Download short Novo Protein Synthesis of c-FLIP De CD95

Resistance of Short Term Activated T Cells to
CD95-Mediated Apoptosis Correlates with De
Novo Protein Synthesis of c-FLIP short
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of June 17, 2017.
Ingo Schmitz, Heiko Weyd, Andreas Krueger, Sven
Baumann, Stefanie C. Fas, Peter H. Krammer and Sabine
Kirchhoff
J Immunol 2004; 172:2194-2200; ;
doi: 10.4049/jimmunol.172.4.2194
http://www.jimmunol.org/content/172/4/2194
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References
The Journal of Immunology
Resistance of Short Term Activated T Cells to CD95-Mediated
Apoptosis Correlates with De Novo Protein Synthesis of
c-FLIPshort1
Ingo Schmitz,*† Heiko Weyd,* Andreas Krueger,2* Sven Baumann,* Stefanie C. Fas,*
Peter H. Krammer,3* and Sabine Kirchhoff4*
T
he elimination of damaged or potentially dangerous cells
by apoptosis is of pivotal importance for the organism.
The role of apoptosis mediated by the death receptor
CD95 (APO-1/Fas) has been investigated intensively, especially in
the immune system (1, 2). Triggering of CD95 by either agonistic
Abs or CD95L leads to oligomerization of CD95 receptors. Subsequently, the adapter molecule Fas-associated death domain
(FADD),5 procaspase-8, and procaspase-10 are recruited to oligomerized CD95, forming a death-inducing signaling complex
(DISC) (3). In the DISC, procaspase-8 and -10 are autoproteolytically cleaved and form the active enzymes (1, 2).
Two signaling pathways of CD95 were identified in different
cell types (4). In type I cells, apoptosis is initiated by activation of
large amounts of caspase-8 at the DISC, followed by rapid cleavage of caspase-3 before the loss of mitochondrial transmembrane
potential (⌬␺M). In contrast, in type II cells, DISC formation is
reduced, and the mitochondrial apoptosis pathway plays a domi-
*Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany; and †Institute of Molecular Medicine, University of Dusseldorf, Dusseldorf,
Germany
Received for publication August 25, 2003. Accepted for publication December
2, 2003.
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 Sonderforschungsbereich 601, Sonderforschungsbereich 405, Deutsches Krebsforschungszentrum/Israeli Minister of Science (Ca 86),
Deutsch-Israelische Kooperation in der Krebsforschung, and the Sander Stiftung.
2
Current address: Laboratory of Lymphocyte Biology, Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA
02115.
3
Address correspondence and reprint requests to Dr. Peter H. Krammer, Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280,
69120 Heidelberg, Germany. E-mail address: [email protected]
4
Current address: COSMIX Molecular Biochemicals, Mascheroder Weg 1b, 38124,
Braunschweig, Germany.
5
Abbreviations used in this paper: FADD, Fas-associated death domain; CHX, cycloheximide; DISC, death-inducing signaling complex; ⌬⌿M, mitochondrial transmembrane potential; LZ-CD95L, leucine zipper-tagged CD95 ligand.
Copyright © 2004 by The American Association of Immunologists, Inc.
nant role. Prominent caspase activation occurs in type II cells only
after the loss of ⌬␺M (4), indicating that mitochondria amplify the
executionary apoptosis caspase cascade in these cells. Activation
of mitochondria by death receptors is mediated by the Bcl-2 family
member Bid. Bid is cleaved by caspase-8, and truncated Bid then
translocates to the mitochondria and induces the release of apoptogenic factors, such as cytochrome c, SMAC/Diablo, Omi/HtrA2,
and endonuclease G (5). In the cytoplasm, cytochrome c binds to
Apaf1, leading to procaspase-9 recruitment. At this protein complex, called apoptosome, caspase-9 is activated (5), which, in turn,
activates executioner caspases, such as caspase-3 and caspase-7.
Accordingly, Bcl-2 overexpression blocks activation of caspases
downstream of mitochondria and subsequent apoptosis only in
type II cells (4).
