Cholera Toxin B-Subunit Prevents Activation and Proliferation of Human CD4 + T Cells by Activation of a Neutral Sphingomyelinase in Lipid Rafts This information is current as of October 1, 2021. Alexandre K. Rouquette-Jazdanian, Arnaud Foussat, Laurence Lamy, Claudette Pelassy, Patricia Lagadec, Jean-Philippe Breittmayer and Claude Aussel J Immunol 2005; 175:5637-5648; ;

doi: 10.4049/jimmunol.175.9.5637 Downloaded from http://www.jimmunol.org/content/175/9/5637

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cholera Toxin B-Subunit Prevents Activation and Proliferation of Human CD4؉ T Cells by Activation of a Neutral Sphingomyelinase in Lipid Rafts1

Alexandre K. Rouquette-Jazdanian,* Arnaud Foussat,* Laurence Lamy,* Claudette Pelassy,* Patricia Lagadec,† Jean-Philippe Breittmayer,* and Claude Aussel2*

The inhibition of human CD4؉ T lymphocyte activation and proliferation by cholera toxin B-subunit (CTB) is a well-established phenomenon; nevertheless, the exact mechanism remained unclear. In the present study, we propose an explanation for the rCTB-induced inhibition of CD4؉ T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipid composition of rafts. First, rCTB inhibits sphingomyelin synthesis; second, it enhances synthesis; and third, it activates a raft-resident neutral sphingomyelinase resembling to neutral sphingomyelinase type 1, thus generating a transient ceramide production. We demonstrated that these ceramides inhibit protein C␣ phosphorylation and its translocation into Downloaded from the modified lipid rafts. Furthermore, we show that rCTB-induced ceramide production activate NF-␬B. Combined all together: raft modification in terms of lipids, ceramide production, C␣ inhibition, and NF-␬B activation lead to CD4؉ T cell inhibition. The Journal of Immunology, 2005, 175: 5637–5648.

holera toxin B-subunit (CTB)3, produced by the bacte- release of IL-10, IL-6, and TNF-␣ by human monocytes (5). CTB rium Vibrio cholerae, exerts profound immunomodula- modulates Ag processing and presentation by macrophages (6–8). http://www.jimmunol.org/ C tory effects on blood cell populations in vitro. CTB trig- CTB inhibits CD3- and PMA/ionomycin-induced murine T cell gers the polyclonal activation of B cells. This activation occurs in proliferation (9, 10). Such proliferation is inhibited even if CTB is the absence of significant proliferation and involves the up-regu- added hours after the start of culture (10). CTB also inhibits the lation of a number of important molecules, namely MHC class II proliferation of 2.10 cells (a human IL-2-dependent, CD4Ϫ T cell (Ia), B7, CD40, ICAM-1, and IL-2R␣ (CD25) (1, 2). CTB induces clone) in response to IL-2 produced endogenously on stimulation selective apoptosis of T CD8ϩ cells. This apoptosis is preceded by with anti-TCR or provided exogenously (11). ETB induces nuclear enhanced expression of CD25 and is independent of Fas (CD95) or translocation of NF-␬B in Jurkat cells (12). To conclude, CTB TNF-␣. It involves a NF-␬B-dependent and caspase-3-dependent displays pleiotropic effects on human PBMCs (hPBMCs), it inhib- by guest on October 1, 2021 pathway (3). A similar effect is exerted by CTB in vivo where oral its CD4ϩ T cell activation and proliferation, but the exact mech- administration of CTB leads to a demonstrable depletion of CD8ϩ anism remained unclear. T cells from both the Peyer’s patch and intraepithelial lymphocyte In this study, our results explain the inhibitory effect of rCTB compartments (4). Heat-labile enterotoxin B-subunit (ETB) from both on the activation and on the proliferation of human CD4ϩ T Escherichia coli, which is a close homologue