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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2660-2670
Functional Fas Expression in Human Thymic Epithelial Cells
By
Nathalie Moulian,
Claire Renvoizé,
Colette Desodt,
Alain Serraf, and
Sonia Berrih-Aknin
From CNRS UPRESA, Hôpital Marie-Lannelongue, Le Plessis
Robinson, France; and INSERM U461, Faculté de Pharmacie,
Chatenay-Malabry, France (CNRS UPRESA and U461 are affiliated to the
Institut Paris-Sud sur les Cytokines).
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ABSTRACT |
Fas, a cell surface receptor, can induce apoptosis after
cross-linking with its ligand. We report that Fas antigen is
constitutively expressed in medullary epithelial cells of the human
thymus. Expression is decreased in cultured thymic epithelial cells
(TEC), similarly to HLA-DR antigen. TEC are resistant to
anti-Fas-induced apoptosis after 4 days of primary culture, and this
resistance is reversed by concomitant addition of cycloheximide.
Cycloheximide also downregulated the expression of Fas-associated
phosphatase-1, which has been found to inhibit Fas-induced apoptosis.
This phosphatase could be involved in the resistance to Fas-induced
apoptosis observed on day 4 of TEC culture. When TEC were subcultured
after 10 to 13 days of primary culture, exposure to interleukin-1- ,
tumor necrosis factor- , and interferon- , alone or together,
reinduced Fas mRNA and protein expression. In coculture with activated
thymocytes, TEC also upregulated Fas protein expression.
Cytokine-activated TEC became sensitive to apoptosis induced by an
agonistic anti-Fas antibody. This apoptosis was inhibited by Z-VAD-fmk
but not by Z-DEVD-fmk and DEVDase activity was slightly increased in
Fas-stimulated TEC, suggesting that DEVDase activity is not sufficient
to induce TEC apoptosis. Taken together, these data show that the Fas
receptor is expressed in medullary epithelial cells of the human thymus and is able to induce apoptosis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
Fas (APO-1/CD95) IS A CELL surface
receptor expressed in a variety of tissues; on cross-linking, Fas can
induce apoptosis in vitro and in vivo.1 The Fas receptor
shows homology with several members of the tumor necrosis factor
receptor (TNFR) family, including CD40 and P55-TNFR. Its major function
appears to be the induction of apoptosis in cells expressing it. Fas
expression in several cell types is upregulated by the cytokines
interferon- (IFN-), interleukin-1 (IL-1), and
TNF- .2-4 The role of the Fas/FasL system is well
characterized in lymphocytes in two mechanisms: activation-induced cell
death of peripheral lymphocytes5-7 and T-cell
cytotoxicity.8
Cross-linking of Fas receptors activates an array of cysteine proteases
(caspases) in a cascade-like fashion. Upon Fas ligand binding, Fas
recruits FADD/MORT1 and RIP via the interaction of their death
domains.9 Caspase-8 (FLICE/MACH) acts very early in
Fas-induced apoptosis and potentially acts as a direct link between the
Fas signaling complex and ICE-like proteases,10,11 each of
the latter playing a distinct role in Fas-induced
apoptosis.12 A tyrosine phosphatase, Fas-associated
phosphatase-1 (FAP-1), acts as a negative switch in the Fas pathway and
interacts with the carboxyl terminus of the Fas receptor.13
The thymus plays a central role in T-cell differentiation and T-cell
repertoire selection. Clonal deletion of immature thymocytes is an
important mechanism ensuring self-tolerance. Some thymocytes generated
in the thymus survive to become mature T cells, but most are deleted by
apoptosis through thymic selection.14,15 T-cell maturation
in the thymus is driven by interactions between developing thymocytes
and thymic stromal cells.16 Thymic epithelial cells (TEC)
are a major cellular component of the thymic stroma and play a key role
in T-cell commitment.17
Fas is widely expressed in mouse thymocytes,18,19 which can
be induced to undergo apoptosis by an agonistic anti-Fas antibody, both
in vitro and in vivo.18,20-22 The involvement of Fas in
thymic negative selection is controversial.23-27
A subpopulation strongly expressing Fas antigen represents 1% to 4%
of total thymocytes in humans.28-30 We recently described two distinct pathways (an antigen-dependent pathway and a
cytokine-dependent pathway) that can upregulate Fas expression in human
thymocytes31; cytokine-activated thymocytes display a
higher susceptibility to Fas-induced apoptosis in vitro than
anti-CD3-activated thymocytes.
Fas is also expressed in nonlymphoid tissues, notably in various
epithelial cells.32 The expression and function of Fas antigen in nonlymphoid cells of the human thymus has not yet been documented. We therefore studied Fas expression in TEC both in situ by
means of immunochemistry and in vitro by using cultured cells.
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MATERIALS AND METHODS |
Thymic tissue and TEC culture.
Normal thymus fragments were obtained from infants aged from 5 days to
2 years undergoing heart surgery at Marie-Lannelongue Hospital (Le
Plessis-Robinson, France). For some experiments, fragments of thymic
tissue were flash-frozen in liquid nitrogen and stored at
 80°C.
