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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1297-1307
Two Signaling Pathways Can Increase Fas Expression in Human
Thymocytes
By
Nathalie Moulian,
Jocelyne Bidault,
Claude Planché, and
Sonia Berrih-Aknin
From CNRS UPRESA, Hôpital Marie-Lannelongue, Le Plessis
Robinson, France.
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ABSTRACT |
Fas, a cell surface receptor, can induce apoptosis after
cross-linking with its ligand. Fewer than 3% of human thymocytes strongly express Fas. We report that Fas antigen expression can be
upregulated by two signaling pathways in vitro, one mediated by
anti-CD3 and the other by interleukin-7 + interferon- . The two
signaling pathways differed in several respects. (1) Fas expression increased in all thymic subsets after cytokine activation, but only in
the CD4 lineage after anti-CD3 activation. (2) Fas upregulation was
inhibited by cyclosporin A (a calcineurin inhibitor) in
anti-CD3-activated but not in cytokine-activated thymocytes. (3)
Cycloheximide (a metabolic inhibitor) inhibited Fas upregulation in
cytokine-activated thymocytes but not in anti-CD3-activated
thymocytes. (4) Cytokine-activated thymocytes were more susceptible
than anti-CD3-activated thymocytes to Fas-induced apoptosis, a
difference mainly accounted for by CD4+ cells. The nature
of the stimulus might thus influence the susceptibility of human
thymocytes to Fas-induced apoptosis.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
Fas (APO-1; CD95) IS A cell surface
receptor that is expressed on a variety of tissues. It shows homology
with several members of the tumor necrosis factor receptor
family.1 Its major function appears to be the induction of
apoptosis in cells expressing it. Ligation of Fas by its ligand (FasL)
or agonistic anti-Fas antibodies induces apoptosis in vitro and in
vivo. In the immune system, the role of the Fas/FasL system is well
characterized in T-cell cytotoxicity2 and
activation-induced cell death of peripheral lymphocytes.3-5
Most mouse thymocytes express Fas and agonistic anti-Fas antibody can
induce mouse thymocyte apoptosis in vitro in the presence of additional
signals (metabolic inhibitors6 or T-cell receptor [TCR] stimulation7), in thymic organ
culture,8 and in vivo.9 The involvement of Fas
in thymic negative selection is controversial. In lpr mice,
which develop systemic autoimmune disease related to a Fas gene
defect,10 and in Fas-null mice, negative selection in
response to endogenous superantigens is normal in the thymus, whereas
activation-induced cell death of activated peripheral lymphocytes is
impaired.11-13 However, Castro et al14 recently reported that thymocytes from lpr mice have reduced
susceptibility to TCR-induced apoptosis and that antigen-specific
thymocyte deletion in normal mice can be inhibited in vivo by blocking
the Fas-FasL interaction. Finally, Kishimoto and Sprent15
reported that TCR-CD28-mediated negative selection of mouse
HSAhi CD4+CD8 thymocytes in
vitro in the presence of high anti-TCR antibody concentrations requires
a Fas-dependent pathway.
Few teams have examined Fas expression and function in the human
thymus. The normal thymus contains a thymocyte subpopulation strongly
expressing Fas (Fashi) and representing 1% to 4% of total
thymocytes.16-18 This thymocyte subpopulation may be a
target for negative selection and Fas might be involved in the
elimination of autoreactive cells, notably because this transitional
population contains a large fraction of dead cells16; in
addition, an antagonistic anti-Fas antibody inhibits the complete
deletion of staphylococcal enterotoxin B-reactive V 3-positive human
thymocytes.17 We have previously found that
Fashi cells accumulate in the thymus of patients with
myasthenia gravis, an autoimmune disease characterized in 85% of cases
by the presence of anti-acetylcholine receptor antibodies and
associated with thymic abnormalities.18 Fashi
thymocytes from myasthenia gravis patients were depleted in vitro by an
agonistic anti-Fas antibody (clone CH-11) and could be involved in the
autoimmune response targeting the acetylcholine receptor.
In humans, Fas is expressed by resting peripheral lymphocytes and its
expression is upregulated by various cytokines (interferon- [IFN-] in a human lymphoma cell line19
and interleukin-2 [IL-2] in peripheral blood
lymphocytes20). In addition, Fas antigen can be induced in
vitro by mitogenic stimulation of naïve T and B cells from
neonatal blood.21
There are several molecular mechanisms transducing the apoptotic signal
after Fas-FasL interaction of Fas and its ligand, but little is known
of how the expression of these molecules is regulated or the
physiologic function of Fas in in the human thymus. We
therefore examined the regulation of Fas expression in human thymocytes
in the presence of various stimuli and described two signaling pathways that can lead to Fas expression upregulation, one
antigen-dependent and the other cytokine-dependent. These two pathways
involve different intracellular mechanisms and lead to different
susceptibility to Fas apoptosis.
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MATERIALS AND METHODS |
Thymocyte isolation and culture.