of CTB, induces the lymphocytes. rCTB specifically binds to the monosialoganglioside GM1, a raft marker, and strongly alters the lipid composition of rafts of CD4ϩ T lymphocytes. First, rCTB inhibits sphingomyelin *Institut National de la Sante´et de la Recherche Me´dicale (INSERM) Unit 576, IFR (SM) synthesis, secondly it enhances phosphatidylcholine (Ptd- 50, Hoˆpital de l’Archet I, Nice Cedex 3, France; and †INSERM Unit 526, Activation Cho) synthesis, and thirdly it activates a raft-resident neutral sphin- des Cellules He´matopoı¨e´tiques, Physiologie de la Survie et de la Mort Cellulaires et Infections Virales, IFR 50 Ge´ne´tique et Signalisation Mole´culaires, Faculte´deMe´- gomyelinase (SMase) resembling to NSM1, thus generating a tran- decine Pasteur, Nice Cedex 2, France sient ceramide production. We demonstrated that these ceramides Received for publication July 29, 2004. Accepted for publication August 8, 2005. inhibit protein kinase C␣ (PKC␣) phosphorylation and its trans- The costs of publication of this article were defrayed in part by the payment of page location into the modified lipid rafts. We also demonstrated that charges. This article must therefore be hereby marked advertisement in accordance rCTB as ETB activates the NF-␬B transcription factor. Further- with 18 U.S.C. Section 1734 solely to indicate this fact. more, we also show that rCTB induces NF-␬B activation via the 1 This work was supported by Institut National de la Sante´et de la Recherche Me´di- production of ceramides. Combined all together, raft modification cale. A.K.R.-J. is a recipient of a doctoral fellowship from the Ministe`re de l’Enseignement Supe´rieur et de la Recherche and from the Association pour la Re- in terms of lipids, ceramide production, PKC␣ inhibition, and cherche sur le Cancer. NF-␬B activation lead to CD4ϩ T cell inhibition. 2 Address correspondence and reprint requests to Dr. Claude Aussel, Institut National de la Sante´et de la Recherche Me´dicale Unit 576, IFR 50, Hoˆpital de l’Archet I, 151 Materials and Methods Route de Saint Antoine de Ginestie`re, B.P. 79, 06202 Nice Cedex 3, France. E-mail address: [email protected] Cells 3 Abbreviations used in this paper: CTB, cholera toxin B-subunit; ETB, enterotoxin Citrate anticoagulated venous blood samples of healthy adult volunteers B-subunit; hPBMC, human PBMC; SM, sphingomyelin; PtdCho, phosphatidylcho- and buffy coats collected from normal donors by the Etablissement Fran- line; SMase, sphingomyelinase; PKC␣, protein kinase C␣; 7-AAD, 7-aminoactino- ␤ ␤ c¸ais du Sang were obtained according to institutional guidelines. hPBMCs mycin D; m- -CD, methyl- -cyclodextrin; NAC, N-acetyl-L-cysteine; GSH, gluta- were isolated from either blood samples or buffy coats by centrifugation on thione; PDMP, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol; FB1, fumonisin B1; PNS, postnuclear supernatant; PVDF, polyvinylidene difluoride; HSB, a Ficoll-Hypaque density-gradient (1.077 g/ml). Interface PBMCs were HEPES saline buffer; DAG, diacylglycerol; C-1-P, ceramide-1-phosphate; pCTB, pu- pelleted, washed, and cultured in RPMI 1640 supplemented with 10% (v/v) rified CTB; DRM, detergent-resistant membrane; FA, fatty acid; PtdEtn, phosphati- heat-inactivated FCS, 50 U/ml penicillin G sodium, 50 ␮g/ml streptomycin dylethanolamine; ASM, acidic SMase; SMS, SM synthase. sulfate, 2 mM L-glutamine, 1 mM sodium pyruvate, 20 mM HEPES, and

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 5638 rCTB-INDUCED SMase ACTIVATION CAUSES CD4ϩ T CELL INHIBITION

