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Cutting Edge: Modulation of Fas-Mediated Apoptosis by Lipid Rafts in T Lymphocytes¹

Patrick Legembre^2,*, Sophie Daburon^*, Patrick Moreau, Jean-François Moreau^* and Jean-Luc Taupin^*

^* Laboratoire Composantes Innées de la Réponse Immunitaire et Différenciation, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5164 and Laboratoire de Biogenèse Membranaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5200, University of Bordeaux 2, Bordeaux, France

	Abstract

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In type I cells, Fas-mediated cell death requires cytoplasmic membrane subdomains called microdomains or lipid rafts. On the contrary, Fas signaling is independent of these structures in type II cells. We report that in human T cells, CD28, CD59, and CD55 are all localized into lipid rafts and that CD28 is concentrated into microdomains enriched in ganglioside GM1, whereas CD59 and CD55 are not. Moreover, CD28 cross-linking leads to the formation of lipid raft clusters which exclude CD59 and CD55, and reciprocally. Coligation of Fas with CD55 or CD59 inhibits the apoptotic signal, whereas CD28 recruitment amplifies the Fas signaling pathway. Therefore, we conclude that 1) different types of microdomains exist on the cell surface, with distinct functional properties and 2) the recruitment of these distinct structures may differentially modulate the Fas pathway. Moreover, our results demonstrate that Fas-induced apoptosis can be controlled at the level of the cytoplasmic membrane.

	Introduction

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Fas (CD95/APO-1) belongs to the TNF receptor superfamily and mediates programmed cell death upon Fas ligand binding. This receptor plays an important role in the down-regulation of the immune response. In the so-called type I cells, Fas engagement triggers initiator caspases (caspase-8 and -10) which in turn directly cleave and activate effector caspases (caspase-3, -6, -7), leading to the dismantling of the cellular structure and to the cleavage of genomic DNA. In the so-called type II cells, the Fas signal relies on the mitochondria amplification loop to induce an efficient apoptotic signal (1). The plasma membrane contains biochemically and microscopically distinguishable lipid microdomains or lipid rafts that are enriched in cholesterol and sphingolipids. These structures are compact and resistant to lysis by nonionic detergents in cold and for this reason they are also called detergent-resistant membranes. They possess a high degree of lateral mobility in the loosely ordered membrane glycerophospholipids and are enriched in signaling molecules into the intracellular leaflet and in GPI-linked proteins into the extracellular leaflet. The association of Fas to the lipid rafts was highly debated. Recently Muppidi and Siegel (2) showed that Fas is excluded from the lipid rafts in type II cells and is concentrated in them in type I cells (2). They demonstrated that the disruption of the raft structure in type I cells, using cholesterol chelators or agents dispersing the lipid raft content, inhibited the cell death signal. Recently we showed that in type II cells the forced redistribution of Fas into the microdomains was capable of amplifying apoptosis (3).

The APCs stimulate T lymphocytes through cross-linking of their Ag-specific TCR and coreceptor CD28. This triggers the recruitment of the TCR into a large cluster of microdomains, leading to the immunological synapse which is required for complete activation of the T lymphocytes (4, 5). It has been reported that the T cell activation may be partly mimicked by recruitment of other microdomain-concentrated molecules like CD59 or CD55, although it is known that this activation is not complete and that these molecules cannot replace the CD28 costimulatory signal (6).

On the basis of these known properties of CD55 and CD59 on T lymphocyte stimulation, our objective was to analyze the effect of the costimulation of Fas and each of these molecules on Fas-mediated apoptosis.

	Materials and Methods

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Abs and other reagents

The anti-human Fas mAb 5D7 (7) used in fluorescence microscopy and the isotype-matched negative controls 1F10 (IgG) and 10C9 (IgM) mAbs (8) were all generated in the laboratory. For functional studies, the anti-human Fas agonistic mAb 7C11 (IgM) and the anti-CD28 mAb (clone CD28.2) were obtained from Immunotech. For the immunoblots, anti-CD28 and anti-caspase-8 were purchased from R&D Systems, anti-p56Lck mAb was from Transduction Laboratories, and anti-Fas antiserum (C-20) from Santa Cruz Biotechnology. The methyl--cyclodextrin (MCD)³ and the -cyclodextrin were obtained from Sigma-Aldrich. Anti-CD59 was purchased from International Blood Group Reference Laboratory and anti-CD55 was from Cymbus Biotechnology.