Normal thymus fragments were obtained from infants (age range, 5 days
to 2 years) undergoing heart surgery at Marie-Lannelongue Hospital.
Thymocytes were mechanically isolated by gently scraping fresh
thymic tissue, filtering the cells through sterile gauze, and washing
them once in Hank's Balanced Salt Solution (HBSS).
Ninety-six-well plates were coated with 10 µg/mL mouse anti-CD3 IgG1
antibody (clone 4B5; Boehringer Mannheim, Meylan, France) or 10 µg/mL
mouse IgG1 (Dako, Trappes, France) overnight at 4°C. In some
experiments, 10 µg/mL anti-CD3 IgG1 antibody and 10 µg/mL anti-CD28
IgG1 antibody (clone CD28.2; Immunotech, Marseille, France) were
immobilized on 96-well plates. Isolated thymic cells were cultured in
96-well plates (0.5 × 106 cells in 200 µL) in RPMI
1640 medium supplemented with 10% fetal calf serum, 2 mmol/L
L-glutamine, 25 mmol/L HEPES, 100 IU/mL penicillin, and 100 µg/mL
streptomycin.
Human thymocytes were cultured in the presence of 1 µg/mL
phytohemagglutinin (PHA; Difco Laboratories, Detroit, MI) or the combination of 5 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co, Saint Quentin Fallavier, France) and 500 ng/mL ionomycin (Sigma). The following recombinant human cytokines were also added to
thymocytes: IL-2, IL-7, and IFN- were from Genzyme Diagnostics (Cergy, France); IL-12 was from Sigma; and IL-6 was from Innotest (Besançon, France). At various culture times, cells were
harvested and labeled for Fas expression. Cell recovery and viability
were measured by using the Trypan blue exclusion method. In some
experiments, thymocytes were cultured with 10 µg/mL cycloheximide
(Sigma) or 0.5 µg/mL actinomycin D (Sigma).
Immunofluorescence studies.
Thymocytes were labeled with monoclonal fluorochrome-coupled anti-CD4,
anti-CD8, and anti-CD25 antibodies (Immunotech). Three-color flow
cytometry was used to examine the relationship between Fas and other
markers. Thymocytes were first incubated with anti-Fas (anti-CD95)
monoclonal antibody (clone UB2; Immunotech) for 30 minutes at 4°C,
then washed twice in HBSS supplemented with 5% fetal calf serum,
stained with biotin-coupled goat antimouse IgG antibody, washed twice,
and incubated with streptavidin, Quantum Red conjugate (Sigma), and
membrane fluorescein (FITC)- or phycoerythrin (PE)-coupled antibodies.
We checked that the three-color staining gave the same results as
one-color staining (anti-CD25) and two-color staining (anti-CD4 and
anti-CD8).
Cell labeling was analyzed on a FACScan flow cytometer (Becton
Dickinson, Grenoble, France) using CellQuest software. Ten thousand to
15,000 events were acquired for each sample. A gate was set on freshly
isolated thymocytes displaying homogeneous forward- and side-scatter
parameters. In this gate, the proportion of dead cells (those
incorporating propidium iodide) was always less than 1% whatever the
culture time and conditions. All flow cytometer analyses were performed
in this gate.
After culture with anti-CD3 or IgG1 antibody, cells were labeled with
20 µg/mL goat antimouse IgG antibody (Jackson ImmunoResearch Laboratories Inc, West Grove, PA) before staining with anti-Fas antibody and then biotin-coupled goat antimouse antibody. This prevented the possible staining of biotin-coupled goat antimouse on
anti-CD3 antibody (which was immobilized on 96-well plates and might be
fixed on thymocytes during the culture).
Anti-Fas antibody assay.
Thymic cells (105 cells/well) were cultured in 96-well
plates with 2 µg/mL anti-Fas IgM antibody (clone CH-11; Upstate
Biotechnology Inc, Lake Placid, NY) or 2 µg/mL mouse IgM (Dako) in
RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mmol/L
L-glutamine, 25 mmol/L HEPES, 100 IU/mL penicillin, and 100 µg/mL
streptomycin. When indicated, 10 µg/mL cycloheximide was added to the
culture. Preliminary kinetic study showed that Fas-induced apoptosis
was maximal after 18 hours of incubation with anti-Fas (clone CH-11). Cells were then harvested and apoptosis was analyzed.
Measurement of FITC-conjugated annexin-V binding.
Apoptosis was analyzed by quantifying phosphatidylserine residues
exposed on the cell membrane. 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 a binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, 5 mmol/L
CaCl2). After 15 minutes of incubation in the dark, a
dual-color analysis was performed on a FACScan flow cytometer. Specific
anti-Fas-mediated apoptosis was calculated by subtracting the
proportion of annexin-positive cells in the presence of control IgM
from the proportion of annexin-positive cells in the presence of
anti-Fas CH-11.