Primary TEC cultures were established as previously
described.33 Briefly, small fragments of human thymic
tissue were washed in RPMI medium and transferred to 75-cm2
culture dishes. The culture medium, RPMI 1640 supplemented with 20%
horse serum (Life Technologies, Cergy-Pontoise, France), 0.2% Ultroser
(Life Technologies), 2 mmol/L L-glutamine, 100 IU/mL penicillin, and
100 µg/mL streptomycin was replaced twice a week. After 10 to 13 days
of primary culture, the confluent monolayers were washed with
phosphate-buffered saline (PBS) and treated with 0.075% trypsin (Life
Technologies) and 0.16% EDTA for 5 minutes at 37°C. In some
experiments, TEC were collected after 2, 4, 7, or 10 days of primary
culture. The epithelial nature of the cells was checked by
immunocytochemical analysis of cytocentrifuged cells, using the
antikeratin monoclonal antibody CK-1 (Dako, Trappes, France). Our
culture conditions select medullary epithelial cells, because cells
collected after 10 to 13 days of culture strongly express antikeratin
CK-1, a marker of the medullary epithelial network. The proportion of
keratin-positive cells was consistently greater than 95%.
After 10 to 13 days of primary culture, TEC were subcultured (5 × 105 cells/well) in 24-well Primaria plates (Polylabo,
Paris, France) and incubated for 24 hours to allow them to adhere.
After two washes with Hank's Balanced Salt Solution (HBSS), the medium
was replaced with RPMI supplemented with 5% horse serum and the
following cytokines: 1 ng/mL recombinant human IL-1 (Sigma Chemical
Co, Saint Quentin Fallavier, France), 10 ng/mL recombinant TNF-
(Genzyme, Cergy Saint Christophe, France), and 500 U/mL recombinant
human IFN- (Genzyme), separately or together. TEC were treated with trypsin-EDTA after 24, 48, or 72 hours. After three washes, the cells
were used for immunofluorescence studies.
Immunofluorescence studies.
TEC were labeled fluorescein isothiocyanate (FITC)-conjugated
anti-HLA-DR (Immunotech, Marseille, France) or anti-Fas antibodies (Dako). TEC were first incubated with anti-Fas (anti-CD95) monoclonal antibody (clone UB2; Immunotech) for 30 minutes at 4°C, then washed twice in Hank's solution (HBSS) supplemented with 5% fetal serum calf, stained with biotin-coupled goat antimouse IgG antibody (Immunotech), washed twice, and incubated with Quantum Red-conjugated streptavidin (Sigma).
Cell labeling was analyzed on a FACScan flow cytometer (Becton
Dickinson, Grenoble, France) using Cell Quest software. A gate was set
on intact cells by using forward- and side-scatter analysis; 104 cells were analyzed in the gate. The proportion of
cells expressing HLA-DR among total cells or the mean fluorescence
intensity (MFI) of Fas staining was measured.
TEC/thymocytes cocultures.
After 10 to 13 days of primary culture, TEC were subcultured in 24-well
plates (0.5 × 106 cells/well). Heterologous
thymocytes were mechanically isolated by gently scraping fresh thymic
tissue, filtering the cells through sterile gauze, and washing them
with HBSS. After two washes of adherent TEC, 5 × 106
thymocytes (in 1 mL RPMI containing 5% horse serum) were added per
well. When indicated, 5 ng/mL phorbol 12-myristate 13-acetate (PMA;
Sigma) and 500 ng/mL ionomycin (Boehringer Mannheim, Meylan, France)
were added to the coculture. After a 3-day coculture period, wells were
washed three times and TEC were collected as previously described.
After staining with anti-Fas antibody, a gate was set on TEC by using
forward- and side-scatter analysis during the acquisition of data on
the FACScan flow cytometer.
Immunohistochemical analysis of thymic sections.
Acetone-fixed frozen sections 6-µm thick were incubated for 30 minutes at room temperature with polyclonal rabbit antikeratin antibody
(Dako) and monoclonal anti-Fas antibody (clone UB2). They were then
washed three times in PBS and incubated with rhodamine-coupled goat
antirabbit antibody (Immunotech) and FITC-conjugated antimouse antibody
(Silenus Laboratories, Hawthorn, Australia) for 30 minutes. After three
washes in PBS, the sections were mounted in PBS/glycerol. Control
sections were incubated with the fluorescent conjugates only.
Anti-Fas antibody assay and measurement of FITC-conjugated annexin-V
binding.
After two washes with HBSS, subcultured TEC were treated with various
amounts of anti-Fas IgM antibody (clone CH-11; Upstate Biotechnology
Inc, Lake Placid, NY) or the same concentrations of irrelevant IgM
antibody (Dako). When indicated, 10 µg/mL cycloheximide (Sigma) was
added to the culture. After 24 hours of exposure to IgM or anti-Fas
CH-11 antibody, the cells were washed twice with PBS and harvested by
trypsin-EDTA treatment; living cells were counted using the Trypan blue
exclusion method. In some experiments cells were washed in PBS and
fixed for 5 minutes in ethanol containing 5% acetic acid. Cells were
then stained with Toluidine blue for 30 seconds and rinsed with water.