1ϫ minimal essential medium nonessential amino acid solution (Invitrogen side angle light scatter. CD25 and CD69 surface expression on CD4ϩ T Life Technologies). The human leukemic T cell line Jurkat was obtained lymphocytes was determined flow cytometry after gating lymphocytes on from American Type Culture Collection. Cells were cloned by limiting the basis of membrane expression of CD4. CD25 and CD69 up-regulation dilution. Clone D was selected on the basis of its IL-2 production when was also examined on Jurkat cells pretreated or not with the indicated drugs activated with PHA and the phorbolester 12-O-tetradecanoylphorbol-13- (as detailed in the figure legends) then stimulated or not with PMA (10 acetate. Jurkat clone D were grown in the same medium as PBMCs. Cells ng/ml) plus ionomycin (100 nM) for 20 h. The mean fluorescence intensity were maintained at densities between 8 ϫ 105 and 1 ϫ 106/ml in a hu- of 5000 cells was determined by flow cytometry (FACScan; BD midified incubator under 5% CO2 (Heraeus). Biosciences). Abs, reagents, and radioactive products Viability measurement of treated cells rCTB was a kind gift of Dr. F. Anjue`re and Prof. C. Czerkinsky (U721, hPBMCs, treated or not with rCTB (10 ␮g/ml) for 72 h, were stained with Nice, France). rCTB was produced in a mutant strain of Vibrio cholerae 01 a FITC-CD4 mAb. Then cells were incubated with both annexin-PE and deleted of its CT genes and transformed with a multicopy plasmid encod- 7-AAD according to the manufacturer’s specifications. 7-AAD can be ex- ing CTB. The rCTB used was purified from the culture medium by a cited by the 488-nm argon laser line and emits in the far red range of the combination of salt precipitation and chromatographic methods, as previ- spectrum; consequently, its spectral emission can be separated from the ously described (13), and has been already used in several studies (14–16). emissions of FITC and PE. The fluorescence parameters allow character- Rabbit polyclonal Ab anti-GM1, 1-phenyl-2-decanoylamino-3-morpho- ization of necrotic cells (annexin-PEϩ/7-AADϩ), apoptotic cells (annexin- lino-1-propanol, HCl (DL-threo-PDMP, hydrochloride/PDMP), and fumo- PEϩ/7-AADϪ), and viable cells (annexin-PEϪ/7-AADϪ) in the chosen nisin B1 (FB1) from Fusarium moniliforme were purchased from Calbio- subset of FITCϩ cells. Moreover, viability of FB1- and PDMP-treated chem. mAb anti-PKC␣ (clone M4, IgG1), rabbit polyclonal Ab anti- Jurkat cells was also compared with control Jurkat using the same phospho-PKC␣ (Ser657) (IgG), rabbit polyclonal Ab anti-p56Lck (IgG), technique. and rabbit polyclonal Ab anti-LAT (IgG) were obtained from Upstate Bio-

technology. Rabbit polyclonal Ab anti-PLC␥1 (sc-81) was purchased from Lipid rafts isolation Downloaded from Santa Cruz Biotechnology. Anti-CD3 mAb (clone X3, IgG2a) and anti- Raft isolation was accomplished using a combination of published proto- CD28 mAb (clone 28.2, IgG1) were produced in our laboratory. CD4- cols (17, 18). After radioactive labeling and/or pretreatment with rCTB (10 PerCP (clone SK3, IgG1), CD69-FITC (clone FN50, IgG1k), CD25-PE ␮g/ml), Jurkat cells (80–100 ϫ 106) were sonicated gently with a Vibracell (clone M-A251, IgG1k), and apoptosis detection kit (annexin V-PE, 7-ami- sonicator (five bursts of 5 s, 5W; Bioblock Scientific) in 1 ml of ice-cold noactinomycin D (7-AAD)) were purchased from BD Pharmingen. Perox- buffer (25 mM HEPES, 150 mM NaCl, 5 mM EDTA, 10 mM sodium idase-labeled anti-rabbit IgG and rabbit anti-mouse IgG coupled to perox- pyrophosphate, 5 mM Na3VO4, and 10 mM NaF) supplemented with a idase were obtained from Rockland, and R-PE-Cy5-conjugated ␣ mixture of protease inhibitors (1 mM -PMSF, 100 U/ml aprotinin,1 http://www.jimmunol.org/ streptavidin was obtained