protection assay.
RiboQuant MultiProbe RNase Protection Assay System (Pharmingen,
San Diego, CA) was used for the detection and quantitation of specific
mRNA species. The following 32P-labeled antisense RNA
probes were prepared using the Human Apoptosis hAPO-3 Template Set
(Pharmingen), which included human Fas and human glyceraldehyde
3-phosphate dehydrogenase (GAPDH). The probes were hybridized with 10
µg total RNA (from treated thyrocytes), 10 µg poly A+ RNA from a
human B-cell lymphoma cell line (BJAB, positive control for Fas
message), 2 µg HeLa RNA (assay positive control), and 2 µg yeast
transfer RNA (background control). After hybridization, the samples
were subjected to RNase treatment followed by purification of
RNase-protected probes. The protected probes were resolved on a 5%
denaturing polyacrylamide gel. The quantity of specific transcripts
present was analyzed by autoradiography and quantified using a 445 SI
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Relative amounts of
Fas message were corrected for RNA loading by comparing with the GAPDH
band intensity for each sample.
Protein isolation and Western blot
Cellular proteins were isolated from TRI Reagent lysates of
thyrocytes treated with or without TSH. After removal of the RNA
fraction, proteins were precipitated from the phenol-ethanol
supernatant, washed, and solubilized in 1% SDS following the vendor’s
protocol. Total protein concentration was determined using BCA Protein
Assay Reagent (Pierce Chemicals, Rockford, IL). SDS-PAGE to separate
proteins was performed using standard techniques. Fourteen micrograms
of total protein were mixed with 2x sample buffer (4% SDS, 10%
ß-mercaptoethanol, 20% glycerol in 0.125 M Tris, pH 6.8)
containing bromophenol blue, boiled for 3 min, and then separated by
electrophoresis through a 12.5% SDS-polyacrylamide gel. A portion of
the gel was stained with Coomassie blue to detect total protein
content, and duplicate lanes were electrophoretically transferred to
nitrocellulose. The membrane was then blocked with 10% milk in PBS
with 0.02% sodium azide (PBS-A) for 2 h at 27 C. Specific
proteins were detected using polyclonal rabbit anti-Fas (C-20 or N-18,
Santa Cruz Biotechnology, Santa Cruz, CA) at 1 µg/ml or a monoclonal
mouse IgG anti-TPO (FKA-10, a gift from Dr. Leslie DeGroot, University
of Chicago) at 1:500 dilution. Incubation of primary antibody (Ab) for
16 h at 4 C was followed by washes with PBS-A with 0.05% Tween-20
then incubation with alkaline phosphatase-conjugated Abs against rabbit
IgG (Sigma Chemical) or against mouse IgG (Jackson ImmunoResearch
Labs., West Grove, PA) for 1–2 h at 27 C. After washing, the blots
were developed using a BCIP/NBT substrate (0.05 mg/ml
5-bromo-4-chloro-3-indolyl phosphate, 0.1 mg/ml nitro blue tetrazolium,
4 mM MgCl2, in 0.15 M Tris/0.1
M NaCl, pH 9.6). The Western blots and protein-stained gel
were scanned into TIFF format files in Adobe Photoshop for Macintosh
(Adobe Systems, Mountain View, CA) using a flatbed scanner. Relative
band intensities were evaluated using IPLab Gel 2.0a (Signal Analytics
Corp., Vienna, VA).
Immunostaining
Thyroid cells were grown on Falcon chamber slides (Becton
Dickinson, Franklin Lanes, NJ) or on glass coverslips. For
immunostaining, slides were washed twice with PBS then fixed with
methanol for 5 min at 4 C and briefly air dried and placed into PBS and
blocked with 5% normal goat serum. Slides were incubated with 1.0
µg/ml affinity-purified rabbit anti-Fas (C-20 or N-18, Santa Cruz
Biotechnology), rabbit antithyroglobulin (Dako Corp., Carpinteria, CA),
or ChromPure rabbit IgG (Jackson ImmunoResearch Labs.) in 1.5% normal
goat serum in PBS for 18 h at 4 C. Mouse monoclonal Ab (mAb) to
Fas, IgG1 clones UB2 and ZB4 (MBL International, Watertown,
MA), and IgM clone CH11 (Upstate Biotechnology, Lake Placid, NY), and
purified mouse IgG1 or mouse IgM (Sigma Chemical) as
controls, were also used at 1.0 µg/ml. After washing with PBS, slides
were incubated with biotinylated Abs specific for rabbit IgG, mouse
IgG, or mouse IgM, followed by detection using an avidin-biotin complex
detection kit with glucose oxidase substrate (Vectastain ABC-GO kit,
Vector Labs., Burlingame, CA). Slides were briefly counterstained
with eosin and mounted with permount (Fisher Scientific, Fair Lawn,
NJ).
