Download in Thymocytes and Mature T Cells Transduction Pathways to Induce

Glucocorticoids Engage Different Signal
Transduction Pathways to Induce Apoptosis
in Thymocytes and Mature T Cells
This information is current as
of August 3, 2017.
Dapeng Wang, Nora Müller, Kirsty G. McPherson and
Holger M. Reichardt
J Immunol 2006; 176:1695-1702; ;
doi: 10.4049/jimmunol.176.3.1695
http://www.jimmunol.org/content/176/3/1695
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References
The Journal of Immunology
Glucocorticoids Engage Different Signal Transduction
Pathways to Induce Apoptosis in Thymocytes and Mature
T Cells1
Dapeng Wang, Nora Müller, Kirsty G. McPherson, and Holger M. Reichardt2
G
lucocorticoids (GCs)3 are a class of steroid hormones
that exert a wide range of anti-inflammatory, immunosuppressive, and antineoplastic activities, including the
ability to induce apoptosis in T and B lymphocytes (1). This property of GCs is widely exploited to treat neoplastic disorders such
as leukemia and lymphoma (1–3). However, despite being one of
the first recognized forms of programmed cell death (4), the molecular mechanism of GC-induced apoptosis is still incompletely
understood. GCs passively diffuse into the cell and bind to the GC
receptor (GR), a member of the nuclear receptor superfamily (5).
Subsequently, the hormone-receptor complex translocates into the
nucleus, where it modulates gene expression either by direct binding to its cognate response elements or via interaction with other
transcription factors. In the case of thymocyte apoptosis, it has
been previously shown that gene activation is essential to this process (6). However, the genes required for initiating the cell death
program are only beginning to be uncovered (7–11).
Concerning the effector phase of GC-induced apoptosis, many
data suggest that it proceeds via the mitochondrial pathway involving Bcl-2 family members. Disruption of Bcl-2 in mice accelerates GC-induced thymocyte apoptosis, whereas lack of Bax
and Bak prevents it (12, 13). In addition, Bim- and Puma-deficient
mice are partially compromised in this process, supporting the idea
that GCs may act by up-regulating proapoptotic BH3-only proteins
Molecular Immunology, Institute for Virology and Immunobiology, University of
Würzburg, Würzburg, Germany
Received for publication August 11, 2005. Accepted for publication November
10, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from Volkswagen Stiftung (I/75 403), Deutsche
Forschungsgemeinschaft (Re1631/1), and Wilhelm Sander-Stiftung (2003.129.1).
2
Address correspondence and reprint requests to Dr. Holger M. Reichardt, Molecular Immunology, Institute for Virology and Immunobiology, University of
Würzburg, Versbacher Strasse 7, 97078 Würzburg, Germany. E-mail address:
[email protected]
3
Abbreviations used in this paper: GC, glucocorticoid; DEX, dexamethasone; GR,
GC receptor; T-ALL, acute T lymphoblastic leukemia.
Copyright © 2006 by The American Association of Immunologists, Inc.
(11, 14, 15). With regard to the involvement of caspases, genetargeting experiments and pharmacological studies have arrived at
conflicting results. Although the use of small peptide inhibitors has
implicated caspase-3 and -8 in GC-mediated thymocyte apoptosis
(16, 17), the respective knockout mice lack any obvious defects in
this process (18, 19). In contrast, caspase-9 as well as Apaf-1deficient mice are impaired in dexamethasone (Dex)-induced thymocyte cell death (20 –22), whereas in vitro experiments are not in
support of these findings (17, 23). Thus, although a large number
of components have been implicated in GC-induced thymocyte
apoptosis, the exact apoptotic program initiated by GCs remains
controversial.
Besides the mitochondria, participation of the lysosome as well
as the endoplasmatic reticulum in various apoptotic pathways is
now well established (24). In particular, cathepsin B has gained
acceptance as a central mediator of cell death. Cathepsins are synthesized as proenzymes, transported into the lysosomal vesicle,
and activated through proteolytic cleavage (25). Although the controlled recycling of cellular macromolecules by cathepsins occurs
within the lysosome, it has been observed that during certain forms
of apoptosis, cathepsin B is also released into the cytosol. The
mechanism of translocation is poorly understood, but there is evidence that it is achieved by caspases (25). Opening of pre-existing
pores, active transport across the membrane, and limited lysosomal
membrane rupture have all been hypothesized to account for the
leakage of cathepsin B into the cytoplasm following apoptotic
stimuli (26, 27). Once in the cytosol, cathepsin B can induce cell
death by activating initiator caspases (26, 28) or directly cleaving
nuclear substrates (26, 27). However, it is unknown whether one of
these effector mechanisms is involved in GC-induced cell death.
