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The Journal of Immunology, 2007, 178: 3944-3953.
Copyright © 2007 by The American Association of Immunologists, Inc.

Fever-Like Hyperthermia Controls T Lymphocyte Persistence by Inducing Degradation of Cellular FLIPshort1

Annika Meinander*,{dagger},{ddagger}, Thomas S. Söderström*,{ddagger}, Aura Kaunisto*,§, Minna Poukkula2,*,§, Lea Sistonen*,{ddagger} and John E. Eriksson3,*,{ddagger}

* Turku Centre for Biotechnology, Åbo Akademi University and University of Turku, Turku, Finland; {dagger} Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland; {ddagger} Department of Biology, Åbo Akademi University, Turku, Finland, and § Department of Biology, University of Turku, Turku, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Fever has a major impact on immune responses by modulating survival, proliferation, and endurance of lymphocytes. Lymphocyte persistence in turn is determined by the equilibrium between death and survival-promoting factors that regulate death receptor signaling in these cells. A potential integrator of death receptor signaling is the caspase-8 inhibitor c-FLIP, the expression of which is dynamically regulated, either rapidly induced or down-regulated. In this study, we show in activated primary human T lymphocytes that hyperthermia corresponding to fever triggered down-regulation of both c-FLIP-splicing variants, c-FLIPshort (c-FLIPS) and c-FLIPlong, with consequent sensitization to apoptosis mediated by CD95 (Fas/APO-1). The c-FLIP down-regulation and subsequent sensitization was specific for hyperthermic stress. Additionally, we show that the hyperthermia-mediated down-regulation was due to increased ubiquitination and proteasomal degradation of c-FLIPS, the stability of which we have shown to be regulated by its C-terminal splicing tail. Furthermore, the induced sensitivity to CD95 ligation was independent of heat shock protein 70, as thermotolerant cells, expressing substantially elevated levels of heat shock protein 70, were not rescued from the effect of hyperthermia-mediated c-FLIP down-regulation. Our findings indicate that fever significantly influences the rate of lymphocyte elimination through depletion of c-FLIPS. Such a general regulatory mechanism for lymphocyte removal has broad ramifications for fever-mediated regulation of immune responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Elevated body temperatures associated with fever have been proposed to have a major impact on immune responses during infections, as fever influences the clonal expansion and proliferation of lymphocytes and enhances the lymphocyte-mediated cytotoxicity in target cells (1, 2, 3). The persistence of lymphocyte populations in turn will be determined by the balance of death and survival-promoting factors in the cells. At normal temperatures, this balance is primarily determined by death and survival signals from growth factors and cytokines. In fever, however, stress and stress-specific cytokine signaling are important for the fate-determining balance (3, 4, 5, 6, 7, 8). Cell stress is an influential factor in development and in fully developed organisms, both under normal and pathological conditions. Stress has the capacity to induce protective measures, such as the heat shock response, a common response from bacteria to mammals, characterized by elevated synthesis of heat shock proteins (Hsps)4, which function as molecular chaperones to prevent stress-induced protein aggregation and misfolding (9, 10). In contrast, if the stress is severe enough it triggers apoptosis, a process necessary for elimination of terminally damaged cells, apart from the numerous other physiological functions assigned to apoptosis (11, 12). The decision to undergo apoptosis can descend either from signals within the cell, the intrinsic apoptotic pathway, or from extrinsic signaling by death receptor triggering (12, 13).

Apoptotic signaling via the extrinsic pathway is involved in the elimination of immune cell populations (14, 15). The extracellular signals are mediated by death ligands that activate their corresponding receptors. Among these receptors, CD95 (Fas/APO-1), a member of the TNFR superfamily, is of particular importance in determining the lifespan of immune cell populations (16, 17). When CD95 ligand (CD95L) binds to CD95, the adaptor protein FADD binds to the intracellular death domain of the ligated receptors (18, 19). The death effector domain of FADD then engages the apoptotic machinery due to its affinity for the apoptotic initiator caspases, procaspase-8 and procaspase-10, which are activated upon oligomerization, inducing cleavage to active caspase fragments (20, 21, 22, 23, 24). In addition to the initiator caspases, the death effector domain in FADD also interacts with the apoptosis modulator cellular FLICE-inhibitory protein (c-FLIP) (25). Together with the activated death receptor, FADD, caspase-8, caspase-10, and the FLIP proteins form the core of the death-inducing signaling complex (DISC) that also contains other proteins, many of which have been implicated as modulators of death receptor signals (26). Depending on the proteins recruited to the DISC, further apoptotic signaling can either be induced or suppressed (26).

Originally c-FLIP was reported to act as a physiological inhibitor of caspase-8, thereby preventing receptor-mediated apoptosis (25). In most cell types, c-FLIP exists as two differently spliced variants, a long form, c-FLIPL, and a short form, c-FLIPS, with isoform-specific functions, not only limited to inhibition of caspase-8 (27, 28). c-FLIPL, which resembles caspase-8 albeit lacking the proteolytically active cysteine in the caspase homology domain, is able to participate in autoprocessing and membrane-restricted activation of caspase-8 (29, 30, 31, 32). c-FLIPL can also be cleaved by caspase-8, and the cleaved p43 form of c-FLIPL has been suggested to promote NF-{kappa}B signaling (33, 34, 35, 36, 37, 38). In contrast, c-FLIPS, which lacks the caspase homology domain, inhibits binding and oligomerization of caspase-8 in the DISC, thereby preventing the activating cleavage of caspase-8 (29) and generation of the cleaved NF-{kappa}B-activating fragment of c-FLIPL. Thus, although first considered to be similar inhibitor proteins, the c-FLIP isoforms have now been established as DISC molecules with distinct and even opposite functions (39, 40).