Death receptor-induced apoptosis can be counteracted by
c-FLIP, also known as FLAME-1/I-FLICE/Casper/CASH/MRIT/
CLARP/Usurpin (6, 7). c-FLIP occurs in two isoforms, a short
form and a long form (c-FLIPshort and c-FLIPlong), which both can
be recruited to the DISC. c-FLIPlong has similar domains as
caspase-8, but an inactive enzymatic site. When stably overexpressed, c-FLIPlong interferes with generation of the large active
subunit of caspase-8 at the DISC level in both CD95 type I and
type II cells (8 –10). However, although c-FLIP has been previously considered mainly to be a caspase-8 inhibitor, recent reports
indicate that at low and physiological concentrations c-FLIPlong
may be required for efficient caspase-8 activation (11, 12). This is
also reflected by c-FLIP-deficient mice, which are characterized by
heart failure and have a strikingly similar phenotype as caspase8-deficient mice. In contrast to c-FLIPlong, the splice variant cFLIPshort structurally resembles viral FLIP (v-FLIP) and contains
two death effector domains, but no caspase-like domain. High levels of c-FLIPshort completely abolish cleavage of caspase-8 at the
DISC (8). The role of c-FLIP proteins in T cells is controversial.
c-FLIPshort clearly contributes to the resistant phenotype of CD3restimulated and CD28-costimulated T cells (13, 14). In contrast,
although some studies show down-regulation of c-FLIPlong during
in vitro culture of primary T cells (15, 16), others did not find
changes in c-FLIPlong expression (9). Therefore, the exact roles of
0022-1767/04/$02.00
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In the early phase of an immune response, T cells are activated and acquire effector functions. Whereas these short term activated
T cells are resistant to CD95-mediated apoptosis, activated T cells in prolonged culture are readily sensitive, leading to activationinduced cell death and termination of the immune response. The translation inhibitor, cycloheximide, partially overcomes the
apoptosis resistance of short term activated primary human T cells. Using this model we show in this study that sensitization of
T cells to apoptosis occurs upstream of mitochondria. Neither death-inducing signaling complex formation nor expression of Bcl-2
proteins is altered in sensitized T cells. Although the caspase-8 inhibitor c-FLIPlong was only slightly down-regulated in sensitized
T cells, c-FLIPshort became almost undetectable. This correlated with caspase-8 activation and apoptosis. These data suggest that
c-FLIPshort, rather than c-FLIPlong, confers resistance of T cells to CD95-mediated apoptosis in the context of immune
responses. The Journal of Immunology, 2004, 172: 2194 –2200.
The Journal of Immunology
Materials and Methods
Preparation of primary T cells and cell culture
Human peripheral T cells were prepared as described previously (17). For
activation, resting T cells (day 0) were cultured at 2 ⫻ 106 cells/ml with 1
␮g/ml PHA for 16 h (day 1). T cells were then washed three times and
cultured for additional 5 days in the presence of 25 U/ml IL-2 (day 6). The
human B lymphoblastoid cell line SKW6.4 was cultured in RPMI 1640
supplemented with 10% FCS, 50 ␮g/ml gentamicin, and 5 mM HEPES.
Abs and reagents
The mAbs against FADD and the polyclonal Abs against Bcl-xL were
purchased from Transduction Laboratories (Lexington, KY). The anti-Bax
Ab was obtained from Upstate Biotechnology (Lake Placid, NY), the antiBcl-2 Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA),
and the anti-caspase-10 Ab was obtained from MBL (Watertown, MA).
The C15 mAb (mouse IgG2b) recognizes the p18 subunit of caspase-8
(20). The anti-c-FLIP mAb NF6 (mouse IgG1) was generated against GSTN-c-FLIP as previously described (9). Anti-APO-1 (anti-CD95) is an agonistic mAb (IgG3, ␬) recognizing an epitope on the extracellular part of
APO-1 (CD95/Fas) (21). The HRP-conjugated goat anti-rabbit IgG was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated goat anti-mouse IgG1 and IgG2b were obtained from Southern Biotechnology Associates (Birmingham, AL). Leucine zipper-tagged CD95
ligand (LZ-CD95) was produced as described previously (22). Cycloheximide was purchased from Sigma-Aldrich (St. Louis, MO) and used at a
concentration of 10 ␮g/ml for the indicated time periods. All other chemicals used were of analytical grade and purchased from Merck (Darmstadt,
Germany) or Sigma-Aldrich.
Immunoprecipitation and Western blot
Immunoprecipitation of CD95 DISC was conducted as previously described (3, 20). Briefly, 2 ⫻ 107 cells, either unstimulated or treated with
1 ␮g/ml anti-APO-1 for 5 min, were lysed in lysis buffer (20 mM Tris-HCl
(pH 7.4), 1% Triton X-100, 10% glycerol, 150 mM NaCl, 1 mM PMSF,
and 1 ␮g/ml leupeptin, antipain, chymostatin, and pepstatin A) for 15 min
on ice and centrifuged (15 min, 14,000 ⫻ g). Subsequently, DISC was
precipitated with protein A beads (Sigma-Aldrich) for 4 h at 4°C.