Cell line and peripheral blood T lymphocytes from healthy donors

We used T leukemia Jurkat cell line called Jurkat 77 obtained from Dr. P. Anderson (Dana-Farber Cancer Institute, Boston, MA). Activated CD4⁺ T lymphocytes from healthy donors’ blood were obtained as follows. PBMC were isolated by Ficoll centrifugation, washed twice, and mixed at a 1:1 ratio with irradiated stimulatory allogeneic PBMC in the presence of PHA (1 µg/ml; Sigma-Aldrich) and rIL-2 (100 U/ml) in medium supplemented with 10% human serum. After 4 days of culture, cells were washed and resuspended with IL-2 alone. Activated CD4⁺ T lymphocytes were then enriched by depletion of CD8⁺ cells using magnetic beads covered with an anti-CD8 mAb (Immunotech). These cells were cultured in RPMI 1640 supplemented with 8% heat-inactivated FCS and 2 mM L-glutamine in a 5% CO₂ incubator at 37°C.

GM1 incubation and flow cytometry analysis

Cells (1 x 10⁶) cells were washed twice with PBS and resuspended in RPMI 1640. Cells were treated or untreated with 120 µg/ml GM1 for 1 h at 37°C, washed with PBS, and stained. For each staining, 2 x 10⁵ cells were incubated for 30 min at 4°C with 10 µg of the indicated Ab per ml in 0.1 ml of PBS supplemented with 1% BSA (PBS/BSA). Cells were washed with PBS/BSA and incubated for 30 min at 4°C with the FITC-conjugated goat anti-mouse IgG. After washing, cells were resuspended in 0.2 ml of PBS and immediately analyzed by flow cytometry with a FACSCalibur flow cytometer (BD Biosciences).

Cell cytotoxicity assays

Cytotoxicity assays using beads as effectors were performed as follows. Polystyrene beads of 6 µm in diameter (Polybead Polystyrene Microsphere; Polysciences) were washed three times with PBS and incubated overnight at room temperature at a ratio of 6 x 10⁵ beads per 50 µl of Ab solution in PBS. After three washes with PBS, the beads were mixed with the ⁵¹Cr-labeled target cells at the indicated ratios and ⁵¹Cr release was measured exactly as previously described (3).

Detergent lysis experiments

The cells were lysed in lysis buffer (25 mM HEPES, 1% Triton X-100, 150 mM NaCl, pH 7.4) containing protease inhibitors (1 mM PMSF, 5 µg/ml aprotinin, 10 µM leupeptin) for 30 min on ice. The lysate was centrifuged at 15,000 rpm for 10 min and the supernatant was conserved for immunoblot analysis. In experiments with MCD, Jurkat cells were incubated for 20 min at 37°C in serum-free medium with methyl--cyclodextrin (2 mM) or with -cyclodextrin (control reagent) at the same concentration, then washed before the addition of the beads in medium containing no FCS. The isolation of microdomains by ultracentrifugation on a sucrose gradient was performed exactly as previously described (7).

Immunoprecipitation

Jurkat cells (2 x 10⁷) were lysed for 30 min at 4°C in lysis buffer supplemented with a mix of protease inhibitors (Sigma-Aldrich). Lysate was centrifuged at 15,000 rpm for 10 min and the supernatant was used to immunoprecipitate proteins. Protein concentration in cellular extracts was determined using the bicinchoninic acid method (Sigma-Aldrich) according to the manufacturer’s protocol. Aliquots of lysate containing 500 µg of proteins were incubated for 4 h at 4°C with 10 µg/ml of the indicated Ab bound to protein A-Sepharose beads (Sigma-Aldrich). Immune complexes were washed extensively in lysis buffer and separated by SDS-PAGE.

Western blot analysis

Proteins were separated by SDS-PAGE on 12% gels in reducing conditions and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences). The membrane was blocked for 1 h with TBST (50 mM Tris, 160 mM NaCl, 0.1% Tween 20, pH 8) containing 5% dried skimmed milk, and all subsequent steps were performed in this buffer. The indicated specific Ab was then incubated overnight at 4°C. After washes, the peroxidase-labeled anti-mouse (Amersham Biosciences), anti-goat (Vector Laboratories), or anti-rabbit (Zymed Laboratories) secondary Ab was added for 1 h. Then the proteins were visualized with the ECL substrate kit (Amersham Biosciences).