The apoptotic signaling in lymphocytes is subjected to a stringent but dynamic regulation. Following T lymphocyte activation, the death receptors of proliferating lymphocytes need to be carefully secured against apoptotic signaling via the CD95L, the expression of which is greatly elevated when the cells are activated (41, 42, 43, 44). In contrast, the T lymphocytes produced during clonal expansion need to regain their sensitivity toward death receptor-mediated apoptosis to be eliminated when the Ags have been cleared out (44). Among a multitude of transcription-dependent (45, 46, 47) and -independent (48, 49, 50) measures to control CD95 responses, the amount of c-FLIP is a major determinant (51, 52, 53, 54, 55). The T lymphocytes are resistant to CD95-mediated apoptosis early upon activation, which has been suggested to be mediated by up-regulation of c-FLIPS expression. The sensitivity toward CD95 signaling is regained in the fully activated lymphocytes by a subsequent decrease in the c-FLIPS levels (51, 52, 53, 56). The excess of activated T lymphocytes needs to be efficiently eliminated at the end of an immune response to avoid autoreactivity. Indeed, high quantities of c-FLIP and impaired lymphocyte apoptosis are associated with several cases of autoimmune diseases (57, 58, 59). Because the amount of c-FLIP is regulated both at the level of synthesis and degradation (27, 60, 61), changes in the intracellular c-FLIP expression provide an efficient and well-regulated mechanism for the T lymphocytes to modulate their susceptibility to apoptotic signaling.

Interestingly, several studies on fever patients and individuals exposed to local or whole body hyperthermia treatments have shown a decrease in the T lymphocyte repertoire (5, 6, 7, 8). We have previously shown in Jurkat cells that severe hyperthermia (42°C) induces sensitization to CD95-mediated apoptosis. The sensitization, which could not be counteracted by overexpression of Hsp70, led to decreased c-FLIP levels and elevated activation of caspase-8 (62). To elucidate whether these observations could have relevance at conditions resembling fever, we studied the effect of hyperthermia at lower temperatures on human primary peripheral T lymphocytes. We show here that activated T lymphocytes are significantly sensitized to CD95-mediated apoptosis when they are exposed to fever-like hyperthermia. Moreover, we demonstrate that the sensitization induced by hyperthermia is a consequence of increased proteasomal degradation of c-FLIPS. Our results implicate that fever plays a major role in regulating the termination of T lymphocyte populations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture and treatments

Primary human peripheral T lymphocytes were collected from several healthy volunteers by venipuncture. The blood samples were diluted 1/2 in PBS, overlaid onto Ficoll-Paque PLUS (Amersham Biosciences), and centrifuged at 1500 rpm for 30 min. Mononuclear cells from the Ficoll-Paque separation were depleted by plastic adherence for 2 h. Nonadhering cells were further separated in a nylon wool column to exclude B lymphocytes. Resting T lymphocytes (day 0) were stimulated with 1 µg/ml PHA (Sigma-Aldrich) for 22 h. After 1 day of activation, cells were washed and T lymphocyte proliferation was supported by addition of 20 U/ml IL-2 (Sigma-Aldrich). The same dose of IL-2 was added at day 3 of activation. Human Jurkat T lymphoma cells (American Type Culture Collection) and primary human peripheral T lymphocytes were cultured in a humidified 5% CO2 atmosphere at 37°C in RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin, and 2 mM L-glutamine. For transfections of FLAG-c-FLIPL and FLAG-c-FLIPS (a gift from J. Tschopp, Institute of Biochemistry, University of Lausanne, Lausanne, Switzerland), as well as FLAG-c-FLIPS{Delta}203–221 (described by Poukkula et al. (63)), Jurkat cells were subjected to a single electric pulse (220 V, 975 µF). The stably overexpressing cells were selected by geneticin (G418, 1.5 mg/ml; Calbiochem). The proteasome inhibitor epoxomicin (epoxo; Calbiochem) was used at 200 nM and the protein synthesis inhibitor cycloheximide (CHX; Sigma-Aldrich) was used at 5 µM. Apoptosis was induced with agonistic anti-CD95 Ab CH-11 (Biosite), 200 ng/ml for 2 h in Jurkat cells, if not otherwise indicated, and 1 µg/ml for 2 or 12 h in primary human T lymphocytes. To induce hyperthermia, heat shock treatments (HS) were performed in a water bath at 40 or 42°C for 30 min if not otherwise indicated. After exposure to hyperthermia, cells were either harvested or returned to 37°C for recovery and/or additional treatments. The CD95L-blocking Ab NOK-1 (BD Pharmingen) was used at 1 µg/ml for 15 min before apoptosis induction.

Detection of apoptosis

Apoptosis in Jurkat cells and primary peripheral blood T lymphocytes was detected by annexin V analysis. Cells were incubated for 10 min on ice with 4 µl/ml human recombinant FITC-conjugated annexin V (Sigma-Aldrich) in annexin V binding buffer (2.5 mM HEPES (pH 7.4), 35 mM NaCl, and 0.6 mM CaCl2) and analyzed on a FACScan flow cytometer (BD Biosciences).

Immunoblotting

For Western blot analysis, cells were lysed in Laemmli sample buffer. Western blotting was performed using Abs against c-FLIP (clone NF6; Alexis), CD95L (clone G247-4; BD Pharmingen), caspase-8 (clone C15; Alexis), Hsp70 (SPA-810; StressGen Biotechnologies), Hsc70 (SPA-815; StressGen Biotechnologies), and ubiquitin (clone FK-1; Biomol). HRP-conjugated secondary Abs were from Southern Biotechnology Associates, Promega, and Amersham Biosciences. The results were visualized using the ECL method (Amersham Biosciences) on x-ray film. For densitometric analysis of Western blots, the x-ray films were scanned and the analysis was done with the ScionImage GelPlot2 software. The values were normalized to the untreated control sample, which was given the value 1.