For Western blot analysis, postnuclear supernatant equivalents of 106
cells or 30 ␮g of protein, as determined by the bicinchoninic acid method
(Pierce, Rockford, IL), were separated by 12% SDS-PAGE, blotted onto a
nitrocellulose membrane (Amersham Pharmacia Biotech, Arlington
Heights, IL), and blocked with 5% nonfat dry milk in PBS/Tween (0.05%
Tween 20 in PBS). After washing with PBS/Tween the blots were incubated overnight with NF6 anti-c-FLIP, C15 anti-caspase-8, anti-FADD, or
anti-Bcl-xL Abs at 4°C. Blots were washed again with PBS/Tween, incubated with HRP-coupled isotype-specific secondary Abs (1/20,000) for 1 h
at room temperature, washed again, and developed with a chemiluminescence reagent (NEN, Boston, MA).
For stripping, blots were incubated for 30 min in a buffer containing 62.5
mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM 2-ME at 60°C. The blots
were washed six times for 10 min each time in PBS/Tween and blocked
again in 5% nonfat dry milk.
Surface staining
Cells (5 ⫻ 105) were incubated with 1 ␮g/ml anti-APO-1 for 15 min at
4°C, washed with PBS, incubated another 15 min with PE-labeled goatanti-mouse Abs (Dianova, Hamburg, Germany), and after further washing
analyzed in a FACScan cytometer (BD Biosciences, Mountain View, CA).
Cytotoxicity assay
For assaying apoptosis 106 cells were stimulated in 24-well plates with 1
␮g/ml anti-APO-1 and 10 ng/ml protein A or were left untreated for 16 h
at 37°C. Cells were centrifuged in a minifuge (Heraeus, New York, NY) at
4000 rpm for 5 min, washed once with PBS, and resuspended in a buffer
containing 0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X-100, and 50
␮g/ml propidium iodide (Sigma-Aldrich). After incubation at 4°C in the
dark for at least 16 h, apoptotic nuclei were quantified by FACScan (BD
Biosciences). Specific apoptosis was calculated as follows: (% experimental apoptosis ⫺ % spontaneous apoptosis)/(100 ⫺ % spontaneous
apoptosis) ⫻ 100.
Determination of mitochondrial membrane potential
To measure ⌬␺M, anti-CD95 (1 ␮g/ml)-treated or untreated cells (5 ⫻
105/ml) were incubated with 5,5⬘,6,6⬘-tetrachloro-1,1⬘,3,3⬘-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; 5 ␮g/ml; FL-1; Molecular Probes,
Eugene, OR) for 20 min at room temperature in the dark, followed by
analysis on a flow cytometer (FACScan).
Results
Cycloheximide sensitizes short term activated T cells by
affecting CD95 signaling upstream of mitochondria
Short term activated primary human T cells (day 1) are resistant to
CD95-mediated apoptosis. However, after prolonged culture, these
T cells (day 6) become sensitive to CD95-triggered apoptosis (17).
Day 1 T cells can be sensitized to CD95-mediated apoptosis by
CHX, an inhibitor of translation, suggesting that resistance to
CD95-mediated apoptosis is dependent on protein biosynthesis
(Fig. 1A). In contrast, CHX did not increase the sensitivity of resting T cells (day 0) or augment that of activated day 6 T cells. To
exclude the possibility that CHX treatment simply up-regulates
CD95 surface expression, resulting in higher sensitivity, we performed FACS analysis of untreated and CHX-treated T cells.
CD95 was expressed in low amounts on day 0 T cells, but was
strongly up-regulated on the cell surface of activated day 1 and day
6 T cells. The mean fluorescence intensities (MFI) are 10.7 for day
0, 69.6 for day 1, and 72.9 for day 6 T cells. Treatment with CHX
led to a slightly decreased expression of CD95 on T cells regardless of their activation state, with MFI of 12.6 for day 0, 48.6 for
day 1, and 49.1 for day 6 CHX-treated T cells (Fig. 1B). Thus,
CHX apparently affected an intracellular factor in the signaling
cascade of CD95.