Immunofluorescence and imaging

Jurkat cells were incubated with beads for 15 min at 37°C, washed once in PBS, and adhered 5 min at room temperature to poly-L-lysine-coated slides (ESCO; VWR). Cells were then fixed in PBS containing 2% formaldehyde for 15 min, washed twice in PBS, and treated for 10 min with 50 mM glycine in PBS to quench the aldehyde groups. Cells were washed in PBS/1% BSA, treated with FITC-labeled cholera toxin B (CTB) and anti-CD28, anti-CD59, or anti-CD55 mAbs in PBS/1% BSA for 30 min at 4°C, rinsed five times, and incubated for 1 h with the secondary Ab Alexa-594-conjugated goat anti-mouse Ab (Molecular Probes) in PBS/1% BSA. Slides were washed with PBS, dried, and mounted with Fluoroprep (Biomerieux). Images were acquired and processed on a confocal microscope (LSM 510; Carl Zeiss) with x63 objective. Image analysis and merging of images was done with Adobe PhotoShop 7.0 software (Adobe Systems).

	Results and Discussion

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CD28, CD55, and CD59 are distributed into the lipid rafts

We previously showed that two microdomain-localized molecules were able to amplify the Fas-mediated apoptosis pathway in activated T lymphocytes (3, 7). To generalize our findings, we decided to study the effect of the recruitment of the two GPI-anchored proteins CD55/DAF and CD59/protectin/MIRL on Fas engagement.

Microdomains migrate in the low-density fractions compared with the nonraft membranes at the equilibrium in a sucrose gradient fractionation, after cell lysis. Fractions 2–4 contained the microdomains and were enriched in p56^Lck, a well-known microdomain-concentrated kinase (Fig. 1). As we previously reported, CD28 was also concentrated into the microdomains (3). CD55 and CD59 were also localized into these fractions, whereas the transmembrane receptor Fas was in the nonraft membranes (fractions 5–7). Then at the resting state, the Jurkat cell line is a type II cell with Fas excluded from the lipid rafts, and CD28, CD55, and CD59 are all distributed into the detergent-insoluble membranes.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. CD28, CD55, and CD59 are concentrated into microdomains. Jurkat cells (1 x 108) were lysed and subjected to density sucrose gradient fractionation. For anti-Lck, anti-CD59, and anti-Fas immunoblots, 10 µg of protein was loaded per lane and for anti-CD28 and anti-CD55 immunoblots, 30 µg of protein was loaded per lane.

The mobilization of Fas into the microdomains has been associated with an increased efficiency of the apoptotic pathway (2, 9). Recently, we confirmed this by demonstrating that recruitment of Fas into the CD28-containing lipid rafts amplified the cell death signal of the Jurkat cell line and of activated CD4⁺ T lymphocytes, both type II cells (3). To determine whether CD59 or CD55 behaved like CD28, we used Ab-coated beads to cross-link Fas with these molecules. As expected, CD28 recruitment increased the Fas-mediated cell death of the T lymphoma cell line Jurkat as well as of activated CD4⁺ T lymphocytes from healthy blood donors (Fig. 2A, upper and lower panels, respectively). In contrast, the recruitment of CD59 or CD55 with Fas did not trigger any amplification of the signal and, on the contrary, inhibited the suboptimal cell death signal triggered by low concentration of 7C11 in Jurkat cells (Fig. 2B). We next analyzed in Jurkat cells the cleavage and activation of the initiator caspase-8 and of the proapoptotic mitochondria activator BH3 interacting domain death domain (BID). Amplification of caspase-8 cleavage was observed in the presence of the CD28 cosignal (Fig. 2C), whereas the corecruitment of Fas and CD59 completely inhibited the basal caspase-8 activation triggered by beads coated with a low concentration of anti-Fas (Fig. 2C). The triggering of BID leads to its cleavage, and can be followed by the disappearance of the resting form in immunoblot (3). Consistently with what was observed with caspase-8, BID cleavage was significantly increased or inhibited upon CD28 or CD59 cotriggering with Fas, respectively (Fig. 2C). We analyzed in CD4⁺ T lymphocytes from healthy donors the activation of the effector caspase-3 and of BID and observed that it was enhanced by Fas/CD28 cotriggering, as shown by the increased cleavage of both proteins (Fig. 2E).