Apoptosis was analyzed by quantifying phosphatidylserine residues
exposed on the external cell membrane. Indeed, one of the plasma
membrane alterations occurring in the early stages of apoptosis is the
externalization of phosphatidylserine at the cell surface; it triggers
specific recognition and removal by phagocytes.34 Annexin-V
is a calcium-dependent phospholipid binding protein with high affinity
for phosphatidylserine and is used for the detection of apoptotic
cells.35 One microliter of human recombinant FITC-conjugated annexin-V (Boehringer Mannheim) and 2 µg/mL propidium iodide were added to 100 µL of cell suspension in binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, 5 mmol/L Ca
Cl2). After 15 minutes of incubation in the dark,
dual-color analysis was performed on a FacsScan flow cytometer. Cells
incorporating propidium iodide, ie, dead cells, were excluded from the analysis.
Caspase inhibitory peptides and DEVDase assay.
In some experiments, TEC were preincubated with 20 µmol/L
Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) or
Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-fmk; Enzyme Systems
Products, Dublin, CA) for 2 hours before adding IgM or
anti-Fas CH-11 antibody. Both Z-VAD-fmk and Z-DEVD-fmk peptides were
cell-permeant; Z-VAD-fmk is a cystein protease inhibitor or broad
specificity, whereas Z-DEVD-fmk inhibits more specifically caspases
from the caspase-3 family, also called DEVDases (DEVD-cleaving caspases).
After 7 hours of incubation with IgM or CH-11, TEC were harvested,
washed three times in PBS, and lysed in a buffer containing 10 mmol/L
HEPES, pH 7.4, 2 mmol/L EDTA, 0.1% CHAPS, 5 mmol/L dithiothreitol (DTT), and a cocktail of protease inhibitors (17 µg/mL
phenylmethyl sulfonyl fluoride [PMSF], 10 µg/mL leupeptin, 10 µg/mL aprotinin, and 10 µg/mL pepstatin). Samples were centrifuged
at 100,000g at 4°C to obtain cytosolic proteins.
Proteolytic reactions were performed in a buffer containing 20 mmol/L
HEPES, pH 7.4, 10% glycerol, and 2 mmol/L DTT. Fifty micrograms of
proteins and 100 µmol/L N-acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pNA) were
added and the mixture was incubated during 3 hours at 37°C. The
formation of p-nitroanilide was measured at 405 nm using a Uvikon 930 spectrophotometer (Kontron Instruments, Saint Quentin en
Yvelines, France). In the same experiment, we used an internal control
of DEVDase activity, ie, K562 target cells killed by
lymphokine-activated killer cells, as previously
described.36
RNA preparation and reverse transcriptase-polymerase chain reaction
(RT-PCR).
Total RNA was extracted using the RNAplus kit (Bioprobe, Paris,
France), then purified with 0.5 vol of 7.5 mol/L ammonium acetate and
2.5 vol of 100% ethanol, and centrifuged at 15,000 rpm for 30 minutes
at 4°C. The pellet was washed in 75% ethanol, dried under a
vacuum, and stored at 80°C after dissolution in water. The
total RNA concentration was determined by measuring absorbance at 260 nm with a Gene Quant spectrophotometer (Pharmacia, Cambridge, UK). The
purity of the RNA preparation was checked by measuring the 260 nm/280
nm ratio.
The oligonucleotide primers used for RT-PCR were from Genset
(Paris, France) and the sequences were as follows: Fas primers, forward
5'-GACAAAGCCCATTTTTCTTCC-3' and reverse
5'-ATTTATTGCCACTGTTTCAGG-3'; FAP-1 primers, forward
5'-GAATACGAGTGTCAGACATGG-3' and reverse 5'-AGGTCTGCAGAGAAGCAAGAATAC-3'; -actin primers, forward
5'-GGGTCAGAAGGATTCCTATG-3' and reverse
5'-GGTCTCAAACATGATCTGGG-3'. A 50-µL reverse transcription reaction mixture containing 1 µg of total RNA, 5 µL of 10× RT buffer (Eurobio, Les Ulis, France), 1.5 mmol/L each dNTP (Eurobio), 40 U of RNasin (Promega, Charbonnières, France), 1 µmol/L reverse primer, and 4 U of avian myeloblastosis virus (AMV)
reverse transcriptase (Eurobio) was incubated at 42°C for 60 minutes. PCR was performed in a total volume of 100 µL containing 10 µL of RT reaction mixture, 10 µL of PCR buffer (Eurobio), 1.5 mmol/L MgCl2, 0.5 µmol/L of each primer, 0.2 mmol/L of
each dNTP, and 2.5 U of EurobioTaq II polymerase (Eurobio). The mixture
was overlaid with mineral oil and amplified in a PHC3 thermal cycler
(Techne, Cambridge, UK) as follows: denaturation at 94°C for 1 minute; annealing at 53°C (Fas), 60°C (FAP-1), or 58°C
(-actin) for 1 minute; and extension at 72°C for 2 minutes. The
final elongation step lasted 10 minutes at 72°C. PCR products were
analyzed on 1.5% agarose gel containing ethidium bromide.
Western blotting.