Mitochondria have been shown to play a central role in apoptosis induction by many stimuli (5). To address at which level CHX
sensitizes in the apoptotic signaling cascade, we measured the
⌬␺M. In agreement with their resistant phenotype, the ⌬␺M of
anti-APO-1-treated day 1 T cells remained unchanged (Fig. 2).
However, in the presence of CHX, the ⌬␺M of anti-APO-1-treated
cells was significantly decreased, although CHX alone had almost
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c-FLIP and its different isoforms in resistance of short term activated T cells remain to be determined.
Stimulation of resting peripheral T cells (referred to as day 0 T
cells) during an immune response leads to their activation (day 1
T cells), resulting in increased expression of several genes, including cytokines and CD95 (2). However, despite high CD95 surface
expression, day 1 T cells are resistant to CD95-mediated apoptosis
(17, 18). Stimulation of previously activated T cells results in activation-induced cell death, which has been shown to involve the
CD95 receptor/ligand system (1, 2). After prolonged culture in the
presence of IL-2 (day 6 T cells), activated T cells develop an
apoptosis-sensitive phenotype (17, 18). As CD95 surface expression changes only slightly between day 1 and day 6 T cells, the
apoptosis-resistant phenotype of day 1 T cells must be caused by
a block in CD95 signal transduction. Our laboratory previously
showed that DISC formation is impaired in these resistant T cells,
whereas the antiapoptotic protein Bcl-xL is highly up-regulated (9,
19). Moreover, resistant day 1 T cells can be sensitized to CD95mediated apoptosis by treatment with the translation inhibitor cycloheximide (CHX) or the transcription inhibitor actinomycin D,
indicating that de novo protein synthesis is required for activated
T cells to maintain a resistant phenotype (17). We now show that
inhibition of protein synthesis does not influence either DISC formation or the expression of antiapoptotic Bcl-2 family members.
However, c-FLIP protein and, importantly, mainly the c-FLIPshort
variant were down-regulated, suggesting that not only the expression levels but also the differential degradation of the c-FLIP isoforms might be involved in the resistant phenotype of short term
activated T cells.
2195
2196
T CELL SENSITIZATION TO APOPTOSIS
FIGURE 1. CHX sensitizes short term activated primary T cells. A, The
sensitivity of resting day 0 (d0), PHA-activated (d1), and T cells subsequently cultured in IL-2-containing medium (d6) toward CD95-mediated
apoptosis was determined in the presence or the absence of 10 ␮g/ml CHX
as described in Materials and Methods. B, The d0, d1, and d6 T cells were
cultured overnight with or without 10 ␮g/ml CHX. Subsequently, the surface expression of CD95 was analyzed by FACS staining. The data shown
in A and B are representative for three independent experiments.
no effect (Fig. 2). Thus, CHX affects the apoptotic machinery upstream of mitochondria. In day 6 T cells, the ⌬␺M was already
decreased upon CD95 stimulation without CHX pretreatment, consistent with the CD95 sensitivity of these cells (Fig. 2).
CHX affects the expression of c-FLIP, but not that of Bcl-2
family proteins
Next we addressed which apoptotic relevant molecules were affected by CHX treatment. Upstream of changes in ⌬␺M are DISC
components such as FADD, caspase-8, caspase-10, and c-FLIP as
well as Bcl-2 family proteins such as Bcl-2, Bcl-xL, and Bax.
CHX-induced sensitization of day 1 T cells could be associated
with up-regulation of proapoptotic molecules or down-regulation
of antiapoptotic molecules. To investigate the expression levels of
DISC components and Bcl-2 family proteins, lysates of untreated
day 0, day 1, and day 6 T cells as well as CHX-treated day 1 T cells
were prepared and analyzed by Western blot. First, the expression
of c-FLIP proteins, which are the most upstream regulators in the
signaling cascade of CD95-mediated apoptosis, was analyzed. cFLIPlong was similarly expressed in day 0 and day 1 T cells. Day
6 T cells showed an ⬃2-fold reduction in protein expression. CHX
treatment reduced c-FLIPlong expression of day 1 T cells to levels
detected in day 6 T cells (Fig. 3A). In contrast, c-FLIPshort was
only expressed in day 1 T cells, not in day 0 or day 6 T cells, and
expression was substantially reduced by CHX (Fig. 3A). Thus, the
expression of c-FLIPshort correlates with resistance to CD95-mediated apoptosis. Caspase-8, one of the initiator caspases in death
receptor signaling, showed similar expression in day 0, day 1, and
day 6 T cells and was only marginally reduced by CHX treatment.