One problem of the current discussion on the mechanisms of
GC-induced apoptosis stems from the fact that different primary
cells and lymphoma cell lines are compared irrespective of their
individual cellular characteristics and gene expression profiles. Although it is generally assumed that GCs induce apoptosis via a
conserved mechanism, this is not supported by any experimental
data. We therefore wondered whether a unique signal transduction
pathway is engaged by GCs to initiate and execute cell death in all
0022-1767/06/$02.00
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Glucocorticoids (GC) induce apoptosis in a variety of cells, but their exact mode of action is controversial. Although initiation relies
on the GC receptor (GR) and de novo gene expression, the effector phase differs among cell types. Proteasomal degradation as well
as caspase-3, - 8, and -9 activity are essential for GC-induced apoptosis in murine thymocytes, but the same enzymes are dispensable in splenic T cells. Live imaging by confocal microscopy revealed that lysosomal cathepsin B, an unrecognized component
of this pathway to date, becomes rapidly activated in thymocytes after GC exposure. This is followed by leakage of cathepsin B
into the cytosol, nuclear condensation, and processing of caspase-8 and -3. According to our model, activation of caspase-3 by
caspase-9 in thymocytes occurs both directly as well as indirectly via a lysosomal amplification loop. Interestingly, acute T
lymphoblastic leukemia cells depend on caspase activity to undergo GC-induced cell death similar to thymocytes. Collectively, the
apoptotic program induced by GCs comprises cell type-specific as well as common features. The Journal of Immunology, 2006,
176: 1695–1702.
1696
types of T lymphocytes or whether distinct pathways exist. Our
data now show that the initiation phase of GC-induced apoptosis is
similar irrespective of the differentiation state of the cell. In contrast, execution of cell death in thymocytes and splenic T cells
differs significantly in the requirement for signal transduction components such as the proteasome, caspases, and cathepsins. In the
future, this could potentially form a basis for new anticancer strategies that specifically target tumor cells while leaving mature T
cells of patients untouched.
Materials and Methods
Animal experimentation
Abs and reagents
The following Abs and reagents used for flow cytometry were obtained
from BD Biosciences: annexin V FITC, annexin V Cy5, anti-mouse TCR␤
Ab (H57-597), anti-Bcl-2 Ab, and active caspase-3 PE apoptosis kit. The
CaspGLOW Fluorescein Active Caspase-8 Staining Kit was purchased
from BioVision. The anti-GR Ab was obtained from Santa Cruz Biotechnology, and the anti-␤-tubulin polyclonal Ab, Dex, propidium iodide, cycloheximide, and mifpristone (RU486) were from Sigma-Aldrich.
Caspase-3 and -8 inhibitors were purchased from R&D Systems, and lactacystin, pan-cathepsin inhibitor, cathepsin B inhibitor, and PD150606 are
from Calbiochem.
Cell isolation and culture
Thymocytes and spleen cells were isolated by passing the freshly isolated
organs through a nylon mesh, followed by repeated washings with PBS.
Splenic T cells were purified by MACS (Miltenyi Biotec), according to the
manufacturer’s instructions. In brief, spleen cells were stained with FITCconjugated H57 Ab, followed by incubation with anti-FITC-coupled magnetic microbeads (Miltenyi Biotec). The labeled cells were passed through
an LS column positioned in a magnetic field, and the T cell fraction was
subsequently eluted. Purity was assessed by flow cytometry and routinely
found to be ⬎95%. After counting, thymocytes and splenic T cells were
resuspended in RPMI 1640 medium containing 10% FCS and standard
serum complements (Invitrogen Life Technologies) at a concentration
of 106 cells/ml and cultured in 96-well plates at 37°C in an incubator
with 5% CO2.
Jurkat J.Gr cells are a derivative of Jurkat E6.1 (American Type Culture
Collection) transduced with a lentivirus expressing the mouse GR linked to
enhanced GFP via an internal ribosomal entry site element (J. van den
Brandt, F. Lühder, D. Wang, K. G. McPherson, R. Gold, and H. M.
Reichardt, submitted for publication). WEHI7.1 cells were obtained from
American Type Culture Collection, whereas TALL-1 cells are from
DSMZ. All cell lines were cultured in RPMI 1640 medium containing 10%
FCS and penicillin/streptomycin, and split every 2–3 days according to the
suppliers’ instructions.
FACS analysis
Flow cytometry was performed on a FACSCalibur machine (BD Biosciences). Cell survival after Dex treatment was determined by costaining
with annexin V and propidium iodide. Cell survival was normalized to
untreated control cultures to correct for spontaneous apoptosis, which varied from 10 to 40%. To this end, the percentage of surviving cells in
cultures that were left untreated for a certain period of time was set to
100%, and the relative cell survival in the various treatment groups was
calculated as a survival ratio. Staining for cleaved caspase-3 and activated
caspase-8 was performed according to the instructions of the manufacturers. All FACS data were analyzed using CellQuest software.