In some experiments, TEC were collected and solubilized in a lysis
buffer containing 150 mmol/L NaCl, 50 mmol/L Tris, pH 8.0, 5 mmol/L
EDTA, 1% Triton X-100, 0.02% NaN3, 1 mmol/L PMSF (Sigma), and 0.15 U/mL aprotinin (Sigma) for 20 minutes at 4°C. Insoluble material was removed by centrifugation at 4°C for 10 minutes. After
boiling, the samples (20 µg total protein) were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel
(7.5%) and proteins were transferred to polyvinylidene difluoride (PVDF) membranes. A second SDS-PAGE gel was colored with
Coomassie blue to check that similar amounts of protein were loaded
into the gel. Blots were saturated in PBS containing 0.1% Tween-20 and
5% dry nonfat milk and incubated for 4 hours at 4°C in PBS containing 0.1% Tween-20, 0.1% dry nonfat milk, and 0.2 µg/mL polyclonal goat anti-FAP-1 antibody (Santa Cruz Biotechnology, Santa
Cruz, CA). Bound antibody was detected by using peroxidase-conjugated antigoat Ig (Santa Cruz Biotechnology). Immunoreactivity was determined using the ECL chemiluminescence reaction (Amersham France S.A., Les
Ulis, France).
Statistical analysis.
Differences between groups were identified by using the Mann-Whitney or
Wilcoxon test (Instat software). A difference was considered
statistically significant if the P value was less than .05.
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RESULTS |
Fas antigen is expressed in situ in human thymus.
Fas expression was analyzed on cryosections of human thymuses in
double-staining experiments with antikeratin to visualize the
epithelial network. We used a polyclonal antikeratin antibody that
stains thymic epithelial cells more strongly in the medulla than in the
cortex. Fas was mainly expressed in the medulla
(Fig 1). Double-staining showed that most
cells expressing keratin were also Fas-positive, whereas cortical
epithelial cells were clearly Fas-negative. Thus, a subset of TEC
express Fas antigen in the human thymus. A subset of human thymocyte
(<3% of total thymocytes) with a strong expression of Fas was
previously described using cytofluorimetry.28-30 We could
rarely distinguish Fas expression in some medullary thymocytes; thus,
Fas expression in the human thymus is mostly epithelial.
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| Fig 1.
Double immunofluorescence distribution of Fas and keratin
in the human thymus. Frozen thymus sections were fixed in acetone and
then double-stained with antikeratin (left) and anti-Fas (right)
antibodies. After three washes they were stained with rhodamine-coupled
antirabbit and FITC-coupled antimouse antibodies. The top plates
(×10) show that Fas expression is mainly observed in the medullary
area of the human thymus. Control sections (×20) were incubated with
the fluorescent conjugates only. The lower plates are enlargements of
the framed areas in the upper plates. The white arrows indicate
medullary epithelial cells expressing keratin and Fas antigen.
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Fas antigen expression falls in cultured TEC.
We examined Fas and HLA-DR expression by immunofluorescence in TEC
after 2, 4, 7, or 10 days of culture. Only about 1% of cultured TEC
expressed HLA-DR after 10 days in primary culture, whereas 51% of TEC
were HLA-DR-positive cells after 2 days
(Fig 2). Fas antigen expression followed
the same pattern and decreased threefold during primary culture,
between day 2 and day 10.
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| Fig 2.
Fas and HLA-DR expression is lost during culture of TEC.
Primary cultures of human TEC were established as described in
Materials and Methods. TEC were collected after 2, 4, 7, or 10 days of
primary culture by trypsin treatment and then labeled with
FITC-conjugated anti-HLA-DR or anti-Fas antibodies. TEC were first
incubated with anti-Fas monoclonal antibody for 30 minutes at 4°C,
then washed in HBSS supplemented with 5% fetal serum calf, stained
with biotin-coupled goat antimouse IgG antibody, washed, and incubated
with streptavidin/Quantum Red conjugate. Cell labeling was analyzed on
a FACScan flow cytometer. Vertical bars on Fas histograms indicate the
Fas MFI level in TEC on day 2. Fas antigen expression fell during
culture, similarly to HLA-DR antigen expression.
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Fas antigen expression by cultured TEC is upregulated by cytokines.
Fas and HLA-DR expression were monitored by means of flow cytometry
after cytokine treatment (IL-1-, TNF-, and IFN-) of TEC
subcultures. All three cytokines individually upregulated Fas
expression (Fig 3A). The Fas MFI in the
presence of one or several cytokines was expressed as a ratio relative
to control values. Fas expression by cultured TEC increased more
strongly when the three cytokines were added together than when they
were added separately (Fig 3A and C): the Fas MFI ratio was 2.4 ± 0.4 after 48 hours in the presence of IL-1-, TNF-, and IFN-,
compared with 1.4 ± 0.05 with IL-1-, 1.4 ± 0.1 with TNF-,
and 1.7 ± 0.1 with IFN-. Like HLA-DR, the effect was maximal
after 48 hours of incubation (Fig 3C and D). HLA-DR expression was
increased by IFN-, as previously described,33 but not by
IL-1- and TNF- (Fig 3D); the combined effect of the
three cytokines was slightly less potent than that of IFN- alone.
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| Fig 3.