Therefore, caspase-8 may serve as a loading control (Fig. 3A). The
expression of the other initiator caspase, caspase-10, and the proapoptotic Bcl-2 family member, Bax, was only slightly decreased
by CHX, as was the loading control ␤-actin (Fig. 3B). FADD
expression was decreased in a more pronounced manner, suggesting that FADD protein turnover is faster than that of other proapoptotic molecules. The expression of the antiapoptotic molecules Bcl-2 and Bcl-xL was slightly decreased comparable to that
of the ␤-actin control. Although the ratio between both c-FLIP
isoforms sometimes varied depending on the blood donor (compare, e.g., Fig. 3, A and B), analysis of the kinetics showed that
c-FLIPshort was down-regulated faster than c-FLIPlong (Fig. 3C).
Taken together, these results imply that loss of c-FLIPshort expression was most likely responsible for sensitization of day 1 T cells
to CD95-mediated apoptosis.
Down-regulation of c-FLIPshort correlates with loss of ⌬␺M
As prominent activation of caspase-8 takes place directly at the
DISC of type I cells, but relies on a mitochondrial amplification
loop in type II cells (4), we next analyzed the correlation between
c-FLIP down-regulation and the downstream effects of CHX on
⌬␺M in a time-course experiment. Day 1 T cells were pretreated
for different time periods (0 –3 h) with CHX. Subsequently, the
cells were incubated for another 2 h with LZ-CD95L in the presence or the absence of CHX (resulting in total time of treatment
with CHX of 0 –5 h). Some of these cells were then tested for ⌬␺M
by JC-1 staining, whereas the remaining cells were lysed and subjected to Western blot analysis. As shown in Fig. 4A, the expression of c-FLIPlong and its p43 cleavage product decreased only
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FIGURE 2. Drop in ⌬␺M in CHX-treated d1 T cells. T cells, activated
overnight with PHA (d1 T cells), were incubated with 10 ␮g/ml CHX for
1 h or were left untreated. After CHX pretreatment, 1 ␮g/ml anti-APO-1
was added, and the cells were cultured for an additional 2 h. Subsequently,
cells were stained with the mitochondrial membrane potential-sensitive dye
JC-1 and analyzed by FACS. The data shown are representative of three
independent experiments.
The Journal of Immunology
2197
expression was virtually absent. Taken together, these results
strongly suggest that the major factor responsible for sensitization
of day 1 T cells is c-FLIPshort, rather than-FLIPlong.
CHX does not influence DISC formation
FIGURE 3. Reduced expression of c-FLIPshort upon CHX treatment. A,
Postnuclear lysates were prepared from freshly prepared T cells (d0), PHAactivated T cells (d1), which were either untreated or treated with 10 ␮g/ml
CHX for 4 h, and long-term activated T cells (d6). Subsequently, lysates
were resolved on 12% SDS-PAGE, blotted onto nitrocellulose, and analyzed with Abs against c-FLIP and capsase-8. B, The d1 T cells were either
untreated or treated with 10 ␮g/ml CHX for 4 h and analyzed by Western
blot with Abs specific for the indicated proteins. C, The d1 T cells were
treated with 10 ␮g/ml CHX for the indicated time periods, lysed, and
analyzed by Western blot as described in A. Blots were probed with Abs
against c-FLIP. Bcl-xL was used as a loading control. The data shown are
representative of at least two independent donors.
slightly after 4 h of CHX treatment and ⬃5-fold when CHX was
administered for 24 h. In contrast, the decrease in c-FLIPshort expression was much more rapid after CHX treatment. After 2–3 h of
CHX treatment, the expression of c-FLIPshort was reduced ⬎2fold, and it was barely detectable by Western blotting after longer
treatment. This is consistent with Fig. 3C. Moreover, loss of cFLIPshort expression correlated with the decrease in ⌬␺M in these
cells (Fig. 4B). The loss of ⌬␺M was highest when c-FLIPshort
As c-FLIP proteins regulate CD95-mediated apoptosis by inhibiting caspase-8 activation at the DISC, we analyzed the DISC composition in day 1 and day 6 T cells in the presence or the absence
of CHX. As a control, the DISC of the type I cell line SKW6.4 was
immunoprecipitated in parallel. As previously reported (9), in day
1 T cells only weak DISC formation could be detected by Western
blot analysis, as indicated by the detection of p43 c-FLIPlong and
c-FLIPshort. In contrast, day 6 T cells formed a DISC comparable
to the SKW6.4 cells, as detected by the presence of FADD and the
intermediate p43/p41 cleavage fragments of caspase-8 (Fig. 5).