In some experiments, thymocytes were first stained with
biotin-conjugated anti-CD4 and PE-conjugated anti-CD8. After two
washes, cells were incubated with streptavidin, Quantum Red conjugate. After two washes with phosphate-buffered saline (PBS), CD4/CD8 staining
was analyzed. Thymocytes were then labeled with FITC-conjugated annexin-V. The proportion of annexin-positive thymocytes was examined in the CD4/CD8 subsets. We checked that CD4/CD8 double staining or
annexin-V-FITC labeling were not modified in three-color experiments.
After culture with control IgM or anti-Fas antibody (clone CH-11),
thymocytes were stained with anti-Fas antibody as previously described
but with clone CH-11 (instead of clone UB2). Then thymocytes were
labeled with FITC-conjugated annexin-V.
Statistical analysis.
Differences between groups were compared by using the Mann-Whitney or
Wilcoxon test (Graph Pad Software, San Diego, CA). A difference was
considered significant if the P value was less than .05.
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RESULTS |
Regulation of Fas expression in human thymocytes by different
activation signals.
Freshly isolated thymocytes from 5 infants' thymuses (age, 7 days to 2 years) were cultured with the following activation factors: immobilized
anti-CD3 (mouse IgG1) antibody (10 µg/mL), PHA (1 µg/mL), and the
combination of PMA (5 ng/mL), a protein kinase C activator, and
ionomycin, a calcium ionophore (500 ng/mL). Control cultures were
performed in the absence of stimuli or with immobilized mouse IgG1 (10 µg/mL). Fashi cells represented 0.45% ± 0.08% of
freshly isolated cells (means ± SEM, n = 5). Thymocytes were
collected after 4, 16, 24, and 48 hours of culture and Fas, CD4, and
CD8 expression was examined by using three-color flow cytometry. CD25
expression was also measured to check the level of cell activation.
The ability of anti-CD3 antibody to induce thymocyte death in vitro and
the need for a costimulatory signal during TCR stimulation are both
controversial.22,23 Human thymocytes cultured for 24 hours
were slightly but not significantly depleted by anti-CD3 antibody: when
106 freshly isolated viable thymocytes were cultured, 0.91 ± 0.10 × 106 and 0.81 ± 0.09 × 106 thymocytes were recovered after 24 hours of incubation
with immobilized IgG1 and immobilized anti-CD3, respectively (5 independent determinations). The corresponding numbers of viable
thymocytes were 0.96 ± 0.10 × 106 in control
conditions, 0.91 ± 0.27 × 106 after PHA
activation, and 0.68 ± 0.07 × 106 after
PMA/ionomycin activation. The cell depletion induced by PMA/ionomycin
was not mediated by the Fas system, because it was not prevented by a
blocking anti-FasL antibody (clone NOK-1; data not shown).
Kinetic studies showed that CD25 and Fas expression were maximal after,
respectively, 16 and 24 hours of activation.
Figure 1 shows the result of a
representative experiment. PHA weakly activated human thymocytes
(measured by CD25 expression) and had no effect on Fas expression.
PMA/ionomycin strongly activated thymocytes: 74.5% ± 1.4% of
cells expressed CD25 after 16 hours, compared with 1.9% ± 0.2% of
cells in control conditions (5 independent determinations). This
activation induced a slight increase in Fas expression: 3.3% ± 0.9% of total cells were Fashi after 24 hours of
activation with PMA/ionomycin, compared with 1.0% ± 0.2% in
control conditions. By contrast, immobilized anti-CD3 antibody
moderately increased CD25 expression after 16 hours of incubation
(18.6% ± 4.2% of cells v 2.0% ± 0.2% in the
presence of mouse IgG1), but enhanced Fas expression (7.0% ± 2.8% of total cells were Fashi after 24 hours of
incubation with anti-CD3 antibody v 1.2% ± 0.2% with
mouse IgG1). The mean fluorescence intensity (MFI) of Fas labeling was
also significantly increased to 43.8 ± 4.2 after 24 hours of
incubation with anti-CD3 antibody, compared with 33.3 ± 2.9 with
mouse IgG1 (P < .05).
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| Fig 1.
Fas expression is differently regulated by activation
signals in human thymocytes. Fas and CD25 expression were analyzed
after culture with the following activators: immobilized anti-CD3
(mouse IgG, 10 µg/mL), PHA (1 µg/mL), and the combination of PMA (5 ng/mL) and ionomycin (500 ng/mL). Control cultures were performed in the absence of agents or with immobilized mouse IgG1 (10 µg/mL). Thymocytes were collected after 4, 16, 24, and 48 hours of culture and
Fas, CD4, and CD8 expression was examined using three-color flow
cytometry. CD25 expresssion was also measured to check the level of
cell activation. A representative experiment is shown. (A) Analysis of
Fas expression after 24 hours of culture with the different activators.
Fas staining is shown as solid profiles and staining controls as open
profiles. Thymocytes were first labeled with anti-Fas antibody, then
with biotin-coupled antimouse antibody, and lastly with Quantum
Red-conjugated streptavidine and anti-CD25 or anti-CD4 and
anti-CD8 antibodies; only the last two steps were performed in staining
controls. Anti-CD3 antibody-activated thymocytes display the
higher increase in the Fashi thymocyte proportion and in
the MFI of Fas staining. (B) Kinetic study of Fas and CD25 expression
on thymocytes cultured with activation factors. CD25 expression was
maximal after 16 hours of activation and Fas expression after 24 hours.