Induction of apoptosis
After weaning to Cellgro media, cultured thyrocytes received
pretreatment with or without 500 IU/ml human IFN- (Boehringer
Mannheim) for 48 h and were grown with or without TSH
supplementation. Cells were then treated for 24 h with 0.4–0.8
µg/ml mouse monoclonal IgM anti-Fas, (clone CH11, Upstate
Biotechnology) or purified mouse IgM (Sigma Chemical) as a control.
Some cells also received 10 µg/ml cycloheximide (CHX) (Sigma
Chemical) with the IgM Abs. Staurosporine treatment at 2
µM was included for positive induction of apoptosis in
thyrocytes (9, 16).
Detection of apoptosis
Apoptosis was evaluated by examination of morphological changes
in the cells such as membrane blebbing and cells rounding up and
becoming nonadherent. In addition, the ApopTag Plus in situ
apoptosis detection kit (Oncor, Gaithersburg, MD) was used to detect
fragmented DNA specific for apoptosis in thyrocytes grown on
coverslips. Briefly, cells were fixed with 4% formalin, then
endogenous peroxidase was quenched, and DNA was labeled with
digoxigenin using terminal deoxynucleotidyl transferase. Subsequent
incubation with peroxidase conjugated antidigoxigenin followed by
staining with diaminobenzidene substrate resulted in brown stained
apoptotic nuclei. Methyl green was used as a counterstain for
unaffected nuclei. Cells that became nonadherent were collected, fixed,
and dried onto slides then stained as described.
Quantitation of cell death
Measurement of cell death used the MTT assay (20). Cultured
thyrocytes were plated in triplicate in 96-well plates at the same cell
density of 5,000–10,000 cells/well in 100 µl. Cells were treated to
induce apoptosis as described. Metabolically active cells were then
detected by adding MTT to a final concentration of 0.5 mg/ml for 4
h at 37 C. Isopropanol with 0.04 N HCl was added (100 µl/well), and
the wells were mixed to solubilize the purple-colored product. Optical
densities were read using a Microplate Reader (model 450, Bio-Rad
Labs., Hercules, CA) at 595 nm with 630 nm reference
wavelength.
Statistic analysis
Mean and SD of results were calculated using
StatView software (Abacus Concepts, Berkeley, CA) on a Power Macintosh
Computer. Means were compared for differences by ANOVA with
significance set at a 95% confidence level.
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Results |
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). The presence of message for TPO and
Tg served as controls for the thyroid origin of this RNA. ß-actin
message was amplified equivalently for each sample, indicating that
approximately equal amounts of RNA were present. No specific bands for
Fas, Tg, TPO, or ß-actin were amplified from duplicate samples that
were not reverse transcribed.
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).
Quantitative analysis of Fas message was performed using a ribonuclease
protection assay system (RiboQuant, Pharmingen), with RNA
concentrations in the analyzed samples corrected based on the amount of
GAPDH transcript detected. The levels of Fas transcript were equivalent
between the samples (0.26 ± 0.04 U) regardless of TSH treatment
(Fig. 2). IFN- pretreatment also did
not appear to substantially alter the amount of Fas mRNA isolated from
thyrocytes as determined by RT-PCR. The ribonuclease protection assay
confirmed this as it measured less than twice as much Fas mRNA in
IFN--treated cells compared with untreated cells (0.27 U with
IFN- vs. 0.19 U without IFN-).
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). The detected
band at 46 kDa corresponds to the published molecular weight of human
Fas antigen of 43–48 kDa (6) and corresponded to a similar sized band
in BJAB cell extracts (data not shown).
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). The FKA-10 mAb
specific for TPO detected a band corresponding to TPO only in
TSH-treated cultures (lanes 1 and 3), but not in cells deprived of TSH
(lanes 2 and 4). This indicated that treatment with TSH had effectively
induced TSH-specific promoter elements but had not altered the levels
of Fas protein. Normal and MNG thyrocytes demonstrated no significant
increase in Fas protein expression as a result of IFN- pretreatment
of thyrocytes as detected by Western blot (data not shown).
Fas protein was demonstrated on thyrocytes in vitro by
immunohistochemical staining using rabbit polyclonal Abs to Fas antigen
and Tg (Fig. 4). Similar patterns and
intensity of staining were observed in all four patients examined.