View larger version (61K): [in this window] [in a new window]   FIGURE 2. Modulation of Fas apoptosis by CD28 and CD59 recruitment. A, B, and D, Polystyrene beads were coated with the indicated mix of Abs, incubated for 4 h with cells, and a 51Cr release assay was performed. A, Top panel, Jurkat cells were incubated with beads coated with a low concentration of anti-Fas 7C11 mAb (0.08 µg/ml) and the indicated Ab. Bottom panel, Activated CD4+ T lymphocytes were incubated with beads coated with 2 µg/ml anti-Fas 7C11 mAb and the indicated Ab. B, Jurkat cells were mixed with beads coated with 0.08 µg/ml 7C11 and the indicated Abs. C, Jurkat cells were incubated for 120 min with beads coated with 7C11 mAb at a low concentration (0.08 µg/ml) and the indicated Abs, then lysed. "C" means isotype control Ab. For each condition, 10 µg of protein per lane was subjected to an immunoblot. D, Top panel, Jurkat cells were mixed with beads coated with a high concentration of 7C11 (1 µg/ml) and the indicated Ab. Bottom panel, Activated CD4+ T lymphocytes were incubated with beads coated with 5 µg/ml 7C11 and the indicated Ab. E, Activated CD4+ T lymphocytes were incubated for 120 min with beads coated with the indicated concentration of 7C11 mAb. Ten micrograms of protein was loaded per lane and an anti-caspase-3 immunoblot was performed on the top panel. On the bottom panel, the disappearance by cleavage of BID was analyzed by immunoblot. The blots and histograms are representative of three independent experiments.

Recruitment of Fas into lipid rafts leads to the modulation of the apoptotic signal

We then quantified the activity of caspase-3 by densitometric analysis of the amount of its cleavage products, which confirmed that CD28 and CD59, respectively, amplified and inhibited Fas-mediated apoptosis via a modulation of caspase activity (Fig. 3, A and B).

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Modulation of Fas-mediated apoptosis requires the structural integrity of lipid rafts. A and B, Polystyrene beads were coated with the indicated mix of Abs and incubated with cells for 45 min. Cells were lysed and 10 µg of proteins was separated in a SDS-PAGE and an anti-caspase-3 immunoblot was performed. A, Top panel, Jurkat cells were incubated with 7C11 (0.08 µg/ml) and isotype control mAb or anti-CD28-coated beads. B, Jurkat was incubated for 45 min with 7C11 at a high concentration (1 µg/ml) and isotype control mAb or anti-CD59-coated beads. A and B, Bottom panels, for each condition a densitometric analysis of caspase-3 activity was performed. p17- and p20-specific bands were quantified by ImageJ software (W. S. Rasband, ImageJ, U. S. National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/). Bars correspond to the ratio of the intensity of cleaved caspase-3 bands of (cells treated with indicated stimulation/cells treated with control mAb in the absence of MCD) x 100.

It is noteworthy that CD59 and CD55 are known to inhibit cell death induced by the complement system. In this study, we show that they may also protect from death receptor-mediated apoptosis. Then these molecules could be involved in the protection of the cell toward various types of apoptosis inducers.

CD59/CD55 vs CD28 segregate into different microdomains with distinct biochemical properties

It has been reported that activation of T lymphocytes via the TCR is complete with CD28 corecruitment and only partial with CD59 aggregation (6). Moreover the same authors reported that GM1-containing lipid rafts were clustered upon CD28 but not upon CD59 cross-linking (6). Therefore, to explain how microdomains were capable of either amplifying or inhibiting the Fas-mediated apoptosis pathway, we asked whether CD59 and CD28 were concentrated into different microdomains.