IL-1-, TNF-, and IFN-, alone and in
combination, upregulate Fas expression in cultured TEC. TEC subcultured
after 10 to 13 days of primary culture were incubated with the
cytokines. At 24, 48, or 72 hours, TEC were collected by trypsin
treatment and labeled with anti-HLA-DR or anti-Fas antibody. (A) A
representative experiment shows that, after 48 hours of incubation,
IL-1-, TNF-, and IFN-, both alone and together, increased Fas
MFI. Vertical bars on Fas staining histograms indicate the Fas MFI
level in TEC cultured in control conditions, ie, in medium. (B) mRNA
was extracted from TEC cultured without () or with (+) 1 ng/mL
IL-1-, 10 ng/mL TNF-, and 500 U/mL IFN-. Lane 1 corresponds to
the molecular weight (MW) marker (pUC18 DNA Marker Hae III
digest; Sigma). mRNA was submitted to RT-PCR. In three independent
experiments, cytokine-activated TEC showed a strong increase in Fas
mRNA levels in comparison to TEC cultured in control conditions,
whereas -actin expression was not modified. (C) The MFI ratio is the
ratio between Fas MFI measured in the presence of one or several
cytokines and Fas MFI measured in control conditions. MFI ratios are
expressed as a function of time. Results are means ± SEM of four
independent experiments. (D) The proportion of HLA-DR-positive cells
was analyzed in the same experiments.
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Moreover, Fas mRNA levels monitored by RT-PCR were low or undetectable
in control conditions and were strikingly increased after 24 hours in
the presence of the three cytokines, suggesting that Fas upregulation
occurred at the transcription level (Fig 3B).
Activated thymocytes induce Fas upregulation in TEC.
Heterologous thymocytes were cocultured with TEC for a 3-day period.
Fas expression was analyzed in TEC only, ie, in cells in the R region
(Fig 4A) according to forward-and
side-scatter parameters. Thus, thymocytes (that have smaller size) were
excluded from the analysis. In the absence of any activation, Fas
expression was not modified in TEC (Fig 4). In the absence of
thymocytes, the addition of PMA, a phorbol ester that activated protein
kinase C, and ionomycin, a calcium ionophore, did not have any effect on Fas expression in TEC. By contrast, when thymocytes activated by
these agents were cocultured with TEC, we measured an increase in Fas
MFI in TEC: it was 52.0 ± 5.0 in TEC cocultured with
thymocytes in medium and 82.7 ± 9.9 in TEC cocultured with
activated thymocytes (n = 3). Thus, activated thymocytes are
able to upregulate Fas expression in TEC.
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| Fig 4.
Fas upregulation is induced in TEC cocultured with
activated thymocytes. TEC subcultured after 10 to 13 days of primary
culture were cocultured with heterologous thymocytes in the presence or
in the absence of 5 ng/mL PMA and 500 ng/mL ionomycin (PMA/I). After 3 days, TEC were collected. (A) Fas expression was analyzed in TEC, ie,
in cells gated in the R region. (B) A representative experiment shows
that, without thymocytes, PMA and ionomycin did not induce any effect
on Fas expression in TEC. When cocultured with thymocytes, Fas
expression is upregulated in TEC when PMA and ionomycin were added. (C)
Data are the mean ± SEM from three independent experiments.
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Susceptibility of TEC to cell death induced by an agonistic anti-Fas
antibody on day 4 of culture.
Because Fas antigen expression decreased gradually during culture, we
examined whether the remaining Fas antigen was functional and whether
TEC in primary culture were sensitive to an agonistic anti-Fas
antibody. TEC on day 4 were treated with 0.5 µg/mL anti-Fas IgM
antibody (clone CH-11) or with 0.5 µg/mL mouse IgM. Susceptibility to
Fas-induced apoptosis was assessed by (1) the number of viable cells
recovered and (2) the proportion of annexin-V-positive cells (Fig 5). Although Fas antigen expression
was still strong on day 4 of primary culture, TEC were not sensitive to
Fas-induced apoptosis at this time. This resistance to Fas-induced
apoptosis was reversed by concomitant addition of 10 µg/mL
cycloheximide, an inhibitor of protein synthesis. In these conditions,
cell density was reduced (Fig 5A), cell recovery was about 70% of that
in the presence of IgM and cycloheximide or in the absence of
cycloheximide (Fig 5B), and the proportion of annexin-V-positive cells
was significantly increased (P < .05, n = 4; Fig 5C and D).
TEC on day 4 of culture were also resistant to 0.5 µg/mL recombinant
soluble Fas ligand and this resistance was similarly reversed by
concomitant addition of cycloheximide (not shown).
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| Fig 5.
Resistance to Fas-induced apoptosis of human TEC on day 4 of primary culture can be raised by concomitant addition of
cycloheximide. TEC were collected on day 3 of primary culture and
subcultured in 24-well plates. After a 24-hour period to allow cells to
adhere, 0.5 µg/mL agonistic anti-Fas IgM antibody (clone CH-11) or
0.5 µg/mL mouse IgM antibody was added in the presence or absence of
10 µg/mL cycloheximide. (A) After cell fixation in ethanol containing
5% acetic acid, cells were stained with Toluidine blue and
photographed. In the absence of cycloheximide, no change in cell
density was observed (data not shown). When cycloheximide was added,
cell density was clearly reduced in the presence of agonistic anti-Fas
antibody (clone CH-11) in comparison to cells treated with IgM. (B)
Living cells recovered from 0.5 × 106 cells subcultured
in 24 wells on day 3 were counted after the culture by using the Trypan
blue exclusion method. In the absence of cycloheximide, the number of
cells collected was not significantly modified by anti-Fas. By
contrast, when cycloheximide was added, the number of cells recovered
was clearly reduced in the presence of anti-Fas. Data are the means ± SEM of three independent experiments. (C) Apoptosis was analyzed by
quantifying phosphatidylserine residues exposed on the external cell
membrane. Annexin-V binding was performed as previously described. A
representative analysis shows that TEC undergo anti-Fas
(CH-11)-induced apoptosis in the presence of cycloheximide, because
the proportion of annexin-V-positive cells is increased relative to
IgM treatment, whereas TEC were resistant in the absence of
cycloheximide. (D) Data are the means ± SEM of four independent
experiments.