By contrast with Fas expression, CD25 expression was strongly increased
by PMA + ionomycin but moderately increased by anti-CD3
activation.
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Because CD28 provides a costimulatory signal for TCR stimulation in T
lymphocytes24 and for TCR-induced thymocyte
apoptosis,23 we examined whether costimulation with
immobilized anti-CD28 antibody modulated the anti-CD3-induced increase
in the Fashi thymocyte proportion. The number of human
thymocytes cultured with anti-CD28 + anti-CD3 antibodies was similar to
the number of thymocytes recovered after anti-CD3 activation (not
shown). As shown in Table 1, anti-CD28
antibody did not modify the anti-CD3-induced increase in Fas
expression in human thymocytes.
Effect of cytokines on Fas expression.
Fas expression in human thymocytes was measured after 24 hours of
incubation with IL-2 (200 U/mL), IL-6 (5 pg/mL), IL-7 (10 pg/mL), IL-12
(1 pg/mL), or IFN- (500 U/mL). Apart from IFN-, none of the
cytokines modified Fas expression (Fig 2).
IFN- slightly but significantly increased the proportion of
Fashi thymocytes (4.0% ± 1.2% v
1.4% ± 0.1% in control conditions, P < .05). The
Fashi cell proportion was strongly increased by the
combination of IL-7 and IFN- (16.0% ± 3.2% v 1.3% ± 0.2% in control conditions, P < .05). Fas labeling MFI
was also significantly increased by IFN- and by IL-7 + IFN- after
24 hours of incubation (33.0 ± 7.3 and 50.8 ± 8.3, respectively, compared with 24.6 ± 6.5 in control conditions; both
P < .05). The effects of anti-CD3 and IL-7 + IFN- on Fas
expression were not additive (not shown).
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| Fig 2.
Modulation of the Fashi thymocyte proportion
by cytokines. Fas expression in human thymocytes was measured after 24 hours of incubation with IL-2 (200 U/mL), IL-6 (5 pg/mL), IL-7 (10 pg/mL) IL-12 (1 pg/mL), IFN- (500 U/mL), or the combination of IL-7 (10 pg/mL) + IFN- (500 U/mL). By contrast with the other
cytokines, IFN- and the combination of IFN- + IL-7
significantly increased the Fashi thymocyte proportion.
*P < .05 versus control.
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Activation-induced upregulation of Fas expression in thymic subsets.
Fas, CD4, and CD8 expression was examined in thymocytes cultured with
anti-CD3 antibody or mouse IgG1. After 24 hours of incubation with
immobilized anti-CD3 antibody (Fig 3A),
there was a significant decrease in the proportion of double-positive
CD4+CD8+ thymocytes (47.8% ± 4.5% v 66.9% ± 5.6% in control conditions, P < .05, n = 5) and an increase in the intermediate subset
CD8+CD4lo (CD8 with a low expression level of
CD4; 28.65 ± 1.9% v 7.2% ± 0.9% in control
conditions, P < .05). CD4 modulation by antigenic activation
has already been observed in human T-cell clones.25 The
proportions of single-positive CD4+ and CD8+
cells were not significantly affected (Fig 3A).
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| Fig 3.
Effect of anti-CD3 activation on thymic subsets and their
Fas expression. After 24 hours of culture with immobilized anti-CD3 antibody or control IgG1, thymocytes were first labeled with anti-Fas antibody, then with biotin-coupled antimouse antibody, and lastly with
Quantum Red-conjugated streptavidine and anti-CD4 and anti-CD8 antibodies; only the last two steps were performed in staining controls. Fas expression was analyzed in total cells and thymic subsets. (A) Representative analysis of CD4 and CD8 expression by
thymocytes cultured with immobilized anti-CD3 antibody or IgG1. In
comparison with the IgG1 control, anti-CD3 significantly reduced the
proportion of CD4+CD8+ thymocytes and
increased the intermediate subset CD8+CD4lo.
(B) Effect of immobilized anti-CD3 antibody on Fas expression by thymic
subsets. Data are means ± SEM of 5 experiments. (C) A
representative analysis of total thymocytes and thymic subsets is
shown. Fas staining is shown as solid profiles and staining controls as
open profiles. For each stain, the proportion of Fashi
thymocytes and MFI of Fas staining are indicated. Anti-CD3
significantly increased the Fashi thymocyte proportion
among CD4+ and CD4+CD8+ cells
but not among CD8+CD4lo or CD8+
cells.