Diffuse staining with rabbit anti-Fas Ab was observed in the majority
of cells, although the intensity varied. Rabbit polyclonal Abs to both
the amino terminus (N-18) and to the carboxyl terminus (C-20) of Fas
produced identical staining results (data not shown). Three mouse mAbs
against Fas, clones UB2 and ZB4, and clone CH11, were also used to
confirm Fas expression. These Abs positively stained thyrocytes in
comparison with mouse control mAbs of the same isotypes. The pattern of
staining with the mouse mAbs was similar to but less intense than that
seen with the rabbit anti-Fas Abs (not shown). Intense anti-Tg staining
was evident in the cytoplasm of nearly all of the cells and served as a
positive control, indicating that the cells were indeed thyrocytes.
Control rabbit Ab produced no background staining of the thyrocytes.
The expression of Fas antigen was equivalent on cells grown with (Fig. 4, upper left) or without TSH (Fig. 4, upper
right), whereas decreased intensity of staining for Tg in cells
grown without TSH (Fig. 4, lower right) compared with cells
grown in the presence of TSH (Fig. 4, lower left) verified
the depletion of TSH in these cultures. Staining for Fas on thyrocytes
from a MNG patient after treatment with IFN- did not demonstrate any
changes in the amount of Fas (data not shown).
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, A and B). Treatment with
staurosporine, which activates apoptosis through caspases directly, was
used as a positive control and induced massive apoptosis in all the
thyrocyte cultures. All of these cells became nonadherent and rounded
with fragmented DNA detected in nearly all nuclei (Fig. 5C).
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augmented the induction of apoptosis by
anti-Fas and CHX (Fig. 5D) to the point that the percent of nuclei with
fragmented DNA was similar to thyrocytes treated with staurosporine. In
contrast, thyrocytes treated with only IFN- in combination with
anti-Fas inducing mAb (Fig. 5A) or treated with control IgM Ab with CHX
and IFN- (Fig. 5B) failed to induce apoptosis. Control treatment
with CHX alone or in combination with IFN- similarly did not induce
apoptosis (similar to Fig. 5, A and B). To determine whether TSH
treatment altered susceptibility to the induction of apoptosis through
the Fas pathway, thyrocytes were deprived of TSH and showed no
difference from those cultured with TSH in their response to anti-Fas
mAb (data not shown). Treatment with CHX was still required to induce
apoptosis in the TSH-deprived thyrocytes, and IFN- alone did not
generate susceptibility to the induction of apoptosis in these
cells.
Quantitation of apoptosis induced through the Fas pathway
The Fas-mediated induction of cell death in thyrocytes was also
measured using the MTT assay to detect metabolically active cells.
Treatment of thyrocytes with anti-Fas mAb failed to show significant
cell death compared with control IgM Ab-treated cells (Fig. 6). The addition of CHX resulted in
approximately 60% cell death in anti-Fas mAb-treated thyrocytes
compared with CHX plus control IgM Ab-treated thyrocytes
(P < 0.01) (Fig. 6). The presence of CHX did not
affect cell viability in control IgM-treated cells, and comparisons
were made between similarly treated cells receiving either anti-Fas mAb
or control IgM Ab. The presence or absence of TSH again did not alter
the response of thyrocytes to treatment with inducing Ab alone or to
combined treatment with CHX and Ab (Fig. 6).
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or IL-1ß before
induction with anti-Fas mAb alone failed to promote death. However,
when either cytokine was added before CHX plus anti-Fas mAb treatment,
overall cell death increased to greater than 90% of treated cells
compared with similarly treated cultures using control Ab
(P < 0.0001 for either IFN- and IL-1ß
vs. control value) (Fig. 7)
and was equivalent to the quantitative cell death observed with
staurosporine treatment (data not shown). This pattern of apoptotic
response to anti-Fas, CHX, and inflammatory cytokines was consistent
for thyrocytes from the three different donors (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Fas was also present in thyrocytes obtained from patients
with Graves’ disease and MNG. The expression of the thyroid-specific
proteins thyroid peroxidase and thyroglobulin was regulated by TSH, as
previously reported (21, 22, 23), and documented the efficacy of TSH
treatment in these cells. Together, this suggests that Fas is normally
present on thyroid cells, and that the expression of Fas antigen is not
altered by TSH. Despite the presence of Fas antigen on thyroid cells, apoptosis of thyrocytes was not induced by the CH11 anti-Fas mAb that cross-links Fas and activates apoptosis through this pathway (24). CHX, an inhibitor of protein translation, has been used to increase the sensitivity of cells to induction of apoptosis by the Fas pathway by selectively reducing the relative concentrations of labile protein inhibitors (25, 26). This agent was able to restore anti-Fas mAb susceptibility in thyrocytes, in a manner similar to that observed in other cell types in which the Fas pathway is blocked by inhibitors (26). Given that Fas appears to be constitutively expressed on most cells, it appears that the major regulation of apoptosis in this pathway is distal to the receptor. Supporting this concept, Fas-mediated apoptosis is specifically blocked in lymphocytes and thymocytes despite the expression of Fas (27, 28). In addition, viral protein inhibitors that compete with the Fas-associating protein with death domain (FADD) and specifically block apoptosis have recently been described (29). Thus, it appears that the type of inhibition observed in thyroid cells is also observed in other cells and circumstances.