Surface expression analysis of CD95

Cells were washed twice with PBS. After washing, cells were blocked for 30 min with 1% BSA in PBS. Cells were then incubated with CH-11 anti-CD95 Ab (Biosite), 1/500 in 1% BSA in PBS for 30 min followed by washing with PBS. Finally, cells were incubated with Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes) for 30 min. After washes, cells were analyzed by flow cytometry on a FACScan flow cytometer (BD Biosciences). Samples without primary Ab were used as negative controls (second Ab control).

DISC analysis

A total of 5 x 107 Jurkat cells were resuspended in 1 ml of prewarmed RPMI 1640 medium. To ligate CD95 and induce DISC formation, 1 µg of Fc-CD95 ligand fusion protein (CD95L:Fc), a gift from P. Schneider (Institute of Biochemistry, University of Lausanne, Lausanne, Switzerland), was added to the cell suspension. The cells were incubated at 37°C for 12 min, and the reaction was stopped by adding 10 ml of ice-cold PBS. After washing, the cells were lysed in 1 ml of lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 0.2% Nonidet P-40, and complete protease inhibitor mixture (Roche)) for 20 min on ice. The cell debris was removed by centrifugation at 15,000 x g for 12 min at 4°C. One microgram of CD95L:Fc was added to control samples without CD95 ligation. Samples were immunoprecipitated with 15 µl of protein G beads (Amersham Biosciences) for 2.5 h at 4°C. The beads were washed five times with lysis buffer, resuspended in Laemmli sample buffer, and finally boiled for 5 min. The immunoprecipitates and corresponding cell lysates were analyzed by Western blotting. A control sample without CD95L:Fc was used to exclude unspecific binding to the protein G beads (Ab control).

RNase protection assay

Hyperthermia was induced in Jurkat cells at 42°C for indicated times and RNA was prepared with the Qiagen kit for RNA isolation according to the manufacturer’s protocol. The RNase protection assay was done with the RiboQuant MultiProbe RNase Protection Assay System (BD Pharmingen) according to the manufacturer’s standard RPA protocol using the hApo-3b probe (cat. no. 45611P). The results were visualized by autoradiography using BAS Imaging Plates (Fuji Film) and a BAS-1800 Imaging Plate Reader (Fuji Film). The bar graph represents mean values (±SD) from autoradiographs quantified with the AIS-Analytical Imaging Software.

Ubiquitination assay

For immunoprecipitation of c-FLIP, the cell pellet from 3 x 107 c-FLIP stably overexpressing Jurkat cells was resuspended in 75 µl of boiling 1% SDS in PBS, and the resulting lysate was heated at 100°C for 5 min. The lysates were then suspended in 1 ml 0.5% Triton X-100 in PBS. DNA was sheared by sonication and the particulate material was centrifuged at 15,000 x g for 10 min. Samples were taken from the cleared lysates for input control. The lysates were precleared for 30 min without Ab at room temperature and then incubated with anti-FLIP Ab 1/10 (NF6 supernatant, a gift from P. Krammer, Deutsche Krebsforschungszentrum, Heidelberg, Germany) and 20 µl of a 50% slurry of protein A-Sepharose beads under rotation for 2 h at 4°C. After incubation, the Sepharose beads were washed four times with 0.5% Triton X-100 in PBS, and the immunoprecipitated proteins were run on SDS-PAGE, and immunoblotted with anti-ubiquitin Ab (FK-1; Biomol).

Statistical analysis

Comparisons of treatments were made using GraphPad Prism software. Unpaired t tests were performed on data from different individuals, i.e., primary T lymphocytes, whereas paired t tests were performed on data from Jurkat cells and Jurkat-based cell lines. Statistical significance is marked with asterisks (***, p < 0.001; **, p < 0.005; and *, p < 0.05) and ns (p > 0.05) stands for not statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Hyperthermia sensitizes primary human T lymphocytes to apoptosis mediated by CD95

We have previously shown that hyperthermia (42°C) sensitizes Jurkat cells to CD95-mediated apoptosis (62), indicating that hyperthermia has a major impact on death receptor signaling. In this study, we wanted to investigate whether this paradigm could be applicable for regulating the homeostasis of primary T lymphocytes. T lymphocytes were activated by stimulating them with PHA and IL-2, and their sensitivity to CD95-mediated apoptosis was tested by treating them with an agonistic anti-CD95 Ab for up to 12 h. During the first 3 days of activation, CD95 ligation was unable to induce apoptosis, regardless of whether the cells had been subjected to hyperthermia at 42°C or not (Fig. 1A). The resistance of T lymphocytes to CD95-mediated apoptosis during early activation is consistent with earlier reports (52, 64). However, CD95-ligation in cells that were activated for 6 days, induced apoptosis in most of the cells pretreated at 42°C already after 2 h, whereas the cells treated with anti-CD95 Ab alone were barely affected at this time point (Fig. 1A). We also wanted to determine whether the sensitizing effect could be applicable for milder, physiological hyperthermia, resembling fever temperatures. Indeed, when the hyperthermia temperature was lowered to 40°C, a similar sensitization to CD95-mediated apoptosis was observed (Fig. 1B).


Figure 1
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  		FIGURE 1. Primary human T lymphocytes are sensitized to CD95-mediated apoptosis by hyperthermia. A, Apoptosis was quantified by annexin V analysis after primary human T lymphocytes were treated with anti-CD95 Ab, for 12 h (days 0–3) or 2 h (day 6), with or without a hyperthermia induction at 42°C (HS). The bar graphs represent mean values (±SD) of samples from two (days 0–3) or seven (day 6) individuals. Only the results at day 6 were of statistical significance, determined with unpaired t tests. B, Apoptosis in day 6 activated primary T lymphocytes treated as in A, but with a temperature of 40°C, was quantified by annexin V analysis. The bar graphs represent mean values (±SD) of samples from three individuals. Statistical significance was analyzed with an unpaired t test. C, The levels of c-FLIP in control and hyperthermia-exposed (42°C) day 6 activated primary T lymphocytes were examined by Western blotting and anti-Hsc70 Ab was used for loading controls. The Western blot shown is a representative of samples from seven individuals. D, The levels of c-FLIP in control and hyperthermia-exposed (40°C) day 6 activated primary T lymphocytes were examined by Western blotting and anti-Hsc70 Ab was used for loading controls. The Western blot shown is a representative of samples from three individuals. Treatments: HS: 30 min; R: recovery at 37°C for 12 h (days 0–3) or 2 h (day 6); anti-CD95: 12 h (days 0–3) or 2 h (day 6) treatment with 1 µg/ml anti-CD95 Ab.