Western blot analysis
Cells were collected by centrifugation and solubilized in radioimmunoprecipitation buffer. Equal amounts of protein were separated on a 15 or 7.5%
SDS-PAGE gel, transferred onto a polyvinylidene difluoride membrane,
and stained with the indicated Abs. All secondary Abs were peroxidase
coupled, and the blots were developed using ECL as a substrate (Amersham). Normalization was achieved by staining with Abs against ␤-tubulin
or p56Lck.
Microscopic detection of cathepsin B activity in living cells
Imaging was performed using a Zeiss LSM 410 confocal microscope and
a ⫻63 oil objective. A total of 1 ⫻ 106 cells per sample of freshly isolated
thymocytes or splenic T cells was treated for 1 h with Dex or PBS, and
subsequently incubated for 30 min with the cathepsin B substrate, a (zArg-Arg)2 derivative of the cresyl violet fluorophore (Metachem). After
incubation, the cells were washed, resuspended in PBS, and immediately
observed under the confocal microscope. At least 200 cells were counted
for each treatment.
For live cell imaging, eight-chamber coverslides (LAB-TEK II; Nunc)
were coated with 0.01% poly-L-lysine (Sigma-Aldrich) for 5 min, extensively washed with PBS, and dried. After incubation with the cathepsin B
substrate, the cells were resuspended in complete RPMI 1640 medium,
supplemented with 20 mM HEPES buffer, and added to the coated chambers. Data were collected for 1.5 h at 5-min intervals. The temperature of
the sample was maintained at 37°C using an objective heater.
Statistical analysis
All data were analyzed using the program Statistica. Comparison of two
experimental groups was achieved using Student’s t test. For comparison of
several groups, a one-way ANOVA followed by a posthoc Tukey honest
significant difference test was performed. The statistical significance of
selected comparisons is depicted in the figures. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01;
ⴱⴱⴱ, p ⬍ 0.001.
Results
GC-induced apoptosis of T lymphocytes is mediated by the GR
and requires de novo gene expression
It has previously been shown that GC-induced thymocyte apoptosis involves gene activation by the GR (6). However, the mechanism underlying initiation of apoptosis in mature T lymphocytes is
not clear. Therefore, we first investigated GC-induced apoptosis in
thymocytes and splenic T cells from heterozygous GRN⫹/⫺
knockout mice (29), in which levels of GR are greatly reduced
(data not shown). The dose-response curve of Dex-induced apoptosis in GRN⫹/⫺ cells was shifted to higher hormone concentrations in both cell types as compared with GR⫹/⫹ control cells,
indicating that the presence of the GR was essential (Fig. 1A). To
investigate whether altered gene expression underlies GC-induced
apoptosis, we used the pharmacological inhibitor RU486, which is
known to block the agonistic activity of the GR by competitive
binding to the ligand-binding domain (30). As expected, RU486
prevented GC-induced apoptosis in thymocytes and splenic T cells
(Fig. 1B). Finally, we investigated the need for de novo gene expression using cycloheximide. This protein synthesis inhibitor
completely prevented Dex-induced apoptosis in thymocytes and
splenic T cells within the first 10 h after addition of the hormone
(Fig. 1C). Taken together, these results suggest that initiation of
GC-mediated apoptosis depends on GR-induced de novo gene expression irrespective of the differentiation stage of the T cell.
Caspase activity is essential for GC-induced apoptosis of
thymocytes, but not splenic T cells
Previous experiments have implicated caspase-3, -8, and -9 in GCinduced thymocyte apoptosis, but their role in mature T cells has
not been explored (16, 17). Therefore, we treated thymocytes and
splenic T cells with 10⫺7 M Dex for 20 h in the absence or presence of the pan-caspase inhibitor Z-VAD-FMK or inhibitors specific for caspase-3 (Z-DEVD-FMK), caspase-8 (Z-IETD-FMK), or
caspase-9 (Z-LEHD-FMK). Apoptosis was subsequently determined by flow cytometry using annexin V FITC and propidium
iodide. In thymocytes, the pan-caspase inhibitor completely prevented Dex-induced apoptosis. Inhibition of caspase-3, -8, and -9
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All animals were kept in individually ventilated cages under specific pathogen-free conditions. GR knockout mice (29) backcrossed to C57BL/6 for
⬎12 generations (GRN) and wild-type C57BL/6 mice (Charles River Laboratories) were bred in our own animal facility and used for the experiments at 6 –12 wk of age. Because homozygous GRN⫺/⫺ mice are not
viable, heterozygous GRN⫹/⫺ mice that express strongly reduced levels of
GR protein were used in this study. All animal experiments were conducted
in accordance with accepted standards of animal care and approved by the
Bavarian state authorities.