Treatment of T cells with the ganglioside GM1 has been reported to decrease the cell surface expression of CD4 (10, 11). It has been reported that exogenously added GM1 to Jurkat cells may cause the internalization via an endocytosis process of microdomain-concentrated proteins (5), and that CD59 was significantly less decreased on the cell surface than another functionally unrelated GPI-linked protein (5). We thus compared the membrane expression of CD28, CD59, and CD55 by flow cytometry on Jurkat cells incubated with GM1 in serum-free medium (Fig. 4A). The expression of the nonraft protein Fas (see Fig. 1), was unaffected by GM1 treatment. Similarly, CD55 and CD59 were not or only slightly (<10% for CD59) altered. In contrast, in the same conditions, 85% of plasma membrane CD28 was lost. This result suggested that the CD28-rich microdomains differed from those containing CD59 or CD55 in their lipid composition.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Microdomain-associated CD55 and CD59 possess similar biochemical properties which differ for CD28. A, Jurkat cells were preincubated with control or GM1-containing medium and washed, and the expression of the indicated proteins was analyzed by flow cytometry. B, Five hundred micrograms of protein from Jurkat cell lysates were immunoprecipitated with the indicated Abs, then immunoblots were performed.

The CTB subunit displays a high affinity for GM1 (12). We used a FITC-labeled CTB to analyze the microdomain distribution of CD28, CD55, and CD59 by fluorescence microscopy. We first individually cross-linked each molecule with mAb-coated beads and stained the cells with the indicated mAbs. When the Ab-coated beads were mixed with Jurkat cells at 4°C, we observed that the CTB-tagged lipid rafts were disseminated homogeneously on the cell surface, as were CD28, CD55, and CD59 (data not shown). In contrast, a 15-min incubation at 37°C induced the formation of one huge cluster of microdomains at the contact area with the beads (Fig. 5B for CD59, C for CD28, and D for CD55), as expected for microdomain-associated proteins. We next analyzed the membrane distribution of each of the three molecules following cross-linking of any of them. CD59 aggregation induced a patch of microdomains that contained most of surface CD55, but did not concentrate CD28 (Fig. 5B). Similarly, CD55 aggregation triggered the formation of a cluster of microdomains that colocalized with CD59 (Fig. 5D). Therefore, CD59-containing rafts concentrate CD55 and reciprocally, confirming the findings described above. In contrast, CD28 aggregation did not recruit CD55 or CD59 in the huge patch formed (Fig. 5C). Therefore, CD28 is localized into microdomains that are distinct from those containing CD55 and CD59. To confirm that the costimulation of Fas with CD28 or CD59 relocates the apoptotic receptor into the lipid raft, beads were coated with 7C11 and CD28 or CD59. The 7C11-coated beads induced the formation of a Fas cluster, but did not alter the distribution of the lipid rafts (Fig. 5D). On the contrary, cross-linking Fas with CD28 or CD59 efficiently led to the redistribution of Fas into the microdomains.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. CD55 and CD59 are colocalized but are not associated to CD28. A–C, Beads were coated with the indicated Abs (10 µg/ml). D, Beads were co-coated with anti-Fas mAb (7C11) and the indicated Ab. The beads were incubated for 15 min at 37°C with the Jurkat cell line. Then cells were stained with FITC-labeled cholera toxin (green) and with specific anti-CD55, anti-CD59, anti-CD28, or anti-Fas mAbs detected with Alexa594-conjugated goat anti-mouse Ab (red).

It would be interesting to measure the ratio of CD28 vs CD55/CD59-enriched microdomains ratio in various tumor cell lines. Indeed, we hypothesize that the tumor formation process may also select for cells with the higher amount of apoptosis-blocking microdomains (e.g., CD59/CD55-enriched lipid rafts). Therefore, drugs acting on this ratio could represent new therapeutic tools to modulate apoptosis sensitivity of lymphoid or more generally or any kind of tumor cells.

	Disclosures

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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 supported by grants from the Association pour la Recherche sur le Cancer, and from the Ligue Contre le Cancer des Landes, de la Charente, de la Dordogne, de la Gironde, des Pyrénées-Atlantiques. P.L. was supported by a grant from the Association pour la Recherche sur le Cancer and by the French Institute of Health and Medical Research (Institut National de la Santé et de la Recherche Médicale).

² Address correspondence and reprint requests to Dr. Patrick Legembre, Laboratoire Composantes Innées de la Réponse Immunitaire et Différenciation, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5164, University of Bordeaux 2, 146 rue Léo Saignat, Bordeaux 33076, France. E-mail address: patrick.legembre{at}u-bordeaux2.fr

³ Abbreviations used in this paper: MCD, methyl--cyclodextrin; BID, BH3 interacting domain death domain; CTB, cholera toxin B.

Received for publication September 14, 2005. Accepted for publication November 8, 2005.

	References

Top Abstract Introduction Materials and Methods Results and Discussion Disclosures References

 

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