 
In agreement with our observations in Jurkat cells (62), the levels of both c-FLIPS and c-FLIPL markedly decreased upon hyperthermia in the primary T lymphocytes (Fig. 1C). Down-regulation of c-FLIP was observed also when cells were treated with hyperthermia at 40°C (Fig. 1D). To test for the specificity of the stress, we analyzed whether other stresses, such as oxidative stress, osmotic shock or heavy metals, could mediate down-regulation of c-FLIP and sensitize cells to death receptor-mediated apoptosis. However, no down-regulation of c-FLIP could be detected during these stresses (data not shown). Likewise, the other stresses did not have a sensitizing effect on CD95-mediated apoptosis (data not shown), indicating that the death receptor-sensitizing effect is specific for hyperthermia.

Alterations in CD95 receptor and ligand expression could contribute to the hyperthermia-induced sensitization and it has been reported that long-term hyperthermia increases the expression of CD95L (47). CD95 was similarly expressed on the surface of control and hyperthermia-exposed primary T lymphocytes (Fig. 2A). Furthermore, we could not detect any changes in the levels of CD95L during the time points used in this study (Fig. 2B) and the CD95L blocking Ab (NOK-1) did not affect the hyperthermia-induced sensitization to CD95 triggering with agonistic anti-CD95 Ab (Fig. 2C). Taken together, these results exclude elevated CD95 surface expression and CD95L up-regulation as causes for the observed sensitization.


Figure 2
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  		FIGURE 2. Hyperthermia does not affect CD95 or CD95L expression. A, The surface expression of CD95 in day 6 activated T lymphocytes was determined by immunostaining and flow cytometric analysis. A sample without primary Ab treatment was used as a negative control (second Ab control). The histogram shown is a representative of samples from three individuals. B, Jurkat cells were exposed to either hyperthermia (HS) at 42°C for 30 min or left to recover (HS+R) for 2 h at 37°C after the 30-min hyperthermia treatment. The levels of CD95L were examined by Western blotting and anti-Hsc70 Ab was used for loading controls. The Western blot shown is representative of samples from three independent experiments. C, Apoptosis was quantified by annexin V analysis after treatment of Jurkat cells with anti-CD95 Ab (200 ng/ml) for 2 h, with or without a 30-min induction of hyperthermia at 42°C (HS) and in the presence and absence of 1 µg/ml NOK-1 blocking CD95L Ab added 15 min before the hyperthermia induction. The bar graphs represent mean values (±SD) of samples from three independent experiments.

 
Hyperthermia-induced down-regulation of c-FLIPS is responsible for the sensitization to CD95-mediated apoptosis

Western blotting showed that the hyperthermia-induced decrease in the c-FLIP levels was rapidly restored during the recovery at 37°C (Fig. 3A). Therefore, we wanted to study whether the intracellular levels of c-FLIP correlated with the hyperthermia-induced sensitization to CD95-mediated apoptosis. Cells were either treated with anti-CD95 Ab immediately after exposure to hyperthermia, or were left to recover for up to 6 h before ligation of CD95. The kinetics of c-FLIP depletion and return corresponded perfectly to the kinetics of hyperthermia-mediated sensitization and desensitization to CD95-mediated apoptosis (Fig. 3B). A similar inverse correlation between c-FLIP levels and CD95 sensitivity could be observed in primary human T lymphocytes (Fig. 3, C and D), indicating that down-regulation of c-FLIP at the time of CD95 ligation is the mechanism responsible for the sensitization in both Jurkat cells and primary human T lymphocytes. The hyperthermia-induced elevation in the amount of apoptosis could still be detected after 48 h of CD95 treatment and cells were not sensitized if hyperthermia was initiated after addition of anti-CD95 Ab (data not shown), providing further evidence for the sensitizing effect to be determined by the levels of c-FLIP at the time of CD95 ligation.


Figure 3
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  		FIGURE 3. Hyperthermia-induced down-regulation of c-FLIP is required for the sensitization to CD95-mediated apoptosis. A, Jurkat cells were left to recover for 2, 4, and 6 h after a 30-min hyperthermia induction at 42°C (HS) before CD95 ligation. The c-FLIP levels at the time of CD95 ligation were detected by Western blotting using Abs against c-FLIP. Equal loading was controlled with Abs against Hsc70. The Western blot shown is a representative of five independent experiments. B, Apoptosis induced with anti-CD95 Ab (200 ng/ml) for 2 h in Jurkat cells without, directly after, or after a 2-, 4-, or 6-h recovery from hyperthermia at 42°C (HS) was quantified by annexin V analysis. The bar graph represents mean values (±SD) from three independent experiments. Statistical significance was determined with paired t tests. The dotted line represents the level of apoptosis induced by CD95 ligation alone. C, Day 6 activated primary T lymphocytes were treated with anti-CD95 Ab (1 µg/ml) for 2 h without, directly after or after a 6-h recovery from hyperthermia at 42°C (HS). Apoptosis in primary T lymphocytes was quantified by annexin V analysis. The bar graphs represent mean values (±SD) of samples from four individuals. Statistical significance was determined with unpaired t tests. D, The c-FLIP levels at the time of CD95 ligation in the day 6 activated primary T lymphocytes without, directly after, or after a 6-h recovery from hyperthermia at 42°C (HS) were detected by Western blotting. Equal loading was controlled with Abs against Hsc70. The Western blot shown is a representative of samples from three different individuals.