GC-INDUCED APOPTOSIS IS CELL TYPE SPECIFIC
The Journal of Immunology
also significantly reduced the extent of cell death, although the
effects were incomplete (Fig. 2A). In contrast, none of the caspase
inhibitors was able to prevent Dex-induced apoptosis in splenic T
cells (Fig. 2A). The presence of the inhibitors alone had no effect
on cell survival (data not shown). A similar difference between
thymocytes and mature T cells was also found in rat cells, confirming that our observations are not species specific (data not
shown).
Cleavage of caspase-3 into its active form is an essential step of
most forms of apoptosis (31). To obtain further evidence for the
observed cell type specificity, we investigated cleavage of
caspase-3 after GC treatment by Western blot and flow cytometry.
Thymocytes and splenic T cells were cultured for 8 h in the presence or absence of 10⫺7 M Dex, and the time course of cleavage
was studied by Western blot. A band corresponding to the processed enzyme was detected in Dex-treated thymocytes as early
as 2 h, but at no time in splenic T cells (Fig. 2B). This result
was confirmed on a single cell basis by flow cytometry taking
FIGURE 2. Caspase activity is essential for GC-induced thymocyte, but
not splenic T cell apoptosis. A, Thymocytes and splenic T cells were
treated with 10⫺7 M Dex in the absence or presence of 100 ␮M pancaspase (D/P), caspase-3 (D/3), caspase-8 (D/8), or caspase-9 (D/9) inhibitor for 20 h. Culture in the presence of vehicle alone (0.5% DMSO) served
as control. Relative survival was determined by annexin V/PI staining and
set as 100% for controls. The error bars represent the SEM for three independent experiments with three replicates each. Statistical significance
was determined by ANOVA, as described in Materials and Methods, and
is solely depicted for the comparison of individual inhibitor treatments with
the Dex-treated cells. B, Thymocytes and splenic T cells were cultured in
the absence or presence of 10⫺7 M Dex for 8 h. Cells were collected at 2-h
intervals, and the proteins were extracted. Full-length caspase-3 (C3) and
its cleaved form were visualized by Western blot; staining for Lck served
as a loading control. One representative experiment of three is depicted. C,
Thymocytes and splenic T cells were treated with 10⫺7 M Dex or PBS
(con) for 10 h, and the percentage of cells in the live gate that positively
stained for cleaved (active) caspase-3 was determined by flow cytometry
using a PE-conjugated mAb. One representative FACS analysis is depicted
for each sample. The percentages refer to the proportion of cells containing
active caspase-3. D, Time course of caspase-3 cleavage in thymocytes
(Thy) and splenic T cells (Tc) treated with 10⫺7 M Dex or PBS measured
by flow cytometry as in C. E, Thymocytes were treated with Dex in the
presence or absence of 100 ␮M caspase-3 (D/3), caspase-8 (D/8), or
caspase-9 (D/9) inhibitor for 10 h, and the percentage of live cells containing active caspase-3 was determined by flow cytometry. The error bars
in D and E represent the SEM for three independent experiments with three
replicates each. Analysis in E was achieved by ANOVA, as described in
Materials and Methods; statistical significance is only depicted for the
comparison of individual inhibitor treatments with the Dex-treated cells.
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FIGURE 1. The execution phase of GC-induced apoptosis in thymocytes and splenic T cell is similar. A, Thymocytes and splenic T cells from
wild-type (GRN⫹/⫹) and heterozygous GR knockout mice (GRN⫹/⫺) were
cultured in the presence of Dex for 20 h. Relative survival compared with
the untreated control culture was determined by annexin V/PI staining. n ⫽
5 for thymocytes; n ⫽ 3 for splenic T cells. B, Thymocytes and splenic T
cells were treated with 10⫺7 M Dex in the absence (D) or presence (D/R)
of 10⫺6 M RU486 for 20 h, and the relative survival was determined at the
10- and 20-h time points. Survival in the untreated control culture (con)
was set at 100%. C, Thymocytes and splenic T cells were treated with 10⫺7
M Dex in the presence or absence of 10 ␮M cycloheximide for 10 h. Every
2 h, the relative survival as compared with the untreated control culture was
determined. The error bars represent the SEM for three independent experiments with three replicates each. Statistical significance was determined using Student’s t test, as described in Materials and Methods.
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GC-INDUCED APOPTOSIS IS CELL TYPE SPECIFIC
advantage of an Ab specifically detecting the cleaved form of
caspase-3. Dex treatment led to an activation of caspase-3 in
⬃80% of all thymocytes within the first 10 h (Fig. 2, C and D). In
contrast, no cleavage of caspase-3 was observed in Dex-treated
splenic T cells, even after prolonged incubation for 24 h (Fig. 2, C
and D, and data not shown). Importantly, cleavage could be prevented by inhibitors of caspase-3, -8, and -9, suggesting that
caspase-3 is positioned downstream of the other two caspases (Fig.