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We then measured the proportion of Fashi cells in the
different subsets (Fig 3B). A representative analysis is shown in Fig 3C. The Fashi cell proportion was significantly enhanced by
anti-CD3 antibody (7.3% ± 1.8% of total cells v 1.6% ± 0.2% in the presence of IgG1) and especially among
CD4+ cells (28.6% ± 4.9% v 3.7% ± 0.5%
with IgG1) and CD4+CD8+ (5.1% ± 1.3%
v 1.2% ± 0.3% with IgG1). Thus, CD4+ and
CD4+CD8+ cells were enriched eightfold and
fourfold, respectively, in Fashi cells. The proportion of
Fashi cells was very low among
CD8+CD4lo and CD8+ cells and was
not significantly modified by CD3 activation.
By contrast with antigenic stimulation, stimulation by IL-7 + IFN-
did not modify the proportion of thymic subsets (not shown) and
increased the proportion of Fashi cells in all subsets
(Fig 4). A representative analysis is shown in Fig 4B. The proportion of Fashi cells among
CD4+, CD4+CD8+, and
CD8+ cells increased 8-, 11-, and 11-fold, respectively
(Fig 4A).
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| Fig 4.
Effect of IFN- + IL-7 on Fas expression in thymic
subsets. Thymocytes were first labeled with anti-Fas, then with
biotin-coupled antimouse antibody, and lastly with Quantum
Red-conjugated streptavidine and anti-CD4 and anti-CD8 antibodies; only
the last two steps were performed for staining controls. (A) Data are
the means ± SEM of 5 experiments. IFN- + IL-7 increased the
Fashi thymocyte proportion in all the thymic subsets
(CD4+, CD4+CD8+, and
CD8+ cells). *P < .05 versus control. ( )
Medium; ( ) IL-7 + IFN-. (B) Representative analysis of the
proportion of Fashi thymocytes and MFI of Fas staining in
each culture condition and thymic subset. Fas staining is shown as
solid profiles and staining controls as open profiles.
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Cyclosporin A inhibits the increase in Fas expression induced by
anti-CD3 but not that induced by IL-7 + IFN-.
The CD3-induced increase in Fas expression in human thymocytes was
examined in the presence of 100 ng/mL cyclosporin A, an immunosuppressive drug known to act on calcium-dependent phosphatase calcineurin and to inhibit TCR-induced gene expression in T
cells.26 After 24 hours of incubation with cyclosporin A,
there were no changes in cell viability, the CD4/CD8 subsets, or Fas
expression (not shown). The increase in Fas expression induced by
anti-CD3 was partially inhibited (~50%) by cyclosporin A on both
CD4+ and CD4+CD8+ cells
(Fig 5B). The changes in the proportions of
the CD4/CD8 subsets induced by anti-CD3 were also partially inhibited
by cyclosporin A (not shown). By contrast, the effect of IL-7 + IFN-
on the increase in Fas expression was not affected by 100 ng/mL
cyclosporin A (Fig 5A).
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| Fig 5.
Effect of cyclosporin A (CsA) on the increase in
Fashi cell proportion induced by anti-CD3 antibody or by
IFN- + IL-7. (A) Representative analysis of Fas expression and Fas
staining MFI in human thymocytes activated for 24 hours with anti-CD3
antibody or IFN- + IL-7 in the presence of cyclosporin A (100 ng/mL). Thymocytes were first labeled with anti-Fas antibody, then with biotin-coupled antimouse antibody, and lastly with Quantum
Red-conjugated streptavidine; only the last two steps were used for
staining controls. Fas staining is shown as solid profiles and staining controls as open profiles. Cyclosporin A partially inhibited the anti-CD3-induced increase in Fas expression but did not modify the
increase in Fas expression induced by IFN- + IL-7. (B) Effect of
cyclosporin A in the different subsets of human thymocytes cultured
with immobilized anti-CD3 antibody. Data are the means ± SEM of 3 experiments. Cyclosporin A partially inhibited the increase in the
Fashi cell proportion among CD4+ and
CD4+CD8+ thymocytes. () IgG1; ()
anti-CD3; ( ) anti-CD3 + CsA.
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Effect of cycloheximide on the activation-induced increase in Fas
expression.
The increase in the Fashi cell proportion induced by
anti-CD3 or IL-7 + IFN- after 24 hours of incubation was probed with
10 µg/mL cycloheximide, an inhibitor of protein synthesis
(Fig 6). No change in cell viability, Fas
expression, or CD4/CD8 subsets was seen after 24 hours of incubation
with 10 µg/mL cycloheximide. The effect of IL-7 + IFN- on the
increase in Fas expression was totally inhibited by cycloheximide
(P < .03 compared with thymocytes cultured with cycloheximide
alone, n = 5), showing that the increase in Fas expression induced by
these cytokines in human thymocytes requires protein synthesis. By
contrast, the effect of anti-CD3 was slightly potentiated by
cycloheximide. Similar results were obtained with 0.5 µg/mL
actinomycin D, an inhibitor of mRNA synthesis (not shown). Thus, the
anti-CD3-induced increase in Fas expression was slightly potentiated
by mRNA or protein synthesis inhibitors, whereas the cytokine-induced
Fas expression increase was completely inhibited.