from DakoCytomation. Methyl-␤-cyclodextrin mg/ml leupeptin, 1 mg/ml pepstatin, 2 mg/ml chymostatin, and 5 mg/ml (m-␤-CD), CTB, biotin-labeled CTB, monosialoganglioside-GM1, ␣ -macroglobulin) and centrifuged at 800 g at 2°C for 10 min to remove N-acetyl-L-cysteine (NAC), glutathione (GSH), cGMP, PMA, ionomycin, 2 av nuclei and large debris. The resulting supernatant called postnuclear su- pepstatin, leupeptin, chymostatin, and anti-PMSF were purchased from pernatant (PNS) was incubated with 0.5% Triton X-100 (PEG(9,10)-oc- Sigma-Aldrich. ␣ -macroglobulin was purchased from Roche. 1␣,2␣(n)- 2 tylphenyl ether) for 30 min at 2°C. The lysate was then adjusted to 1.33 M [3H]cholesterol (1.3–1.85 TBq/mmol), methyl-[3H]choline chloride (2.22– sucrose by the addition of 2 ml of 2 M sucrose and placed at the bottom of 3.14 TBq/mmol), 9,10(n)-[3H]palmitic acid (37 MBq/mmol), [methyl- an ultracentrifuge tube (Ultra-Clear; Beckman Instruments). A step sucrose 3H]thymidine (740 GBq/mmol), [␥-32P]ATP, and [N-methyl-14C]SM (2.04 gradient (0.2–0.9 M with 0.1 M steps, 1 ml each) was placed on top. The Gbq/mmol, 55 mCi/mmol) were purchased from Amersham Biosciences. weight percentage of sucrose was checked at room temperature using an In vitro T lymphocyte proliferative responses Abbe-3L refractometer (Bioblock Scientific). The tubes were centrifuged at ϳ ϫ by guest on October 1, 2021 38 000,rpm ( 250,000 g, using the radial distance maximal (rmax: 158.8 Positive selection of CD4ϩ T lymphocytes from freshly isolated hPBMCs mm) for conversion with normogram) for 16–18 h (L8-70M Ultracentri- was first performed using a fluorescence activated cell sorter (FACStarϩ; fuge; Beckman Instruments) in a SW41Ti rotor (Beckman Instruments) at BD Biosciences). Reanalysis of the sorted population showed a purity 2°C. One-milliliter fractions were harvested from the top. Rafts were re- higher than 98%. Purified CD4ϩ T lymphocytes were extensively washed covered from the low-density fractions 2 and 3 while the heavy/H fractions then resuspended in prewarmed culture medium at a cellular concentration (soluble material) were recovered from the high-density fractions 8 and 9 of 1 ϫ 106/ml. Cell suspension was cultured in triplicate sets in flat-bottom at the bottom of the ultracentrifuge tube. 96-well plates in the volume of 200 ␮l/well. Cells pretreated or not with either rCTB (10 ␮g/ml) and/or others reagents (as detailed in the figure Immunoblot analysis legends) were stimulated or not by either PMA (10 ng/ml) plus ionomycin Aliquots (50 ␮l) of each sucrose density-gradient fraction were solubilized (100 nM) or soluble anti-CD3 mAb (5 ␮g/ml) plus anti-CD28 mAb (5 in 50 ␮lof2ϫ Hoessli buffer (150 mM Tris-HCl (pH 8.5), 20% glycerol, ␮g/ml) for 72 h. The cultures were pulsed with 1 ␮Ci/well [3H]thymidine 5 mM EDTA, 5% SDS, and 10% 2-ME) and then resolved by 10% SDS- during the last 16 h. Cells were harvested with a semiautomatic cell har- PAGE under reducing conditions, and proteins were transferred onto poly- vester (Skatron Instruments) onto glass fiber filter paper. Filters were dried vinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). and counted by liquid scintillation in a Beckman Tricarb scintillation spec- Membranes were blocked for2hatroom temperature in a blocking buffer trometer. Results are expressed as mean cpm Ϯ SEM of triplicate cultures. containing 5% (w/v) nonfat dry milk in TBS (10 mM Tris-HCl and 140 Cell surface receptors staining mM NaCl (pH 7.4)) and then incubated for 1 h with the appropriate Ab diluted 1000-fold in the same buffer. The membranes were washed exten- hPBMCs or Jurkat cells (1 ϫ 106) were washed