2E). Taken together, these results suggest that thymocytes and mature T cells significantly differ in their requirement for caspase
activity to undergo apoptosis after GC treatment.
Proteasomal degradation and cathepsin activity are required for
GC-induced apoptosis of thymocytes, but not splenic T cells
Cathepsin B rapidly becomes activated after GC exposure and
is part of a lysosomal amplification loop
Based on the inhibitor studies, cathepsin B plays an essential role
in GC-induced cell death in thymocytes. To confirm this finding,
we studied cathepsin B activation by confocal microscopy using a
substrate that is converted into a fluorescent product by the active
enzyme (34). Within 90 min after Dex exposure, about one-half of
all thymocytes stained positive for active cathepsin B, whereas this
was only rarely seen in control cultures (Fig. 4, A and B). In contrast, active cathepsin B was found in many splenic T cells directly
after isolation, but, importantly, there was no change observed
after addition of Dex for up to 6 h (Fig. 4A and data not shown).
In keeping with this finding, enumeration of cells containing active
cathepsin B revealed a significant difference between Dex- and
PBS-treated thymocytes, but not splenic T cells (Fig. 4B and data
not shown). Staining with a green fluorescent lysotracker further
suggests that cathepsin B activation indeed occurs in lysosomes as
expected (data not shown). To study the position of cathepsin B in
the apoptotic pathway relative to the involved caspases, we studied
its activation in the presence of various pharmacological inhibitors
by confocal microscopy. Cathepsin B inhibitor prevented the accumulation of fluorescent cells, while caspase-3 and -8 inhibitor
FIGURE 3. Pharmacological inhibitors allow to dissect the mechanism
of GC-induced apoptosis. Thymocytes and splenic T cells were treated
with 10⫺7 M Dex in the presence or absence of 8 ␮M lactacystin, 10 ␮M
pan-cathepsin inhibitor, 100 ␮M cathepsin B inhibitor, or 100 ␮M
PD150606 for 10 h. Every 2 h, the relative survival as compared with the
untreated control culture was determined by annexin V/PI staining. The
error bars represent the SEM for three independent experiments with three
replicates each. Statistical analysis was performed using Student’s t test.
did not have any significant effect (Fig. 4C). This suggests that
cathepsin B activation precedes the activation of caspase-3 and -8.
In contrast, inhibition of caspase-9 completely abolished cathepsin
B activity, indicating that the lysosome acts downstream of
caspase-9. Given the known ability of caspase-9 to directly activate caspase-3, we hypothesize that the lysosomal program forms
an amplification loop that functions in parallel to the direct effects
of the apoptosome.
Finally, we investigated the kinetics of Dex-induced thymocyte
apoptosis by live imaging of cathepsin B activity using confocal
microscopy. Single cells were selected and observed over a period
of 75 min starting 90 min after GC treatment (Fig. 4D). Within 15
min, strongly fluorescent dots accumulated within the cell, representing active cathepsin B. After 30 min, the active enzyme was
released from the lysosomes, the fluorescence intensity increased,
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It was previously shown that GC-induced apoptosis in thymocytes
involves proteasomal degradation (32, 33). Given that caspase activity is dispensable for GC-induced apoptosis of mature T cells,
we wondered whether this was also true for proteasomal processes.
To this end, we treated thymocytes and splenic T cells with 10⫺7
M Dex in the absence or presence of lactacystin and followed
apoptosis over the first 10 h (Fig. 3). As expected, lactacystin
efficiently prevented cell death in thymocytes (33). However, the
same compound had no effect on GC-induced apoptosis in splenic
T cells. Similar results were obtained with the proteasome inhibitor
MG-115 (data not shown), confirming that proteasome activity
was indeed dispensable for GC-induced apoptosis in mature T
cells.
Recently, lysosomal proteases from the family of cathepsins
have been implicated in various forms of programmed cell death
(25, 27, 34). We therefore tested their requirement for GC-induced
apoptosis in thymocytes and splenic T cells (Fig. 3). The pancathepsin inhibitor Z-FG-NHO-Bz and the cathepsin B inhibitor
Z-FA-CH2F (35) both significantly reduced the extent of Dex-induced apoptosis in thymocytes. In contrast, the same inhibitors
were only weakly effective in splenic T cells. Importantly, the
cathepsin inhibitors are known not to cross-react with caspases
(16, 36, 37). For comparison, the calpain inhibitor PD150606,
which blocks nonlysosomal cysteine proteases, had no effect on
GC-induced apoptosis in either cell type (Fig. 3). Taken together,
these results reveal for the first time a role for cathepsin activation
in GC-induced thymocyte apoptosis.