Interestingly, full-length caspase-8 was hardly detectable in the
DISCs of primary T cells, implying a more efficient cleavage in
these cells. Taken together, these data suggest that, in terms of
DISC formation, day 1 T cells behave like type II cells, whereas
day 6 T cells behave like type I cells. Down-regulation of c-FLIP
proteins by CHX led to reduced recruitment to the DISC. However, CHX treatment had no detectable effect on DISC formation,
suggesting that CHX acts downstream of DISC formation.
Increased caspase-8 activation in the presence of CHX
Next we addressed activation of caspase-8 in day 1 T cells after
CD95 engagement. Therefore, day 1 T cells were left untreated or
were incubated with CHX for 4 h, a condition under which the
expression of c-FLIPlong and c-FLIPshort is reduced and abolished,
respectively. Subsequently, these cells were incubated with LZCD95L for different time periods. The activation of caspase-8 in
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FIGURE 4. Loss of ⌬␺M correlates with reduced c-FLIPshort protein
levels. The d1 T cells were incubated with 10 ␮g/ml CHX for the indicated
time periods or were cultured without CHX (0). For the last 2 h within the
indicated time frames, cells were treated with 1 ␮g/ml LZ-CD95L to induce apoptosis. Cells were split for Western blot analysis and ⌬␺M measurement by JC-1 staining. A, For Western blot analysis lysates were resolved on 12% SDS-PAGE, blotted onto nitrocellulose, and analyzed with
anti-c-FLIP, anti-caspase-8, anti-Bcl-xL, and anti-tubulin Abs. The data
shown are representative of two independent experiments. B, To calculate
the specific loss of ⌬␺M, d1 T cells that were treated with CHX in the same
way but cultured without LZ-CD95L were analyzed by JC-1 staining as
well. Specific ⌬␺M was calculated as follows: (% experimental ⌬␺M ⫺ %
spontaneous ⌬␺M)/(100 ⫺ % spontaneous ⌬␺M) ⫻ 100. The mean of two
experiments is shown.
2198
these cells was then analyzed by Western blot, using monoclonal
anti-caspase-8 Abs. CHX treatment of day 1 T cells led to a significant increase in caspase-8 activation after CD95 engagement,
as judged by the formation of the active subunit p18 (Fig. 6). The
appearance of active caspase-8 preceded the maximal loss of ⌬␺M
(Fig. 4B). Thus, although DISC formation remained unaltered,
CHX treatment appeared to increase DISC activity.
Discussion
The immune response can be divided into three phases, namely
activation, effector, and down phases. Upon Ag encounter, T cells
are activated, proliferate, and subsequently exert their effector
functions, e.g., elimination of invading pathogens. During these
phases of the immune response, T cells are resistant to induction of
CD95-mediated apoptosis (1, 2). After clearance of the Ag, most
of the effector T cells die, and only a few survive as memory cells.
Death by apoptosis in the down-phase of an immune response has
been linked to limitations in cytokine availability (23, 24) as well
FIGURE 6. Enhanced proteolytic processing of caspase-8 into its active
p18 subunit in CHX-treated d1 T cells. PHA-activated T cells (d1) were
treated for 4 h with 10 ␮g/ml CHX and subsequently with 1 ␮g/ml LZCD95L for the indicated time periods. Cellular lysates were prepared, resolved on 12% SDS-PAGE, and blotted onto nitrocellulose. Western blot
analysis was performed with Abs against caspase-8. Detection of tubulin
served as a loading control. The data shown are representative of two
independent experiments.
as to activation-induced cell death, which is mainly mediated by
the CD95 system (2). In vitro, short term activated T cells (day 1)
are resistant to CD95-mediated apoptosis, whereas T cells during
prolonged culture become sensitive (day 6) (17). We show in this
study that short term activated T cells can be sensitized to apoptosis by the translation inhibitor CHX. This sensitization is not
due to enhanced CD95 surface expression, differential expression
of Bcl-2 family members, or increased DISC formation. However,
sensitization correlated with a reduction of c-FLIP expression, in
particular of the short form, c-FLIPshort. Loss of c-FLIPshort expression also correlated with a reduction of ⌬␺M and increased
formation of the active subunit p18 of caspase-8.