 
To determine the specific roles of the two c-FLIP isoforms in the observed CD95 sensitization, we used Jurkat cell lines stably overexpressing either c-FLIPS or c-FLIPL (Fig. 4A). The overexpressed c-FLIP is down-regulated upon hyperthermia, but with slower kinetics than the endogenous c-FLIP, resulting in higher overall exogenous c-FLIP levels in cells exposed to hyperthermia (see Fig. 7 and data not shown). Overexpression of c-FLIPS was able to rescue the cells from the hyperthermia-induced sensitization, whereas overexpression of c-FLIPL did not affect the extent of cell death compared with wild-type cells (Fig. 4B). These results indicate that while both isoforms of c-FLIP were down-regulated upon hyperthermia (Figs. 1C and 3A), the sensitization to CD95-mediated apoptosis induced by hyperthermia was primarily mediated by down-regulation of c-FLIPS.


Figure 4
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  		FIGURE 4. The sensitizing effect of hyperthermia is mediated by down-regulation of c-FLIPS rather than c-FLIPL. A, The protein levels of c-FLIPS or c-FLIPL in control (C) and hyperthermia (HS) treated Jurkat parental cells (WT) and Jurkat stable transfectants overexpressing c-FLIPS or c-FLIPL can be seen from the Western blot. The Western blot shown is a representative of six independent experiments. B, The level of apoptosis in Jurkat parental cells and Jurkat stable transfectants overexpressing c-FLIPS or c-FLIPL was quantified by annexin V staining. The bar graph represents mean values (±SD) from three independent experiments. Statistical significance was determined with paired t tests. Treatments: HS: 30 min at 42°C; R: recovery at 37°C for 2h; anti-CD95: 2 h treatment with anti-CD95 Ab (200 ng/ml).

 

Figure 7
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  		FIGURE 7. The ubiquitination and the proteasomal degradation of c-FLIP are increased upon hyperthermia. A, To detect the effect of proteasome inhibition on the down-regulation of c-FLIP, Jurkat cells were exposed to hyperthermia (42°C for 30 min) with or without a 30-min pretreatment with 200 nM epoxomicin (epoxo). The levels of c-FLIP were detected by Western blotting and anti-Hsc70 Ab was used for loading controls. Western blots were quantified by densitometric analysis and the results were counted as relative protein levels compared with the control sample of either c-FLIPL or c-FLIPS. Statistical significance was determined with paired t tests. The bar graph represents mean values (±SD) from three independent experiments. B, To investigate the ubiquitination of c-FLIPS and c-FLIPL, wild-type Jurkat cells and stably c-FLIP overexpressing Jurkat cell lines were treated with hyperthermia at 42°C for 1 h (HS). The cells were lysed with denaturating SDS. The ubiquitinated proteins were immunoprecipitated from the lysate with NF6 anti-FLIP Ab and protein A-Sepharose. Ubiquitinated c-FLIP was detected by Western blotting with anti-ubiquitin Ab. The amount of protein in the cell lysates before immunoprecipitation is shown in the lower panel and anti-Hsc70 Ab was used for loading controls. The Western blots shown are representatives of samples from three independent experiments. C, Hyperthermia was induced in Jurkat cell lines stably overexpressing either wild-type c-FLIPS (Jurkat-c-FLIPS) or mutant c-FLIPS lacking the C-terminal splicing tail (Jurkat-c-FLIPS{Delta}203–221) at 42°C for the indicated times. The levels of the overexpressed c-FLIPS variants were detected by Western blotting and equal loading was controlled with anti-Hsc70 Ab. Western blots from three independent experiments were quantified with densitometric analysis and the samples were normalized to the untreated control samples (value 1). The dotted line indicates down-regulation of c-FLIP to 50%. D, Ubiquitination assays in wild-type Jurkat cells and in Jurkat cell lines expressing either wild-type c-FLIPS (Jurkat-c-FLIPS) or mutant c-FLIPS lacking the C-terminal splicing tail (Jurkat-c-FLIPS{Delta}203–221) were performed similarly as in B. The Western blot shown is a representative of samples from two independent experiments. E, The level of apoptosis in Jurkat parental cells and Jurkat stable transfectants overexpressing c-FLIPS{Delta}203–221 were quantified by annexin V staining. The bar graph represents mean values (±SD) from three independent experiments. Statistical significance was determined with paired t tests. Treatments: HS: 30 min at 42°C; R: recovery at 37°C for 2h; anti-CD95: 2 h treatment with anti-CD95 Ab (200 ng/ml).

 
Upon CD95 ligation c-FLIP is recruited to the DISC, where it inhibits the activation of caspase-8 (25). Immunoprecipitation of the CD95-DISC revealed a significant decrease of both c-FLIPS and c-FLIPL in the activated DISC of cells exposed to hyperthermia before receptor triggering (Fig. 5). However, induction of hyperthermia did not markedly affect the recruitment of caspase-8 to the DISC (Fig. 5). The absence of c-FLIP in the DISC is most likely caused by down-regulation and not by impaired recruitment, as the reduction in the c-FLIP levels was detected both in cell lysates and in the DISC immunoprecipitates.


Figure 5
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  		FIGURE 5. c-FLIP is lost from the CD95-DISC when cells are subjected to hyperthermia before receptor ligation. The CD95-DISC was analyzed in Jurkat cell lysates by immunoprecipitation with CD95L:Fc fusion protein. The levels of c-FLIP and caspase-8 in the DISC immunoprecipitates and cell lysates were detected by Western blotting, using Abs against c-FLIP and caspase-8. The Western blot shown is a representative of three independent experiments. Treatments: Ab control: sample without CD95L:Fc; HS: 30 min at 42°C; R: 12 min recovery at 37°C; CD95L:Fc: 1 µg/ml CD95L:Fc 12 min at 37°C. The band indicated with an asterisk (*), seen in all immunoprecipitation samples is protein G from the beads. In the immunoprecipitation blot, c-FLIPS is the weak band under the protein G band seen only in the CD95L sample. Because the full-length form of c-FLIPL is cleaved very fast in the DISC, only the p43-c-FLIPL cleaved form of c-FLIPL can be detected in the DISC-immunoprecipitation.