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| Fig 6.
Effect of cycloheximide on the increase in Fas expression
induced by anti-CD3 antibody or IFN- + IL-7. Fas expression was analyzed in human thymocytes after 24 hours of culture in control conditions or with anti-CD3 antibody or IFN- + IL-7 in the
presence or absence of 10 µg/mL cycloheximide. (A) A representative
analysis is presented. Thymocytes were first labeled with anti-Fas
antibody, then with biotin-coupled antimouse antibody, and lastly with
Quantum Red-conjugated streptavidine; only the last two steps were used for staining controls. Fas staining is shown as solid profiles and
staining controls as open profiles. (B) The anti-CD3-induced increase
in the Fashi cell proportion was not significantly modified
by cycloheximide. (C) The cytokine-induced increase was fully inhibited
by cycloheximide. Data are the means ± SEM of 5 experiments. ()
Medium; () IL-7 + IFN-.
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Sensitivity to cell death induced by an agonistic anti-Fas antibody.
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.27 Annexin-V is a calcium-dependent phospholipid binding protein with high affinity for phosphatidylserine and is used
for the detection of apoptotic cells.28 After 18 hours of
incubation with 2 µg/mL anti-Fas antibody (clone CH-11), cells were
examined for FITC-conjugated annexin-V. Because Fas-induced apoptosis
is enhanced by metabolic inhibitors,6,7 10 µg/mL cycloheximide was added to thymocytes when indicated. Freshly isolated
thymocytes were not sensitive to anti-Fas-induced apoptosis, even in
the presence of cycloheximide (not shown).
Thus, we observed the sensitivity to Fas-induced apoptosis of human
thymocytes after 24 hours of antigenic stimulation or cytokine
activation (Fig 7). We also measured
apoptosis in thymocytes previously cultured for 24 hours in control
conditions, ie, with IgG1 antibody or medium alone. The results
obtained with medium alone were always similar to those obtained with
the IgG1 control. The analysis was performed in viable cells using
forward- and side-scatter parameters, as previously described in the
Materials and Methods; in this gate, the proportion of cells that
incorporated propidium iodide was always less than 1%, and we thus
report annexin-V-FITC binding only (Fig 7A). In the absence of
cycloheximide, less than 10% of cells were sensitive to Fas apoptosis,
whatever the stimulus. In the presence of 10 µg/mL cycloheximide,
cells cultured with IgG1 or anti-CD3 antibody were weakly susceptible
to Fas-induced apoptosis (6.2% ± 1.4% and 8.0% ± 2.5%,
respectively, n = 5; Fig 7B). By contrast, 22.5% ± 2.3% of
thymocytes cultured for 24 hours with IL-7 + IFN- underwent
Fas-induced apoptosis (n = 5).
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| Fig 7.
Anti-Fas-induced apoptosis in preactivated thymocytes.
(A) Representative analysis of the apoptotic effect of an agonistic anti-Fas antibody on human preactivated thymocytes. After 24 hours of
culture with IgG1 antibody, anti-CD3 antibody, or IFN- + IL-7, human thymocytes were collected and 105 cells were
incubated with agonistic anti-Fas antibody (clone CH-11, 2 µg/mL) or
IgM antibody (2 µg/mL), with or without 10 µg/mL cycloheximide.
Cells were harvested 18 hours later, washed with PBS, and stained with
FITC-conjugated annexin-V to quantify apoptosis. Specific
anti-Fas-mediated apoptosis was calculated by subtracting the
proportion of annexin-positive cells in the presence of control IgM
from the proportion of annexin-positive cells in the presence of
anti-Fas CH-11. In the absence of cycloheximide, less than 10% of
thymocytes underwent Fas-specific apoptosis. In the presence of
cycloheximide, thymocytes activated by IFN- + IL-7 were more
susceptible than anti-CD3-activated thymocytes to Fas-specific
apoptosis. (B) Data are the means ± SEM of 5 independent experiments.
() IgG1; ( ) anti-CD3; () IL-7 + IFN-.
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Fas-induced apoptosis in thymic subsets.
Three-color labeling with anti-CD4 and anti-CD8 antibodies and
annexin-V was performed on thymocytes cultured with IgM or anti-Fas
(clone CH-11) in the presence of cycloheximide. The proportion of
annexin-positive cells was analyzed among CD4+,
CD4+CD8+ (including
CD8+CD4lo), and CD8+ cells, and the
proportion of CD4+, CD4+CD8+, and
CD8+ cells undergoing Fas-specific apoptosis among the
total thymocyte population was calculated
(Fig 8A). Whatever the conditions,
CD8+ cells undergoing Fas-specific apoptosis represented
less than 1% of total cells. In IgG1 and anti-CD3 conditions,
CD4+CD8+ cells undergoing Fas-specific
apoptosis composed the major subset of apoptotic thymocytes. When cells
were preactivated by cytokines, the proportion of apoptotic
CD4+ and CD4+CD8+ thymocytes were
similar; apoptotic CD4+ cells were about three times more
numerous among cytokine-activated cells than among anti-CD3-activated
cells (11.6% ± 1.1% and 3.2% ± 0.4% of total cells,
respectively, n = 3).