In contrast to previous reports (18, 19) our studies show pretreatment
with either IFN- or IL-1ß did not significantly alter the
expression of either Fas message or protein, and did not enhance
sensitivity to Fas-induced cell death in the absence of CHX. This
suggests that that the rate-limiting step in apoptosis is likely to be
a labile inhibitor that blocks signal transduction and not the
cytokine-induced expression of Fas. Because staurosporine, which
activates apoptosis directly through caspase activation (9), induced
apoptosis in the absence of CHX, it is likely that the inhibitor is
operative in the Fas-specific signaling that occurs before the common
pathway. Therefore, the inhibitor in thyroid cells most likely operates
after Fas receptor engagement but before the activation of
caspases.
Exposure to TSH results in thyroid hyperplasia in vivo (30). This is thought to occur primarily through the proliferation of thyrocytes (reviewed in Ref.31). However, as a growth factor for thyrocytes, TSH also may be expected to decrease the sensitivity of thyrocytes to the induction of apoptosis. In this study, exposure to TSH did not alter the sensitivity of thyrocytes to Fas-induced apoptosis in vitro. These results suggest that the primary mechanism of TSH in promoting the growth of the thyroid is through proliferation, but does not rule out possible suppression of apoptotic pathways other than Fas.
Several recent studies have examined the expression of Fas on thyrocytes. In agreement with our findings, two studies have documented that Fas is constitutively expressed on both normal thyroid tissue (tissue adjacent to thyroid tumors) and on thyrocytes from Graves’ disease patients (5, 18). These studies present somewhat conflicting results in that the histology in Tanimoto et al.’s study (5) indicated that hyperplastic thyroid tissue from Graves’ glands had increased expression of Fas, whereas Kawakami et al. (18) indicated that TSH treatment of thyrocytes in vitro inhibited the expression of Fas antigen. Our results are in concordance with Tanimoto et al.’s immunohistochemical analysis (5), and a careful analysis of the flow cytometric technique used by Kawakami et al. (18) showed only very small differences in Fas expression (2-fold decreases) in vitro due to TSH that are likely to be without functional significance. Another group (19) recently reported that Fas protein was not present in normal thyroid tissue, and indicated that Fas protein expression was induced by IL-1ß. This study used only a single Ab technique to detect Fas antigen and did not examine the expression of Fas mRNA. In light of this, we used two different techniques and several different Abs to identify Fas protein expression, and two different techniques to detect and quantify Fas antigen mRNA; all of these techniques confirmed the expression of Fas in the thyroid. Another potential difference between our work and Giordano et al.’s study (19) that may account for the lack of expression of Fas is that they employed nontoxic goiter tissue as their source of normal thyroid. We have not had the opportunity to examine thyrocytes from this particular disorder, and therefore cannot comment on whether the expression of Fas in these cells mimics our findings in nondiseased thyrocytes.
The studies of Kawakami et al. (18) and Giordano et
al. (19), also suggest that thyrocytes need to be exposed to
cytokines, such as IL-1ß or IFN-, to allow the induction of
apoptosis through the Fas pathway. In contrast, our studies indicate
that Fas is normally expressed on thyrocytes, but that an inhibitor of
the Fas pathway blocks apoptosis. Also, although these two cytokines
independently could not overcome the inhibitor, each did appear to
enhance apoptosis in the presence of CHX. A reason for this difference
may be the assays used to detect cell death. The two other studies used
propidium iodide staining and flow cytometry to detect cells with
hypodiploid DNA content. This assay is nonspecific, because it does not
differentiate between cells undergoing necrosis compared with
apoptosis. It is possible that the cytokines had nonspecific toxic
effects on the thyrocytes that caused necrotic death that was presumed
to be apoptosis. In our experiments, treatment with CHX and anti-Fas
induced apoptosis that was more complete than the results demonstrated
by flow cytometry in the other two studies. This may suggest that the
changes in DNA content these investigators observed were not entirely
due to the induction of apoptosis in these cells. The presence of the
inhibitor may also explain why the expression of Fas ligand on thyroid
cells, as presented in this study, does not induce explosive autologous
apoptosis.