 
Hyperthermia does not affect de novo synthesis of c-FLIP

The hyperthermia-induced down-regulation of both forms of c-FLIP occurred very rapidly; c-FLIPS was down-regulated to 50% already after ~15 min and c-FLIPL after 30 min (Fig. 6A). Because hyperthermia is known to affect gene expression patterns (9), we wanted to study whether hyperthermia induced changes in de novo synthesis of c-FLIP mRNA. For this purpose, we performed RNase protection assays. No inhibition of c-FLIP expression at the mRNA level was evident at the early time points (Fig. 6B), when hyperthermia induced down-regulation of the c-FLIP protein (Fig. 6A). CHX chases, performed to determine the turnover of the c-FLIP isoforms, revealed that the half-life of c-FLIPL in Jurkat cells was ~2 h, whereas the half-life of c-FLIPS was between 30 min and 1 h (Fig. 6C). If the down-regulation of c-FLIP would be due to a hyperthermia-induced general shutdown in translation, the kinetics of protein loss would be the same as the half-life of the protein. Because c-FLIP was lost faster than indicated by its half-life, inhibition of protein synthesis as the mechanism c-FLIP elimination can be excluded.


Figure 6
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  		FIGURE 6. Translation and transcription of c-FLIP are not affected by hyperthermia treatment. A, To analyze the rate of the hyperthermia-induced down-regulation of c-FLIP, Jurkat cells were treated with hyperthermia (HS) at 42°C for indicated times. The c-FLIP amounts were detected by Western blotting and anti-Hsc70 Ab was used for loading controls. The graph represents mean values (±SD) from three independent Western blots that were quantified with densitometric analysis and the samples were normalized to the untreated control samples (value 1). The dotted line indicates down-regulation of c-FLIP to 50%. B, RNase protection assays were performed on Jurkat cell samples exposed to hyperthermia (HS) at 42°C and left for recovery (R) at 37°C as indicated. The bar graph represents mean values (±SD) from two independent experiments, counted as fold induction of control samples. C, The half-life of c-FLIPL and c-FLIPS in Jurkat cells was determined by CHX chases. A total of 5 µM CHX was used for indicated times and the decrease in the c-FLIP levels was monitored by Western blotting. Anti-Hsc70 Ab was used for loading controls. The c-FLIP amounts were quantified from Western blots with densitometric analysis. The graph represents mean values (±SD) from three independent experiments. The levels of c-FLIPL and c-FLIPS were normalized to the untreated control samples (value 1). The dotted line indicates down-regulation of c-FLIP to 50%.

 
Hyperthermia enhances ubiquitination and proteasomal degradation of c-FLIP

Several studies showing that c-FLIP is degraded via the ubiquitin-proteasome pathway (63, 65, 66, 67, 68, 69), prompted us to examine whether proteasomal degradation of c-FLIP was enhanced upon hyperthermia. Indeed, the hyperthermia-induced down-regulation of c-FLIP was inhibited in Jurkat cells pretreated with the specific proteasome inhibitor epoxomicin (Fig. 7A). In agreement with our previous study (63), these results demonstrate a rapid proteasome-dependent turnover of c-FLIP. Because the half-life of c-FLIPS is extremely short, the protein was markedly stabilized by epoxomicin treatments (Fig. 7A). The hyperthermia-induced down-regulation of c-FLIPL was also inhibited by epoxomicin, although the effects were less obvious in comparison to c-FLIPS (Fig. 7A), due to the lower turnover c-FLIPL (63). Ubiquitination assays confirmed that the ubiquitination of both c-FLIPL and c-FLIPS were increased upon hyperthermia treatments (Fig. 7B).

We have previously shown that the rapid turnover of c-FLIPS is determined by its C-terminal region, because the mutated c-FLIPS, lacking this part (c-FLIPS{Delta}203–221), was unable to be ubiquitinated and degraded via the proteasome (63). To investigate the mechanism of the hyperthermia-induced ubiquitination of c-FLIPS, we used the same c-FLIPS{Delta}203–221 mutant. Hyperthermia did not affect the levels or the ubiquitination of c-FLIPS lacking the C-terminal splicing tail, whereas it induced both down-regulation and ubiquitination of the exogenously expressed wild-type c-FLIPS (Fig. 7, C and D). The lysines 192 and 195, which are primary targets for c-FLIPS ubiquitination during differentiation of K562 cells (63), were not, however, involved in the hyperthermia-induced ubiquitination of c-FLIPS (data not shown). Increased ubiquitination of endogenous c-FLIP could be seen in all hyperthermia-treated samples, because the NF6 Ab used for immunoprecipitation recognizes also endogenous c-FLIP (Fig. 7, B and D). Taken together, these results indicate that the C-terminal region regulates both the hyperthermia-induced ubiquitination and degradation of c-FLIPS. Similarly to wild-type c-FLIPS overexpression (Fig. 4B), overexpression of the ubiquitination-resistant mutant protected from the hyperthermia-induced sensitization to CD95-mediated apoptosis (Fig. 7E). The induced proteasomal degradation of c-FLIP during hyperthermia implicates the ubiquitin-proteasome pathway as an efficient route to dynamically regulate the c-FLIP levels to sensitize cells to death receptor ligation.