The Journal of Immunology
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Dex for 10 h, and the percentage of live cells containing active
caspase-8 was determined. As already suggested by the inhibitor
studies, caspase-8 was exclusively activated in thymocytes, but not
in splenic T cells following GC treatment (Fig. 5, A–D). Culture of
thymocytes in the presence of caspase-8 inhibitor completely prevented its activation, as did caspase-9 inhibitor, cathepsin B inhibitor, and lactacystin (Fig. 5C). These results confirm the previously made observation that cathepsin B precedes caspase-8
activation (Fig. 4C), and at the same time place the proteasome and
caspase-9 upstream of caspase-8. In support of this notion, both
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FIGURE 4. Cathepsin B activation is an early event during GC-induced
thymocyte apoptosis. A, Thymocytes and splenic T cells were cultured in
the presence (Dex) or absence (PBS) of 10⫺7 M Dex for 1 h. Subsequently,
the cells were incubated with the substrate for 30 min, and cathepsin B
activity was observed by confocal microscopy. One representative experiment of three is depicted. B, Enumeration of thymocytes positively staining for active cathepsin B, as shown in A. The error bars represent analysis
of ⬎200 representative cells each. C, Thymocytes were treated with 10⫺7
M Dex in the presence or absence of inhibitors of cathepsin B (CatB),
caspase-3 (C3), caspase-8 (C8), or caspase-9 (C9) for 1 h. Treatment with
the vehicle alone (0.2% DMSO) served as a control. Cells positively staining for active cathepsin B were determined and enumerated, as described
for B. Analysis was achieved by ANOVA, as described in Materials and
Methods. Statistical significance is only depicted for the comparison of
individual inhibitor treatments with the Dex-treated cells. D, Thymocytes
were treated with 10⫺7 M Dex for 1 h, followed by incubation with the
substrate for 30 min. Subsequently, cathepsin B activity was observed in
single cells at 5-min intervals for the following 75 min. The kinetics and
distribution of cathepsin B activity for a representative thymocyte are depicted. The observed cell is marked by an open arrow. The bar equals 5 ␮m
in all panels.
and cathepsin B became redistributed into the cytosol. Within another 15 min, nuclear condensation was observed. Then, the enzymatic activity became confined to the contracting remnants of
the cell until it was finally almost undetectable (Fig. 4D). Importantly, a similar release of cathepsin B into the cytosol after Dex
exposure was never observed in mature T cells, confirming that the
involvement of the lysosomal apoptotic pathway is restricted to
thymocytes (data not shown).
Proteasomal degradation precedes caspase-8 activation
Our data indicate that caspase-8 activation occurs after cathepsin B
has been released into the cytosol, but before caspase-3 cleavage.
To obtain independent evidence for this sequence of events, we
directly studied activation of caspase-8 by flow cytometry using
the cell-permeable, nontoxic substrate FITC-IETD-FMK, which
irreversibly binds to the activated enzyme. Thymocytes and
splenic T cells were cultured in the presence or absence of 10⫺7 M
FIGURE 5. Caspase-8 activation occurs downstream of proteasomal
degradation and lysosomal processes. A and B, Thymocytes and splenic T
cells were treated with 10⫺7 M Dex or PBS (con) for 10 h, and the percentage of cells in the live gate that stained positively for activated
caspase-8 was determined by flow cytometry. One representative FACS
analysis is depicted each. The percentages refer to the proportion of cells
containing active caspase-8. C, Caspase-8 activation was determined in
thymocytes treated with Dex in the presence or absence of 100 ␮M
caspase-8 (D/8), caspase-9 (D/9), cathepsin B inhibitor (D/B), or 8 ␮M
lactacystin (D/L), as described for A. D, Caspase-8 activation was determined in splenic T cells cultured in the presence or absence of 10⫺7 M
Dex, as described for B. E, Thymocytes were treated with Dex in the
presence or absence of 100 ␮M cathepsin B inhibitor (D/B) or 8 ␮M
lactacystin (D/L), and the percentage of live cells containing active
caspase-3 was determined by flow cytometry, as described for Fig. 2. The
error bars in C–E represent the SEM for three independent experiments
with three replicates each. Analysis was achieved by ANOVA, as described in Materials and Methods. Statistical significance is only depicted
for the comparison of individual inhibitor treatments with the Dex-treated
cells.
1700
cathepsin B inhibitor and lactacystin also inhibited caspase-3
cleavage (Fig. 5E).