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FAP-1 regulation in TEC.
Because expression of FAP-1, a Fas-associated phophatase, protects
cells from Fas-induced apoptosis,13 we wondered whether FAP-1 expression was altered by cycloheximide treatment. TEC collected on day 3 of primary culture and left to adhere for 24 hours were further cultured for 24 hours in the presence or absence of 10 µg/mL
cycloheximide. They were then harvested and FAP-1 mRNA was examined.
Cycloheximide strongly reduced FAP-1 mRNA levels, whereas it did not
affect -actin mRNA levels (Fig 6). Fas
mRNA levels were not significantly affected by the cycloheximide
treatment (data not shown). In similar experiments, TEC protein
extracts (20 µg protein in each condition) were analyzed in Western
blot assay. We observed a major protein band (apparent molecular
weight, 200 kD) detected by anti-FAP-1 antibody; it was clearly
reduced in the presence of cycloheximide. These results show that FAP-1 downregulation coincides with the acquisition of susceptibility to
Fas-induced apoptosis in human TEC.
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| Fig 6.
Downregulation of FAP-1 expression in TEC in the presence
of cycloheximide. TEC on day 4 of primary culture were incubated for 24 hours with 10 µg/mL cycloheximide (CHX) or in medium (M). (A) mRNA
was extracted and submitted to RT-PCR. In two independent experiments,
treatment of TEC with cyloheximide induced a clear decrease in FAP-1
mRNA levels, whereas -actin expression was not modified. (B) Western
blot analysis of FAP-1 expression. TEC were solubilized, and 20 µg
total protein in each condition was analyzed on SDS-PAGE 7.5% and
transferred to PVDF membranes. The blot was incubated with anti-FAP-1
antibody and then with peroxidase-conjugated antigoat Ig.
Immunoreactivity was determined using the ECL chemiluminescence
reaction. A major protein band (apparent molecular weight, 200 kD) was
decreased in the presence of cycloheximide.
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An agonistic anti-Fas antibody induces apoptosis of
cytokine-activated TEC.
To determine if human TEC activated by cytokines and reexpressing Fas
antigen were susceptible to Fas-induced apoptosis, subcultured TEC were
incubated for 48 hours with IL-1-, TNF-, and IFN-, alone or in
combination, and then treated with various concentrations of IgM or
anti-Fas antibody (clone CH-11). TEC were collected by trypsin
treatment after 24 hours and annexin-V binding was examined. TEC
cultured in the absence of cytokines were not susceptible to apoptosis
induced by agonistic anti-Fas antibody (Fig
7) or recombinant soluble Fas ligand (not shown); this resistance was not reversed by concomitant addition of cycloheximide (not shown). By
contrast, TEC previously treated with IL-1-, TNF-, and IFN-, especially in combination, underwent apoptosis. Thus, the level of
anti-Fas-induced apoptosis was thus related to the level of Fas
expression. Apoptosis was maximal with 0.5 µg/mL agonistic anti-Fas
antibody.
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| Fig 7.
Cytokine-activated TEC are sensitive to anti-Fas-induced
apoptosis. TEC subcultured after 10 to 13 days of primary culture were
incubated in the presence of 1 ng/mL IL-1-, 10 ng/mL TNF-, and
500 U/mL IFN-, alone or in combination, for 48 hours. After two
washes, various concentrations of agonistic anti-Fas antibody or
control IgM were added. After 24 hours, cells were collected by trypsin
treatment and labeled with annexin-V-FITC and propidium iodide. Dead
cells, ie, cells incorporating propidium iodide, were excluded from the
analysis. A representative analysis is shown. (A) The proportion of
annexin-V-positive cells among total living cells is expressed as a
function of the concentration of IgM or anti-Fas. Increasing
concentrations of IgM were inactive, whereas Fas-mediated apoptosis was
concentration-dependent. (B) The analysis of annexin-V-FITC binding in
TEC previously activated by cytokines and incubated with 0.5 µg/mL
anti-Fas CH-11 or IgM is presented.
|
|
Effect of Z-DEVD-fmk and Z-VAD-fmk peptides on anti-Fas-induced
apoptosis in TEC and DEVDase assay in Fas-stimulated TEC.