The sensitization mediated by c-FLIP down-regulation is independent of Hsp70

When cells are subjected to stressful conditions, such as elevated temperatures, the heat shock response is activated, leading to enhanced expression of Hsp70 (9, 10). Such cells, expressing high levels of Hsp70, are referred to as thermotolerant, and have been shown to be less sensitive to several stimuli otherwise lethal for the cell (70, 71). We have shown previously that death receptor signaling is capable of bypassing the protective effects of elevated Hsp70 levels (62). As there are a number of studies suggesting that Hsp70 has the capacity to modulate death receptor-mediated apoptosis in thermotolerant cells (71, 72, 73, 74), we wanted to further examine whether Hsp70 plays a role in CD95-induced apoptosis. To acquire thermotolerance, we induced hyperthermia in Jurkat cells and left the cells to recover. After 12 h of recovery, no sensitizing effect of hyperthermia to CD95 ligation was detected (Fig. 8A). At this time point, the c-FLIP expression levels were restored to normal, but the levels of Hsp70 were increased during the recovery from hyperthermia (Fig. 8B). Next, we exposed the thermotolerant cells to a second induction of hyperthermia for 30 min. The second hyperthermia exposure down-regulated the c-FLIP levels that had been restored during the recovery from the first insult (Fig. 8B), and despite the high levels of Hsp70, these cells were again sensitized to CD95 ligation similarly to the cells treated with anti-CD95 Ab immediately after the first hyperthermia induction (Fig. 8A). Based on these results, we conclude that the sensitizing effect of c-FLIP down-regulation overrides the possible protective effect of Hsp70.


Figure 8
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  		FIGURE 8. Down-regulation of c-FLIP sensitizes thermotolerant cells to CD95-mediated apoptosis. A, Hyperthermia was induced in Jurkat cells at 42°C for 30 min (HS) and the cells were left to recover for the indicated time periods to induce thermotolerance. Thereafter, cells were exposed to another hyperthermia and/or treated with anti-CD95 Ab (200 ng/ml) for 2 h. Apoptosis was quantified by annexin V analysis. The bar graph represents mean values (±SD) from three independent experiments. Statistical significance was determined with paired t tests. The dotted line represents the level of apoptosis induced by CD95 ligation alone. B, The c-FLIP and Hsp70 levels in Jurkat cells at the time of CD95 ligation were detected with Western blotting. Hsp70 was used as a marker for activation of the heat shock response and thermotolerance. Equal loading was controlled with Abs against Hsc70. The Western blot shown is a representative of four independent experiments. Treatments: HS: 30 min at 42°C; R: recovery 37°C for 12 h; anti-CD95: 2 h treatment with anti-CD95 Ab (200 ng/ml).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The regulation of lymphocyte survival and persistence is critical for immune responses during infections. The endurance of T lymphocytes is mainly regulated by the balance between death and survival signaling from cytokines and growth factors (15). Fever is a characteristic feature of inflammation and immune responses, and elevated body temperatures have been proposed to affect the balance between cell death and survival (3, 4). Hyperthermia and fever have been used as a clinical approach to enhance the effect of different therapies. Accordingly, there are several reports showing improved disease course in cancer patients with elevated body temperatures due to fever caused by bacterial infections (7, 75, 76, 77). In addition, severe changes in T lymphocyte populations following fever, local or whole body hyperthermia, and heat stroke have been shown (5, 6, 7, 8). We found that hyperthermia had dramatic effects on cell death induced via CD95 in T lymphocytes. Therefore, our interest was to solve the molecular mechanism for the hyperthermia-induced sensitivity. Because the sensitizing effect of hyperthermia on CD95-mediated apoptosis was lost when the levels of c-FLIP were restored during recovery from hyperthermia, we conclude that down-regulation of c-FLIP was responsible for the sensitization. Additional evidence supporting the conclusion that the hyperthermia-mediated sensitizing effect depends on the levels of c-FLIP at the time of CD95 ligation was obtained in experiments showing that the hyperthermia treatment needs to be initiated before the CD95 ligation and that the cells are equally sensitized to long-term CD95 ligation (data not shown). Experiments with cell lines overexpressing different isoforms of c-FLIP showed that only overexpression of c-FLIPS, not of c-FLIPL, was able to rescue cells from hyperthermia-induced sensitization to CD95-mediated apoptosis, indicating that c-FLIPS was the main responsible isoform.

The levels of c-FLIP in the CD95-DISC were dramatically decreased after hyperthermia, whereas the amounts of caspase-8 were not affected. The down-regulation of c-FLIP in the cell lysates was similar to that in the DISC, suggesting that the absence of c-FLIP in the DISC was due to diminished total c-FLIP amounts and therefore, the hyperthermia-induced sensitivity to CD95-ligation was not due to impaired recruitment of c-FLIP to the DISC. The loss of c-FLIP from the activated DISC changed the ratio between caspase-8 and c-FLIP, allowing efficient activation of caspase-8 in the absence of its inhibitor. This is in agreement with our previous results showing increased overall caspase-8 activity in cells exposed to hyperthermia before CD95 ligation (62). Similarly to our study, a change in the ratio between caspase-8 and c-FLIP within the DISC resulting in enhanced caspase activation and more efficient apoptotic signaling has been shown with 5-fluorouracil treatment (78). It has recently been shown that caspase-2 is activated upon hyperthermia (79) and that caspase-2 is able to prime the cleavage of caspase-8, promoting TRAIL-mediated apoptosis (80). Thereby, caspase-2 could possibly be involved also in the hyperthermia-induced increase in caspase-8 activation and sensitization to CD95-mediated apoptosis.