in cold PBS supplemented sively in TBS containing 0.4% (v/v) Tween 20. Detection was performed with 0.1% BSA (pH 7.4) then incubated for 30 min in the dark at 4°C with with HRP-conjugated anti-rabbit or anti-mouse and ECL reagents (Amer- the appropriate fluorochrome-conjugated mAb according to the manufac- sham Biosciences) according to manufacturer’s instructions. turer’s instructions. For GM1 indirect-staining, Jurkat cells were first in- For phospho-PKC␣ immunoblotting, aliquots of fraction B (fraction cubated with biotin-labeled CTB (10 ␮g/ml). Then cells were washed and 2 ϩ 3) solubilized in the same volume of 2ϫ Hoessli buffer were loaded incubated (30 min at 4°C) in 100 ␮l of 1/25 dilution of RPE-Cy5-conju- on 10% SDS-PAGE and then transferred to PVDF membranes as described gated streptavidin. Cells were washed again and fixed with 0.37% above. Membranes were blocked for2hatroom temperature in a blocking paraformaldehyde. buffer containing 5% (w/v) BSA in TBS and subsequently incubated with rabbit polyclonal Ab anti-phospho-PKC␣ (Ser657)for2hatroom temper- Cytometric analysis of T cell activation markers ature. Phospho-PKC␣ signals were detected with HRP-conjugated goat anti-rabbit, followed by ECL. Membranes were then stripped in a buffer Freshly isolated hPBMCs were washed then resuspended in prewarmed (pH 6.7) containing 62.5 mM Tris-HCl, 2% SDS, and 0.7% 2-ME and culture medium at a cellular concentration of 1 ϫ 106/ml. Cell suspension reblotted with Ab against PKC␣. was dispensed in triplicate sets into flat-bottom 96-well plates in the vol- ume of 200 ␮l/well. hPBMCs pretreated or not with the indicated drugs (as content manipulation detailed in the figure legends) were stimulated or not by soluble anti-CD3 mAb (5 ␮g/ml) plus anti-CD28 mAb (5 ␮g/ml) for 20 h. Then, cells were To reduce cellular sphingolipid content, Jurkat cells were cultured as con- costained with a PE-CD25 mAb, a FITC-CD69 mAb, and a PerCP-CD4 trol cells in a medium supplemented with either FB1 (10 ␮M final con- mAb. hPBMCs were gated on lymphocytes according to their forward and centration) or PDMP (10 ␮M final concentration) for 4 days; FB1 and The Journal of Immunology 5639

PDMP were included in each medium change. Inhibition of sphingolipid Diacylglycerol (DAG) kinase assays synthesis was monitored by analyzing surface expression of GM1 by flow Total cellular ceramide levels were quantified by the DAG kinase assay as cytometry. GM1 replenishment of sphingolipid-depleted Jurkat cells was 32 conducted by incubating cells in serum-free RPMI 1640 containing GM1 P incorporated upon phosphorylation of ceramide to ceramide-1-phos- (0.5 mg/ml for 30 min) at 37°C. phate (C-1-P) by DAG kinase from Escherichia coli (23). After rCTB treatment (10 ␮g/ml) for different period of time, Jurkat cells (5 ϫ 106) were washed twice with ice-cold PBS. After centrifugation (1000 ϫ g,5 Glycerophospholipids and SM content analysis min, 4°C), lipids were extracted with 1 ml of chloroform/methanol/hydro- chloric acid (1 N) (100/100/1, v/v/v), 170 ␮l of buffered saline solution Jurkat cells were washed then incubated for 16–18 h in a HEPES saline (135 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.5 mM buffer (HSB) (pH 7.4), containing 137 mM NaCl, 2.7 mM KCl, 1 mM glucose, and 10 mM HEPES (pH 7.2)), and 30 ␮l of 100 mM EDTA. The Na2HPO4,12H2O, 2.5 mM glucose, 20 mM HEPES, 5 mM MgCl2,1mM ␮ lipids of the organic phase were transferred to a new glass tube and dried CaCl2, and 0.1% BSA at 37°C in the presence of 4 Ci of either 3 3 under a stream of N2. Lipid extracts were then subjected to mild alkaline [ H]palmitic acid or [ H]choline