Although the expression of c-FLIPshort was completely abolished by CHX treatment, only a fraction of day 1 T cells became
susceptible to CD95-mediated apoptosis. Thus, it appears that the
resistance and sensitivity of short term activated T cells are regulated at multiple levels. c-FLIPshort seems responsible for a resistance mechanism that depends on de novo protein synthesis. In
addition, CD95 signaling is regulated by the capability of DISC
formation (9) and the expression of Bcl-xL (19, 25). The fact that
day 1 T cells do not efficiently form a DISC (Fig. 5) (9) indicates
that these cells behave like type II cells, which are dependent on
mitochondrial events to execute apoptosis (4). However, mitochondria are protected in short term activated T cells by the expression of Bcl-xL (19, 25). This resembles the situation in long
term activated T cells upon costimulation where inducible expression of c-FLIPshort and Bcl-xL confers resistance to CD95-mediated apoptosis by inhibiting DISC activity and by protection of
mitochondria, respectively (14, 26). A third level of regulation is
mediated by CD28-induced inhibition of CD95 ligand expression
(14). Without costimulation, long term activated T cells acquire
the ability to efficiently form a DISC and loose expression of BclxL, and thereby are sensitized to CD95-mediated apoptosis. This
switch from protected type II cells to sensitive type I cells is dependent on the presence of IL-2 (25). This complex regulation of
CD95 resistance and sensitivity is important to facilitate an efficient immune response and to prevent the accumulation of activated T cells after Ag clearance.
Apoptosis sensitivity of T cells might be further controlled
by the complex regulation of the relative expression levels of
c-FLIPlong and c-FLIPshort. Although both c-FLIP isoforms had
been previously regarded solely as apoptosis inhibitors, recent results have challenged this view by showing that c-FLIPlong might
even promote caspase-8 activation and apoptosis, which presumably depends on the expression level (11, 12). The proapoptotic
activity of c-FLIPlong is mediated by its heterodimerization with
caspase-8 through the caspase-like domain that is present in cFLIPlong, but not c-FLIPshort. It has been demonstrated that the
affinity of the caspase-8/c-FLIPlong heterodimer to FADD is considerably higher than that of the caspase-8 homodimer (11). Thus,
c-FLIPlong, in contrast to c-FLIPshort, might allosterically modulate
caspase-8 activation and might act as an apoptosis inhibitor only at
high concentrations, achievable mostly after ectopic expression.
Of note, in primary T cells c-FLIPlong levels are ⬃100 times lower
than those of caspase-8 (9), which also suggests that c-FLIPlong is
not a critical mediator of T cell resistance. In our experiments the
expression of c-FLIPlong was only slightly reduced, but could still
be observed after CHX treatment for several hours (Figs. 3B and
4A). This together with a constant expression of c-FLIPlong in T
cells of different activation stages (9) strongly argue against a central role of c-FLIPlong in resistance of T cells to CD95-mediated
apoptosis. The present and previously published data of our laboratory suggest that c-FLIPshort rather than c-FLIPlong is the prime
inhibitor of CD95-mediated apoptosis in T cells.
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FIGURE 5. CHX does not influence DISC formation. PHA-activated
(d1) and day 6-activated T cells (2 ⫻ 107) were treated for 4 h with 10
␮g/ml CHX and subsequently stimulated with 2 ␮g/ml anti-CD95 (clone
anti-APO-1) for 5 min at 37°C or were left untreated. After lysis of the cells,
unstimulated CD95 and DISC were immunoprecipitated by anti-CD95 Ab
(clone anti-APO-1) and analyzed by Western blotting using anti-caspase-8,
anti-c-FLIP, and anti-FADD mAbs. The positions of anti-APO-1 IgG H chain,
procaspase-8, c-FLIP proteins, FADD, and the respective cleavage fragments
are indicated. As a control, DISC formation of 107 SKW6.4 cells, which were
stimulated with anti-APO-1 or were left untreated, was analyzed in parallel.
The data shown are representative of two independent experiments.
T CELL SENSITIZATION TO APOPTOSIS
The Journal of Immunology
Acknowledgments
We thank Wolfgang Mueller, Marc Matuszewski, and Wendelin Wolf for
excellent technical assistance. I.S. thanks Klaus Schulze-Osthoff for critically reading the manuscript and for his support.
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