The apoptotic program induced by GCs in lymphoid tumor cells
partially resembles the one in murine thymocytes
FIGURE 6. The death signal-transducing pathway in T-ALL cells resembles the one in thymocytes. A, Comparative analysis of GR and Bcl-2
protein levels in Jurkat J.Gr, TALL-1, and WEHI7.1 cells and murine
thymocytes by Western blot. Staining with an anti-␤-tubulin Ab served as
a loading control. B, J.Gr cells were treated for 24 h with 10⫺7 M Dex in
the presence or absence of 10 ␮M pan-caspase (D/P), caspase-3 (D/3),
caspase-8 (D/8), caspase-9 inhibitor (D/9), or vehicle (0.1% DMSO, con).
Relative survival was determined by annexin V/PI staining. C and D,
TALL-1 cells were treated for 72 h and WEHI7.1 cells for 24 h with 10⫺5
M Dex in the presence or absence of the same caspase inhibitors as in B,
and the relative survival was determined. The error bars represent the SEM
for three independent experiments with three replicates each. Analysis was
achieved by ANOVA, as described in Materials and Methods. Statistical
significance is only depicted for the comparison of individual inhibitor
treatments with the Dex-treated cells.
To investigate whether GC-induced lymphoid tumor cell death
shares characteristics of thymocyte or splenic T cell apoptosis, we
exposed them to Dex in the presence or absence of caspase inhibitors. Similar to murine thymocytes, cotreatment of all three cell
lines with any of the caspase inhibitors fully prevented induction
of apoptosis (Fig. 6, B–D). Interestingly, the inhibitors were already effective at 10 ␮M, suggesting that the cell lines are more
sensitive to inhibition of caspases than primary lymphocytes. Unfortunately, the effects of cycloheximide, lactacystin, and cathepsin B inhibitor could not be assessed because they turned out to be
toxic already at very low concentrations (data not shown). Taken
together, it appears that, at least with regard to the involvement of
caspases, the apoptotic program induced by GCs in T-ALL cell
lines resembles the one in thymocytes.
Discussion
More than a quarter of a century ago, the phenomenon of GCinduced lymphocyte apoptosis was first recognized (4), but our
knowledge about the signal transduction pathways engaged by
GCs in this process is still incomplete (1–3). Gene-targeting and
inhibitor studies have implicated GR-mediated gene activation,
pro- and antiapoptotic Bcl-2 family members, as well as caspases
in GC-induced cell death, but to date these components have not
been assembled in a cohesive scheme. Little attention has also
been given to the cell type specificity of GC-induced apoptosis.
Most investigators in the past have focused on analyzing apoptosis
in thymocytes, although it has been shown that the pan-caspase
inhibitor Z-VAD-FMK fails to prevent Dex-induced cell death of
mature, peripheral T cells (41). The relevant knockout mouse models such as caspase-3 and -9 as well as Apaf-1-deficient mice have
been exclusively analyzed for Dex-induced apoptosis of thymocytes, but not mature T lymphocytes (19 –22). Although GC-induced cell death has been studied in peripheral T cells of conditional caspase-8 knockout mice (18), this was only done using
activated cells known to be partially protected from GC action.
Thus, a systematic comparison of different cell types for their sensitivity to GCs has not been performed to our knowledge. We have
now found that the execution phase of GC-induced apoptosis differs between thymocytes and splenic T cells. Several components
that are critical to this process were identified in thymocytes,
whereas none of them seem to play a role in GC-induced apoptosis
of splenic T cells. Importantly, we could define a set of events
exclusively occurring in thymocytes. These include activation of
caspase-3, -8, and -9, proteasomal degradation, and the requirement for a lysosomal pathway involving cathepsin B. This shows
for the first time that GCs induce distinct apoptotic programs in
thymocytes and mature T cells (Fig. 7). However, whereas a vast
amount of data is available concerning GC-induced thymocyte apoptosis, we still have no clue as to the pathway used by GCs to
induce cell death in peripheral T cells. Besides the analyses described in this study, further attempts to demonstrate a role for
serine proteases or the apoptosis-inducing factor, a mediator of
caspase-independent cell death (42), failed (data not shown).
Recent work from several laboratories has identified a number
of genes, such as TDAG8, dig-2, Bim, and PUMA, which become
activated during GC-induced apoptosis. Although the importance
of these proteins was confirmed by the analysis of knockout and
transgenic mice, RNA interference, and pharmacological approaches, their exact role in the initiation and execution of the
apoptotic program induced by GCs remains elusive (7–9, 11, 14,
43, 44). The role of caspases is also contentious. Caspase-3, -8, and
-9 have been ascribed a role in GC-induced thymocyte apoptosis
by some investigators, but not by others (17–23, 45). Attempts to
solve this dilemma include the postulate of bifurcated pathways.