chloride. Lipids were extracted and ana- hydrolysis (0.1 M KOH in methanol for1hat37°C) to remove glycero- lyzed from either whole cells (a) or fractions obtained after phospholipids. Five hundred microliters of chloroform, 270 ␮l of buffered ultracentrifugation (b). saline solution, and 30 ␮l of 100 mM EDTA were added. After drying the (a) rCTB-treated cells or control cells were rapidly sedimented, super- organic phase with N2, in vitro phosphorylation of extracted ceramides was natants were discarded, and cell lipids were extracted with chloroform/ performed as described by the manufacturer (RPN 200 kit; Amersham methanol according to Bligh and Dyer (19) then separated by monodimen- Biosciences). A total of 1 ␮Ci of [␥-32P]ATP (4000 Ci/mmol) was used to sional thin-layer chromatography on plates LK6D Silica Gel 60 A start the reaction. After 30 min at room temperature, the reaction was (Whatman) in a solvent system composed of chloroform/methanol/acetic stopped by extraction of lipids with 1 ml of chloroform/methanol/hydro- acid/water (75/45/12/3). Authentic phospholipid standards (Sigma- chloric acid (1 N) (100/100/1, v/v/v), 170 ␮l of buffered saline solution, Aldrich) were run in parallel and detected with iodide vapors. Radioactivity and 30 ␮l of 100 mM EDTA. The lower organic phase was dried under N . in lipid spots was determined by using an automatic linear radiochromatog- 2 The samples were resuspended in 30 ␮l of chloroform/methanol (95/5, v/v) Downloaded from raphy analyzer, Tracemaster 20 (Berthold), equipped with an 8-mm win- and spotted on plates LK6D Silica Gel 60 A. C-1-P was resolved by TLC dow and the integration software supplied by the manufacturer. ␮ using chloroform/methanol/acetic acid (75/25/5, v/v/v) as solvent and mi- (b) An aliquot (50 l) of each different fraction obtained after ultracen- grated as a single spot at R ϭ 0.25. Linearity of the assay was established trifugation on sucrose density-gradient were extracted and analyzed as de- F using purified C16-ceramide (Sigma-Aldrich). Radioactivity in lipid spots scribed above. was determined by using an automatic linear radiochromatography ana- lyzer, Tracemaster 20 (Berthold), equipped with an 8-mm window and the integration software supplied by the manufacturer.

SM synthesis measurement http://www.jimmunol.org/ Jurkat cells (2 ϫ 106) were maintained in 500 ␮l of HSB. At time 0, 4 ␮Ci of either [3H]palmitic acid or [3H]choline chloride were added, with or Semiquantitative RT-PCR without rCTB, at the end of the treatment, and lipids were extracted and After cell treatment, total RNA was isolated from Jurkat cells using TRIzol analyzed as described above. Reagent (Invitrogen Life Technologies) based on method derived by Chomczynski and Sacchi (24). RNA (150 ng) was then reverse transcribed Ϫ Cholesterol analysis using the SuperScript II RNAase H (Invitrogen Life Technologies) following the manufacturer’s instructions and resuspended 3 ␮ ␮ [ H]Cholesterol in toluene solution was first evaporated under N2 and dis- in 150 l final volume. cDNAs (5 l) or water as control were amplified solved in ethanol just prior its use. Jurkat cells were washed then incubated by PCR in a final volume of 25 ␮l using the Platinum TaqDNA 3 for 16–18 h in HBS containing 4 ␮Ci of [ H]cholesterol. Raft purification (Invitrogen Life Technologies) and 300 nM of forward and reverse prim- by guest on October 1, 2021 was performed as detailed above. To determine the distribution of [3H]cho- ers. RT-PCR was typically performed for 35 cycles (denaturation at 95°C lesterol, an aliquot (50 ␮l) of each different fraction obtained after ultra- for 20 s, annealing at 68°C