Our results demonstrate the Fas death pathway is normally blocked by a protein inhibitor in thyrocytes and can be activated when the inhibitor is suppressed. The susceptibility of thyrocytes to Fas-mediated apoptosis may be a limiting factor for thyroid destruction in thyroiditis, because it could modulate lymphocyte cytotoxicity in vivo. An understanding of the expression and function of the inhibitor of the Fas pathway in thyroid cells may therefore provide insights into the factors involved in promoting thyroiditis-induced thyroid damage.
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Acknowledgments |
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Footnotes |
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Received June 2, 1997.
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References |
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L. Tourneur, B. Malassagne, F. Batteux, M. Fabre, S. Mistou, E. Lallemand, P. Lores, and G. Chiocchia Transgenic Expression of CD95 Ligand on Thyroid Follicular Cells Confers Immune Privilege upon Thyroid Allografts J. Immunol., August 1, 2001; 167(3): 1338 - 1346. [Abstract] [Full Text] [PDF]
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 M. N. Nikiforova, J. R. Stringer, R. Blough, M. Medvedovic, J. A. Fagin, and Y. E. Nikiforov Proximity of Chromosomal Loci That Participate in Radiation-Induced Rearrangements in Human Cells Science, October 6, 2000; 290(5489): 138 - 141. [Abstract] [Full Text]
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S. Francavilla, P. D’Abrizio, N. Rucci, G. Silvano, G. Properzi, E. Straface, G. Cordeschi, S. Necozione, L. Gnessi, M. Arizzi, et al. Fas and Fas Ligand Expression in Fetal and Adult Human Testis with Normal or Deranged Spermatogenesis J. Clin. Endocrinol. Metab., August 1, 2000; 85(8): 2692 - 2700. [Abstract] [Full Text]
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 N. Mitsiades, V. Poulaki, S. Tseleni-Balafouta, D. A. Koutras, and I. Stamenkovic Thyroid Carcinoma Cells Are Resistant to FAS-mediated Apoptosis But Sensitive to Tumor Necrosis Factor-related Apoptosis-inducing Ligand Cancer Res., August 1, 2000; 60(15): 4122 - 4129. [Abstract] [Full Text]
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 S. M. Quirk, R. M. Harman, S. C. Huber, and R. G. Cowan Responsiveness of Mouse Corpora Luteal Cells to Fas Antigen (CD95)-Mediated Apoptosis Biol Reprod, July 1, 2000; 63(1): 49 - 56. [Abstract] [Full Text]
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F. Batteux, P. Lores, D. Bucchini, and G. Chiocchia Transgenic Expression of Fas Ligand on Thyroid Follicular Cells Prevents Autoimmune Thyroiditis J. Immunol., February 15, 2000; 164(4): 1681 - 1688. [Abstract] [Full Text] [PDF]
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S. L. Vickers, R. G. Cowan, R. M. Harman, D. A. Porter, and S. M. Quirk Expression and Activity of the Fas Antigen in Bovine Ovarian Follicle Cells Biol Reprod, January 1, 2000; 62(1): 54 - 61. [Abstract] [Full Text]
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D. A. Porter, S. L. Vickers, R. G. Cowan, S. C. Huber, and S. M. Quirk Expression and Function of Fas Antigen Vary in Bovine Granulosa and Theca Cells During Ovarian Follicular Development and Atresia Biol Reprod, January 1, 2000; 62(1): 62 - 66. [Abstract] [Full Text]
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P. L. Arscott, T. Stokes, A. Myc, T. J. Giordano, N. W. Thompson, and J. R. Baker Jr. Fas (CD95) Expression is Up-Regulated on Papillary Thyroid Carcinoma J. Clin. Endocrinol. Metab., November 1, 1999; 84(11): 4246 - 4252. [Abstract] [Full Text]
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 J. D. Bretz, P. L. Arscott, A. Myc, and J. R. Baker Jr. Inflammatory Cytokine Regulation of Fas-mediated Apoptosis in Thyroid Follicular Cells J. Biol. Chem., September 3, 1999; 274(36): 25433 - 25438. [Abstract] [Full Text] [PDF]
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