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Induction of lymphocyte apoptosis is the therapeutical principle
underlying the treatment of lymphoproliferative malignancies such
as lymphoma and leukemia with synthetic GCs (3). To investigate
the signal transduction pathway engaged by GCs in lymphoid tumor cells, we studied three different acute T lymphoblastic leukemia (T-ALL) cell lines. Jurkat J.Gr cells are a derivative of the
human cell line Jurkat E6.1, which had been transduced with a
GR-encoding lentivirus (van den Brandt et al., submitted for publication). TALL-1 is also of human origin and has previously been
shown to respond to GC treatment with growth arrest (38).
WEHI7.1 is a mouse T-ALL cell line frequently used to study
GC-induced apoptosis (11, 39, 40).
First, we studied GR protein levels in all three lines. Whereas
expression in J.GR and TALL-1 cells was comparable to murine
thymocytes, the level of GR protein in WEHI 7.1 cells was much
lower (Fig. 6A). Furthermore, TALL-1 cells express high levels of
Bcl-2, which was undetectable in both WEHI 7.1 and J.Gr cells
(Fig. 6A). These findings may explain the comparably high resistance of TALL-1 and WEHI7.1 cells to GC-induced apoptosis, i.e.,
both lines require treatment with 10⫺5 M Dex to achieve a decent
level of apoptosis, whereas in the case of J.Gr cells 10⫺7 M Dex
is sufficient.
GC-INDUCED APOPTOSIS IS CELL TYPE SPECIFIC
The Journal of Immunology
pathways act in concert, caspase-3 is sufficiently activated to initiate cell death.
T lymphocyte apoptosis induced by synthetic GC derivatives is
an important component of therapeutic protocols used to treat various forms of lymphoma and leukemia. Because under these regimens not only the neoplastic cells, but also the remaining healthy
lymphocytes are affected, strategies would be desirable that allow
the transformed cells to be targeted more selectively. In this context, it is interesting that apoptosis induced in three T-ALL cell
lines shares its dependence on caspase activity with thymocytes,
but not mature T cells. Although involvement of other components
such as the proteasome and cathepsin B could not be addressed in
this study due to the toxicity of the inhibitors, it still suggests that
parallels exist in the apoptotic program induced by GCs in thymocytes and T-ALL cells. From this, one would expect that addressing components of the signal transduction pathways used
only by thymocytes and lymphoma cells, but not mature peripheral
T cells may represent a possible strategy for a more selective cancer therapy. Although suitable candidates have not yet been identified in this work, our results feed the hope that such a concept
might work and be developed in the future.
Acknowledgments
We thank Marco Herold for critical reading of the manuscript,
Melanie Schott and Katrin Voss for excellent technical help,
Thomas Hünig for fruitful scientific discussions, Denise Tischner for advice with the statistical analyses, and François Tronche for providing the
GRN mice.
Disclosures
Lepine et al. (23), for example, suggested a model in which
caspase-8 is activated in parallel to caspase-9, with both converging on caspase-3. Although our concept of GC-induced thymocyte
apoptosis differs a little from their model (see below), it also assumes branched pathways and suggests an amplification loop analogous to that shown for death receptor-induced apoptosis in type II
cells (46).
A novel aspect of this study is the identification of cathepsin B
activation being an early and essential step in the execution phase
of GC-induced thymocyte apoptosis. It was previously shown that
lysosomes and cathepsin B play an important role in the induction
of apoptosis in a variety of cell types (24, 47). However, the function of cathepsin B in GC-mediated cell death remained largely
unexplored. One study has demonstrated increased cathepsin B
mRNA levels 24 h after Dex treatment in thymocytes, but given
the generally fast apoptotic response to this stimulus, the observed
effect is unlikely to be important (48). We have now defined an
important role for cathepsin B activity and consequently the lysosome at an early stage of GC-induced thymocyte apoptosis. The
general model of lysosomal cell death suggests that cathepsin B
becomes activated and subsequently released (25). This leads to its
redistribution into the cytosol, in which cell death is initiated either
through caspase activation or direct cleavage of nuclear substrates
(26, 27). In the case of TNF-␣-mediated hepatocyte apoptosis, this
establishes an amplification loop to enhance cell death (25). Our
findings on GC-induced apoptosis in thymocytes agree with this
model. In summary, we propose the following pathway for GCinduced apoptosis of thymocytes (Fig. 7). Initiation of the apoptotic program proceeds via the mitochondria and results in caspase-9
activation. This directly leads to caspase-3 cleavage through the
apoptosome. However, in contrast to other forms of apoptosis, a
lysosomal amplification loop is required. This involves activation
and cytosomal leakage of cathepsin B, followed by caspase-8 activation, and also converges on caspase-3. Only when the two
The authors have no financial conflict of interest.
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FIGURE 7. Working model of GC-induced apoptosis in thymocytes.
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