for 1 min, extension at 72°C for 1 min). Primers centrifugation on sucrose density-gradient was mixed with Picofluor and were designed using the PRIMER Express Software 1.5 (Applied Biosys- counted by liquid scintillation in a Beckman Tricarb scintillation tems). The following 5Ј and 3Ј primers were as follows: human CD69, 5Ј spectrometer. primer (5Ј-CGTAGCAGAGAACAGCTCTTTGC-3Ј) and 3Ј primer (5Ј- ACAGGACAGGAACTTGGAAGGA-3Ј), and human CD25, 5Ј primer (5Ј-GGGACTGCTCACGTTCATCA-3Ј) and 3Ј primer (5Ј-TTCAACAT Assays for neutral- and acidic-SMase GGTTCCTTCCTTGTAG-3Ј). ␤-actin was used as loading control. The activity of neutral- and acidic-SMase was calculated by using a com- bination of published protocols (20–22). To prepare a stock solution of 50 EMSAs ␮M radioactive SM , 55 ␮l (1375 ␮Ci, 25 nmol) of [N-methyl- 14C]SM (55 mCi/mmol, 10 ␮Ci/400 ␮l in toluene/ethanol, 1/1, v/v) were Total cellular extracts were prepared in Totex lysis buffer (20 mM HEPES

placed in a glass tube, and the organic solvent was removed under N2. The (pH 7.9), 350 mM NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM MgCl2, dried [14C]SM was solubilized in 500 ␮l of 1% (w/v) ␤-octylglucoside by 0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 10 ␮g/ml brief sonication with a bath-type sonicator. Fifty-microliter aliquots of se- aprotinin). Supernatants from a 15,000 ϫ g, 10 min centrifugation at 4°C lected fractions (2 ϩ 3 and 8 ϩ 9) were assayed for the presence of dif- were collected. The NF-␬B probe used for mobility shift assay was con- ferent SMase activities. Reactions were started by adding 50 ␮l of substrate stituted of a synthetic double-stranded oligonucleotide containing the solution. For the measurement of the neutral-SMase activity, this solution NF-␬B site of the Ig␬ promoter (5Ј-GATCCAAGGGACTTTCCATG-3Ј). consisted in 10 ␮l of the stock solution of [14C]SM (0.5 nmol), 10 ␮lofa The 32P-end-labeled probe (T4 kinase) was incubated with Totex extract ␮ buffer consisting in 250 mM HEPES (pH 7.5), 50 mM MgCl2, and 0.5% samples (20 g) for 25 min at room temperature. Complexes were then (v/v) Triton X-100 and 30 ␮l of deionized water. After incubation at 37°C separated by electrophoresis on a 5% nondenaturating polyacrylamide gel for 3 h, the reaction was stopped by adding 800 ␮l of chloroform/methanol in 0.5 ϫ Tris-borate EDTA. Dried gels were subjected to autoradiography. (2/1, v/v) and 200 ␮l of deionized water. A 100-␮l aliquot of the aqueous 14 upper phase containing [ C]phosphorylcholine released from [N-methyl- Luciferase assays 14C]SM was collected and counted by liquid scintillation. The reaction was linear within this frame, and the amount of [N-methyl-14C]SM hydrolyzed Jurkat cells were transiently transfected by electroporation (320 V, 960 ␮F) during an assay did not exceed 10% of the total amount of radioactive SM with 10 ␮g of a luciferase reporter gene controlled by a minimal thymidine added. For calculation of the specific activities, values were corrected for kinase promoter and six reiterated ␬B sites (␬Bx6 luc). At volume of the aqueous phase, volume of the sample, protein content, re- 36 h after transfection, cells were stimulated as indicated. Cells were action time, and specific activity of the substrate. For the assay of acidic- washed twice in PBS (pH 7.2) and lysed in 100 ␮l of reporter lysis buffer SMase activity, the substrate solution consisted in 10 ␮l of the stock so- (Promega). Luciferase activity was assayed by luminometry (Lumat; lution of [14C]SM (0.5 nmol) and 10 ␮l of 0.5 M sodium acetate buffer (pH EG&G Berthold) using the Promega luciferase assay system. Normaliza- 4.8), consisting of 10 mM EDTA, 0.5% (v/v) Triton X-100, and 30 ␮lof tion of transfection efficiency was done using a cotransfected ␤-galactosi- deionized water. The initiation and termination of the reaction and the dase expression vector. Luciferase activity was determined in triplicate and determination of the water-soluble radioactivity released from [N-methyl- expressed as fold increase relative to basal activity seen in untreated un- 14C]SM was proceed as described above. stimulated mock-transfected cells. 