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| Fig 8.
Fas-specific apoptosis in thymic subsets. (A) After 18 hours of incubation with agonistic anti-Fas antibody (clone CH-11, 2 µg/mL) or IgM antibody (2 µg/mL) in the presence of 10 µg/mL cycloheximide, double-staining of CD4/CD8 subsets was
performed in thymocytes before labeling with annexin-V. The
proportion of () CD4+, ()
CD4+CD8+ (DP), and () CD8+
cells that underwent Fas-specific apoptosis was calculated. Data are
the means ± SEM of 3 independent experiments. The proportion of
apoptotic CD4+ cells was three times higher in
cytokine-activated thymocytes than in anti-CD3-activated thymocytes.
(B) Fas staining was performed before annexin-V labeling, and the
proportion of cells undergoing Fas-specific apoptosis was examined in
Fas+/ and Fashi cells. A representative
experiment is shown. Annexin-V stainings in the presence of anti-Fas
CH-11 are presented and specific anti-Fas-mediated apoptosis was
calculated by subtracting the proportion of annexin-positive cells in
the presence of control IgM from the proportion of annexin-positive cells in the presence of anti-Fas CH-11. Fashi cells were
always enriched in apoptotic cells, but cytokine-activated Fashi cells contained more apoptotic cells than did
anti-CD3-activated cells.
|
|
To examine if the level of Fas expression determines the sensitivity to
Fas-induced apoptosis, we studied Fas-specific apoptosis in the
presence of cycloheximide among Fas+/ and
Fashi thymocytes (Fig 8B). Whatever the pretreatment,
Fashi cells contained more annexin-V-positive cells
compared with Fas/+ cells. However,
Fashi cells were twice as susceptible to Fas-specific
apoptosis after cytokine-activated thymocytes as after anti-CD3
activation.
| |
DISCUSSION |
Fas antigen expression by human thymocytes can thus be upregulated by
two signaling pathways, one antigen-dependent and the other
cytokine-dependent. We showed that these two activation pathways differ
(1) in the thymic subsets affected by the increase in the proportion of
Fashi cells, (2) in their sensitivity to cyclosporin A and
thus their dependence on calcineurin phosphatase activity, and (3) in
their sensitivity to metabolic inhibitors and that (4)
cytokine-activated and anti-CD3-activated cells differ in their
susceptibility to Fas-induced apoptosis.
Intracellular regulatory mechanisms of Fas expression in human
thymocytes.
Antigenic stimulation by an anti-CD3 antibody was effective in
enhancing the proportion of Fashi thymocytes, as previously
shown by Yonehara et al.17 This effect involved a small
proportion of thymic cells, mainly in the CD4 lineage. We had
previously shown that Fashi cells that accumulate in the
thymus of MG patients, which are mainly CD4+ and
CD4+CD8+, are enriched in cells autoreactive
against the acetylcholine receptor.18 This finding is thus
compatible with the antigen-dependent Fas upregulation in human
thymocytes. In the same experiment, we compared CD25 and Fas expression
regulation in the presence of various activation signals. We observed
that the regulation of CD25 and Fas expression followed different
kinetic profiles. Furthermore, PMA/ionomycin activation, which strongly
increased CD25 expression in human thymocytes, slightly enhanced Fas
expression, suggesting that Fas and CD25 are not regulated by the same
intracellular pathways.
We used cyclosporin A, an inhibitor of calcineurin activation to
identify intracellular signals underlying the activation-induced increase in Fas expression by human thymocytes. Activation of calcineurin, a calcium- and calmodulin-dependent phosphatase, is known
to be an essential event in T-cell activation via the TCR.26 The increase in Fas expression induced by the TCR
stimulation was partially inhibited by cyclosporin A, suggesting that
calcineurin controlled this increase. This result is compatible with
cyclosporin inhibition of Fas upregulation induced by TCR activation in
murine T-cell hybridomas29 and by cross-linking of CD4
molecules in human peripheral lymphocytes.30 In contrast,
the effect of IL-7 + IFN- was not affected by cyclosporin. Thus,
antigen-dependent cyclosporin-sensitive and cytokine-dependent
cyclosporin-insensitive intracellular pathways can both lead to Fas
upregulation in human thymocytes.
The increase in Fas expression was differently sensitive to
cycloheximide and actinomycin D when induced by antigenic or cytokine activation. Fas upregulation induced by the combination of IL-7 and
IFN- requires de novo mRNA and protein synthesis. By contrast, Fas
upregulation induced by antigenic activation was not inhibited by the
metabolic inhibitors, suggesting that it uses preexisting thymocyte
proteins.
Involvement of Fas in thymic apoptosis.