Tripeptide Z-VAD-fmk is a cystein protease inhibitor of broad
specificity, whereas Z-DEVD-fmk inhibits more specifically cystein proteases from the caspase-3 family (DEVDases). In preliminary experiments, Z-VAD-fmk and Z-DEVD-fmk alone did not have any effect on
TEC apoptosis. To study the effects of these caspase inhibitors on
anti-Fas-induced apoptosis, TEC were first cultured for 48 hours with
the combination of IL-1-, TNF-, and IFN-. After two washes,
TEC were incubated for 2 hours with 20 µmol/L Z-VAD-fmk or
Z-DEVD-fmk, and 0.5 µg/mL agonistic anti-Fas antibody or mouse IgM
was then added. After 24 hours, the cells were collected by trypsin
treatment. As expected (Fig 8), the
agonistic anti-Fas antibody depleted viable cells relative to the
control IgM antibody. Z-DEVD-fmk did not modify the number of cells
recovered or the proportion of annexin-V-positive cells. By contrast,
Z-VAD-fmk restored the number of cells collected and inhibited
anti-Fas-induced apoptosis. Using a specific colorimetric substrate
(DEVD-pNA), DEVDase activity was measured in cytokine-activated TEC
that were cultured during 7 hours in the presence of 0.5 µg/mL IgM or
0.5 µg/mL CH-11. We also evaluated the proportion of apoptotic cells among living cells using annexin-V staining. As shown in
Table 1, after 7 hours of incubation with
agonistic anti-Fas antibody, TEC already undergo Fas-specific
apoptosis, because the proportion of annexin-V-positive cells among
living cells was increased in the presence of CH-11. DEVDase activity
was slightly increased (2.1 and 2.4 times in 2 independent
experiments). In comparison, in a granule-mediated apoptosis in which
caspase-3 family was clearly involved,36 DEVDase activity
was increased 12.1 and 15.6 times, respectively, in K562 target cells
killed by lymphokine-activated killer cells compared with K562 alone.
Thus, the slight activation of DEVDase in Fas-stimulated TEC is well
correlated to the noninhibition of apoptosis induced by z-DEVD-fmk and
is probably not sufficient to induce TEC apoptosis.
View larger version (42K):
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| Fig 8.
Z-VAD-fmk, contrary to Z-DEVD-fmk, inhibits Fas-mediated
apoptosis in cytokine-activated TEC. A representative experiment (from
3 independent experiments) is shown. Human TEC were activated for 48 hours with 1 ng/mL IL-1-, 10 ng/mL TNF-, and 500 U/mL IFN-.
After two washes, cells were preincubated with 20 µmol/L Z-VAD-fmk or
20 µmol/L Z-DEVD-fmk where indicated, before adding 0.5 µg/mL IgM
or 0.5 µg/mL anti-Fas. Twenty-four hours later, cells were harvested
and the number of living cells was measured using the Trypan exclusion
dye method (A); annexin-V-FITC binding was also measured (B).
|
|
| |
DISCUSSION |
We report that Fas antigen is constitutively expressed by medullary
epithelial cells. Fas expression is lost by cultured TEC but can be
restored by cytokines. TEC were sensitive to anti-Fas-induced apoptosis in the presence of cycloheximide on day 4 of culture (when
Fas expression was still high) and when subcultured on day 10 to 13 if
they had been activated by cytokines. These results point to functional
expression of Fas antigen by human medullary TEC.
Fas and Fas ligand expression in the human thymus.
Fas antigen expression has been examined in mouse and human thymic
lymphoid cells. Most mouse thymocytes are
Fas-positive,18,19 whereas only a minor subset strongly
express Fas in the human thymus.28-30 Fas expression in the
epithelial compartment also seems to differ between mice and humans.
Indeed, Fas expression was not found in the epithelial network of mouse
thymus. French et al37 characterized the expression of Fas
and its ligand in the mouse thymus by using in situ hybridization and
immunohistochemistry. They detected Fas expression in thymocytes and
strong Fas ligand expression in thymic epithelial cells and dendritic
cells. We found weak expression of Fas ligand mRNA in human thymus by
means of RT-PCR, but no protein expression was found in situ (not
shown).These results suggest that the Fas/Fas ligand system is
differently expressed in the mouse and human thymus and could have
different functions.
We found that Fas antigen expression decreased during primary human TEC
culture, reaching minimal levels after 10 days. This effect was unlikely to be due to in vitro selection of cortical epithelial cells, which do not express the Fas receptor, because epithelial cells collected after 2, 4, 7, or 10 days of primary culture
were strongly labeled by an antikeratin antibody (clone CK1) that
mainly stains the medullary epithelial network (not shown). Because Fas
was detected in situ, its expression could be downregulated in the
absence of the thymic microenvironment, as previously shown for HLA-DR
in these cells.33 This is supported by the moderate
upregulation of Fas expression in TEC cocultured for 3 days with
activated human thymocytes. Fas expression in vivo could be maintained
by contact with thymocytes. Fas is mostly expressed in the medulla,
where most mature thymocytes are found. Mature human
CD3high thymocytes (both double- and single-positive
CD4+/CD8+ cells) can secrete consistent amounts
of cytokines38 and play a key role in the differentiation
of medullary TEC.39 Thus, medullary mature thymocytes might
interact with medullary TEC, resulting in the maintenance of Fas expression.
Role of Fas in epithelial cells of the human thymus.
Most nonlymphoid tissues constitutively coexpressing Fas and its ligand
in adult mice40 and humans41 are characterized by apoptotic cell turnover, possibly regulated by the Fas system. Fas
is directly involved in the regression of the vaginal epithelium after
ovariectomy and during the estrous cycle in mice.42
Moreover, Fas ligation induces apoptosis in various epithelia in vitro, including ovarian surface epithelial cells,43 thyroid
epithelial cells,4,44 and colon epithelial
cells.45 Our findings show that Fas can also induce
apoptosis of thymic epithelial cells in vitro.