Hyperthermia is known to affect overall gene expression (9). Although c-FLIP, especially c-FLIPS, has a very short half-life of ~30 min, silencing of the c-FLIP gene expression would rapidly down-regulate c-FLIP protein levels. However, we could not detect any reduction in c-FLIP mRNA levels upon hyperthermia, indicating that the transcription of c-FLIP was constant also during hyperthermia. Furthermore, an inhibition of c-FLIP translation was excluded as a determinant of the immediate hyperthermia-induced down-regulation, because the hyperthermia-induced down-regulation of both c-FLIP isoforms is prominently faster than their half-lives. It is, however, plausible that, over a longer time period, hyperthermia-induced shutdown of protein synthesis can contribute to the loss of c-FLIP.

c-FLIP has been shown to be ubiquitinated and degraded via the proteasome (63, 65, 66, 67, 68, 69). In light of our previous study, showing that the ubiquitin-proteasome pathway is a crucial determinant for the c-FLIPS protein levels (63), a likely mechanism for the hyperthermia-induced down-regulation of c-FLIP is increased proteasomal degradation. This hypothesis was supported by experiments showing that the proteasome inhibitor epoxomicin inhibited the hyperthermia-mediated down-regulation of c-FLIP. Ubiquitination assays further proved that the ubiquitination of both c-FLIPL and c-FLIPS is increased upon hyperthermia. The reduction of the hyperthermia-induced ubiquitination, the loss of the hyperthermia-induced down-regulation, as well as the resistance to CD95-mediated apoptosis of the ubiquitination-resistant c-FLIPS mutant verified the involvement of the ubiquitin-proteasome pathway. Our results also demonstrate that the C-terminal splicing tail is required for the hyperthermia-induced ubiquitination of c-FLIPS and, consequently, that the splicing tail is a general determinant of c-FLIPS stability. However, the hyperthermia-induced ubiquitination of c-FLIPS seems to target also lysines other than K192 and K195 and, accordingly, these lysines are not completely indispensable for c-FLIPS ubiquitination. Replacement of a target lysine with an arginine has been shown to transfer ubiquitination to another lysine also in other substrates (81). Thereby, we suggest that the lysine specificity might not be important for the increased ubiquitination of c-FLIPS during hyperthermia. Recently, Chang et al. (69) showed that the ubiquitination of c-FLIPL is regulated by JNK1-mediated activation of the ubiquitin ligase Itch. Hyperthermia is known to trigger several signaling pathways (82), and because the c-FLIP depletion upon hyperthermia treatment is very rapid, it will be important to examine whether c-FLIPS is regulated by similar kinds of signaling mechanisms as the one suggested for c-FLIPL.

The sensitivity toward apoptotic signaling in T lymphocytes has been shown to be modulated by the intracellular c-FLIP levels (51, 52, 53). Although the c-FLIP amounts are decreased upon T lymphocyte activation (day 6), we show that elevated temperatures further deplete c-FLIP, allowing for efficient CD95-dependent elimination of the activated T lymphocytes. The levels of c-FLIP are reduced upon hyperthermia also earlier during T lymphocyte activation (days 1–3), but not to the extent that would allow triggering of the apoptotic signal, because the c-FLIP quantities are much higher at this time point (data not shown). Interestingly, Cippitelli et al. (47) recently showed that long-lasting hyperthermia increases CD95L expression during T cell activation. We were not able to detect such changes in the CD95L levels in Jurkat cells during shorter hyperthermia treatments. Neither did CD95L blocking Ab affect the hyperthermia-induced sensitization to ligation of CD95. The elevated temperatures could on long term, however, both stimulate the external signaling (elevated CD95L expression) and amplify the inside signal from the receptor (elevated caspase-8 activity due to c-FLIPS depletion). The combined signals represent an efficient mechanism to eliminate unspecific cells, but it could also have a stimulatory effect on specific CD95-resistant cells, as caspase-8 has been, apart from its proapoptotic functions, shown to also participate in nonapoptotic NF-{kappa}B activation (83). In fact, it is tempting to speculate that the fever-mediated mechanism described here could provide added specificity and strength to the immune signal.

It is critical that excess activated T lymphocytes are eliminated to avoid formation of unwanted autoreactive or allergy promoting lymphocyte populations (57, 58, 59). Our results indicate that hyperthermia is an important determinant for appropriate apoptotic signaling in activated T lymphocytes. Therefore, fever would constitute a clear beneficial effect, relevant for elimination of unwanted lymphocytes. Related to this beneficial effect, a further subject for speculation is whether the declining incidence of fever among children in industrialized countries due to decreased infection rates (and perhaps, the forceful use of antipyretics) could be linked to the steadily increasing prevalence of allergies and autoimmune disease (84, 85), as a reflection of unspecific lymphocyte activation. Our study explains the molecular mechanism behind previous clinical observations showing a reduced number of T lymphocytes during fever and in hyperthermia-treated patients (5, 6, 7, 8). The link between fever and lymphocyte elimination warrants further studies on the hyperthermia-mediated effects on c-FLIP levels in T lymphocytes during normal and pathological conditions.


   Acknowledgments
 
We thank Peter Krammer for NF6 supernatant, Jürg Tschopp for c-FLIP constructs, and Pascal Schneider for CD95L:Fc fusion protein. We also thank Helena Saarento and Gunilla Henriksson for technical assistance and the members of our laboratories for constructive criticism during the course of this study.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 financed by the Academy of Finland, TEKES, the Finnish Life Insurance Companies, the Sigrid Jusélius Foundation, the Finnish Cancer Organizations, and Åbo Akademi University. A.M., T.S.S., A.K., and M.P. were supported by Turku Graduate School of Biomedical Sciences. Back

2 Current address: European Molecular Biology Laboratory, Developmental Biology Unit, D-69117 Heidelberg, Germany. Back

3 Address correspondence and reprint requests to Prof. John E. Eriksson, Turku Centre for Biotechnology, Åbo Akademi University and University of Turku, FI-20521 Turku, Finland. E-mail address: john.eriksson{at}abo.fi Back

4 Abbreviations used in this paper: Hsp, heat shock protein; c-FLIP, cellular FLICE-inhibitory protein; c-FLIPS, c-FLIPshort; c-FLIPL, c-FLIPlong; DISC, death-inducing signaling complex; CHX, cycloheximide; HS, heat shock treatment. Back

Received for publication April 10, 2006. Accepted for publication December 26, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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