Thymocytes that are not positively selected may die from
neglect31 and may compose the bulk of cells that die of
apoptosis in the thymus.32 As previously described in mouse
thymocytes,7 spontaneous apoptosis of human thymocytes was
not modified by an antagonistic anti-Fas antibody (clone ZB4; not
shown), suggesting that Fas-mediated apoptosis would not mediate death
by neglect in the human thymus.
Fas-mediated apoptosis of freshly isolated mouse thymocytes was shown
to require cycloheximide6 or actinomycin D.7 By contrast, even in the presence of cycloheximide, freshly isolated human
thymocytes never underwent Fas-specific apoptosis in our experimental
conditions. The presence of cycloheximide was required for Fas-induced
apoptosis to occur in cytokine-activated thymocytes. Thymocyte
resistance to Fas-induced apoptosis in the absence of metabolic
inhibitors could be mediated by FAP-1 (Fas-associated phosphatase-1),
which can inhibit Fas signaling33 and which is
downregulated by actinomycin D.34 In human T cells,
Fas-mediated apoptosis is mediated by intracellular glutathione, levels
of which are reduced by cycloheximide35; this process could
also contribute to thymocyte resistance to Fas-mediated apoptosis.
We showed here that cytokine-activated thymocytes were more susceptible
than anti-CD3-activated thymocytes to Fas-mediated apoptosis in vitro
in the presence of cycloheximide. Soluble FasL has been described as
inactive in mouse-activated splenic T cells36 or as
inhibitory in human-activated peripheral T cells,37 whereas membrane FasL can induce apoptosis. Thus, resistance to apoptosis induced by the anti-Fas antibody (clone CH-11) in anti-CD3-activated cells could be due to inactivity of agonistic anti-Fas antibody (or
soluble FasL) on these cells.
CD8+ cells that underwent Fas-specific apoptosis always
formed a very minor subset. Apoptotic CD4+ cells were four
times as numerous in cytokine-activated thymocytes as in
anti-CD3-activated thymocytes, whereas the number of apoptotic CD4+CD8+ cells was similar in the two
conditions. Thus, the difference in susceptibility to Fas apoptosis
between cytokine-activated and anti-CD3-activated thymocytes is mainly
due to CD4+ thymocytes.
Fashi cells were always enriched in annexin-positive cells
relative to Fas+/ thymocytes. Freshly isolated
FashiCD3int thymocytes were similarly shown to
contain a large fraction of dead cells.16 However,
Fas-specific apoptosis was more efficient in cytokine-activated
Fashi cells than in anti-CD3-activated Fashi
cells. Cytokine activation might thus provide an additional signal to
induce apoptosis in human thymocytes.
By contrast, Fisher et al7 showed that antigenic
stimulation and Fas signal must occur simultaneously to induce Fas
apoptosis in mouse thymocytes, a synergistic effect not mediated by Fas upregulation. This difference could be related to the high basal level
of Fas antigen expression in mouse thymocytes, with about 80% of adult
mouse thymocytes expressing Fas.38 Fas expression and
susceptibility to Fas-specific apoptosis are thus clearly different in
human and mouse thymocytes.
Fas was suggested to be involved in the negative selection in the human
thymus.16,17 Fashi thymocytes are enriched in
CD4+ cells in the human thymus.16-18 TCR
stimulation increases Fas expression, mainly in cells of the CD4
lineage (CD4+ and CD4+CD8+
thymocytes); moreover, Fashi thymocytes express an
intermediate level of CD3 antigen16,18 and antigenic
activation downregulates CD3 expression.25 Thus, in vivo
Fashi thymocytes could have been previously activated by an
antigenic stimulation. IL-7/IFN- activation seems to provide
additional signal rendering thymocytes (particularly CD4+
thymocytes) susceptible to Fas-induced apoptosis. In addition, Kishimoto and Sprent15 recently described a semimature
subset (CD4+CD8-HSAhi) in the mouse thymus that
is susceptible to negative selection in a Fas-dependent way. Taken
together, these results suggest that a thymocyte subset in the CD4
lineage could be the target of Fas-mediated negative selection in the
thymus. Cells able to produce FasL are poorly characterized in the
human thymus, but both epithelial and dendritic cells were shown to
express FasL in situ in the mouse thymus.39 Thus, thymic
stromal cells could mediate apoptosis of a thymocyte subset through
Fas/FasL interaction.
In conclusion, two different signaling pathways can increase Fas
expression by human thymocytes in vitro. Antigenic stimulation upregulates Fas antigen expression in vitro, and this effect mainly involves the CD4 lineage. The cytokine pathway upregulates Fas in all
thymic subsets and induces susceptibility to Fas-mediated apoptosis,
whereas anti-CD3 activation is less efficient in this respect. These
findings indicate that Fas has the potential to participate to the
apoptosis of a human thymic subset in vivo.
| |
FOOTNOTES |
Submitted January 9, 1998;
accepted April 7, 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 the Singer-Polignac Foundation.
Address reprints 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.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Drs F. Lacour-Gayet and A. Serraf for providing
thymuses. We are grateful to Dr J. Curnow for critical reading of the
manuscript.
| |
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Abstract
Introduction
Abstract