Regarding the physiologic role of Fas in the thymus
epithelial compartment, Fas could regulate the turnover of TEC,
especially when thymus involutes after childhood. Although no
substantial expression of Fas ligand protein is detected in the human
thymus, activated rat thymocytes46 and activated human
thymocytes and TEC (not shown) can express Fas ligand. Both Fas and its
ligand can be produced by TEC in vitro, but we did not observe cell
death in those cells in the absence of agonistic anti-Fas antibody. Further studies are needed to determine if Fas ligand produced by
activated TEC is also secreted into the extracellular medium, because
membrane-associated and secreted Fas ligand seem to have different
capacities to induce apoptosis,47 and soluble Fas ligand
can block Fas-induced cell death.48
Intracellular partners of the Fas receptor in the induction of cell
death.
On day 4 of primary culture, when they still expressed significant
amounts of Fas antigen, TEC were resistant to Fas-induced apoptosis,
and cycloheximide reversed this resistance. Metabolic inhibitors such
as actinomycin D and cycloheximide can sensitize some cells to
anti-Fas-mediated apoptosis in both mice18 and humans,44,49 suggesting that a labile protein might inhibit Fas-mediated cell death. We found that cycloheximide downregulated FAP-1, a phosphatase able to inhibit Fas-mediated apoptosis. Similarly, Mori et al49 showed that actinomycin D induced
sensitivity to Fas-induced apoptosis and downregulated FAP-1 mRNA in
Kaposi's sarcoma cells. FAP-1 may thus be involved in TEC resistance
to Fas-induced apoptosis on day 4 of primary culture. Other molecules could be involved. Intracellular glutathione, levels of which are
reduced by cycloheximide, can mediate Fas resistance in human T
lymphocytes.50 Fournel et al51 showed that
human T cells required IL-2 to acquire susceptibility to Fas-mediated
apoptosis, and the investigators suggested that IL-2 may decrease FAP-1
expression. TEC subcultured with or without cytokines contained
significant levels of FAP-1 mRNA (not shown), and cytokine-activated
TEC were sensitive to Fas-mediated apoptosis, showing that FAP-1
expression was not sufficient to induce resistance to Fas-mediated cell
death in cytokine-activated cells. Thus, activation by cytokines can overcome the resistance to Fas-induced cell death induced by FAP-1. In
immature20 and mature52 T lymphocytes,
additional signals were shown to interfere with the Fas pathway. We
show here that Fas-induced apoptosis of cultured TEC requires (1) Fas
expression and (2) another signal that can be provided by the removal
of a labile protein able to inhibit the Fas signal (eg, the phosphatase FAP-1) or by cell activation by cytokines. In lymphoid and nonlymphoid cells, additional signals that interfere with the Fas pathway (other
than the one produced by FAP-1) need to be clarified.
Caspase-8 plays a pivotal role in Fas-induced
apoptosis10,11 and links the Fas signaling complex and
other ICE-like caspases. Caspase-3 is a major cysteine protease
activated after Fas triggering.53 However, in caspase-3
knock-out mice,54 Fas-induced apoptosis and
poly(ADP-ribose) polymerase (PARP) cleavage were not impaired. We found
that Fas-induced apoptosis of cytokine-activated TEC was inhibited by
Z-VAD-fmk, a cysteine protease inhibitor, but not by Z-DEVD-fmk, a
specific inhibitor of caspases from the caspase-3 family or DEVDases.
Moreover, DEVDase activity is slightly increased in Fas-stimulated TEC,
compared with a granule-mediated apoptosis in which DEVDase is
implicated and compared with Fas-stimulated cells whose apoptosis is
clearly related to DEVDase activity as Jurkat cells (in which DEVDase
activity is 26 times increased in the presence of agonistic anti-Fas
antibody55). This suggests that Fas-induced apoptosis of
TEC is mediated by cysteine proteases but DEVDase activity is probably
not sufficient to induce Fas-mediated apoptosis in TEC.
In conclusion, human medullary TEC express Fas in situ and are able to
undergo Fas-induced apoptosis. This original system of Fas-mediated
cell death in nonlymphoid cells could serve as a model to examine the
intracellular signals conferring susceptibility to Fas-mediated
apoptosis or to its execution.
| |
ACKNOWLEDGMENT |
The authors are grateful to Dr E. Dulmet and S. Planté (Service
d'Anatomo-Pathologie, Hôpital Marie-Lannelongue, Le
Plessis-Robinson, France) for technical advice in the histological
experiments, to Dr J. Bréard (INSERM U461) for the generous gift
of Z-DEVD-fmk and Z-VAD-fmk and for helpful discussions, and to N. Riché (INSERM U461) for technical assistance.
| |
FOOTNOTES |
Submitted June 23, 1998; accepted November 30, 1998.
Supported by grants from Association Française contre les
Myopathies (AFM), Centre National de la Recherche Scientifique (CNRS),
and Caisse Nationale d'Assurance Maladie des Travailleurs Salariés (CNAMTS). N.M. received a postdoctoral grant from FRM (Fondation pour la Recherche Médicale).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Nathalie Moulian, PhD, Laboratoire
d'Immunologie Cellulaire et Moléculaire, CNRS UPRESA,
Hôpital Marie-Lannelongue, 133 avenue de la Résistance,
92350 Le Plessis Robinson, France.
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ABSTRACT
INTRODUCTION