5640 rCTB-INDUCED SMase ACTIVATION CAUSES CD4ϩ T CELL INHIBITION

Results mycin-induced proliferation. Fig. 1B clearly shows that the GM1-rCTB interaction but not GM1-anti-GM1 Abs interaction epitopes recognized by either rCTB or Ac anti-GM1 are different. inhibits PMA/ionomycin-induced CD4ϩ T proliferation There is no competition between rCTB and Ac anti-GM1 for the binding of GM1. This result may explain why Ac anti-GM1 does Previous works have demonstrated that CTB is able to inhibit T not block the action of rCTB. The epitope recognized by Ac anti- cell activation and proliferation induced by either polyclonal mi- ϩ GM1 may be not involved in the inhibition of CD4 T lympho- togens or by specific Ags (9–11). To see whether GM1 binding cytes because they do not potentiate the effects of rCTB. alone is sufficient or not to inhibit PMA/ionomycin-induced CD4ϩ ϩ T proliferation, CD4 T lymphocytes were pretreated with various The integrity of cholesterol-rich raft is not required for rCTB- concentrations of either purified CTB (pCTB), rCTB, or rabbit induced inhibition of CD4ϩ T lymphocytes polyclonal anti-GM1 Abs, then cells were stimulated with PMA/ ionomycin, and proliferation was measured. Fig. 1A unambigu- The monosialoganglioside GM1 is certainly the most com- ϩ ␤ ously demonstrates that PMA/ionomycin-induced CD4 T cell monly used lipid raft marker. Cholesterol extraction by m- -CD proliferation is inhibited by both pCTB and rCTB in a dose-de- disrupts cholesterol-rich rafts and raft-resident molecules leave pendent manner. Our result unambiguously demonstrates that the rafts. However, a recent study (26) showed that depletion of ϩ ␤ inhibition of the proliferation of CD4 T lymphocytes is not due 73% of cell cholesterol with m- -CD significantly affects the to cAMP produced by contaminant cholera toxin A-subunit be- recovery in detergent-resistant membranes (DRMs) of GM1 cause rCTB gives the same results as pCTB. Furthermore, we in- acetylated or acylated with C8 or unsaturated fatty acids (FAs) vestigated whether cGMP inhibit the effects of pCTB. Indeed, if but not of GM1 acylated with C18,C22,orC24 FAs. To see

whether cholesterol-rich raft integrity is required for rCTB- Downloaded from pCTB is contaminated by cholera toxin A-subunit, it will produce ϩ cAMP, and it is well known that cGMP and cAMP have antago- induced CD4 T cell inhibition, we first treated hPBMCs with ␤ nistic action on proliferation of T lymphocytes (25). As shown in m- -CD to disrupt cholesterol-rich rafts. Then cholesterol-de- Fig. 1A, cGMP does not prevent the pCTB-induced inhibition of pleted hPBMCs were pretreated with rCTB or pCTB and cells the PMA/ionomycin-induced proliferation of CD4ϩ T lympho- were stimulated with PMA/ionomycin. As shown in Fig. 2A, ϩ ␤ cytes, thus indicating that pCTB does not exert its effect via AMPc. cholesterol depletion of CD4 T lymphocytes with m- -CD does not prevent rCTB and pCTB to inhibit PMA/ionomycin- In contrast with rCTB, anti-GM1 Abs have no effects on PMA/ http://www.jimmunol.org/ ionomycin-induced CD4ϩ T cell proliferation. In conclusion, induced CD69 and CD25 up-regulation. The same results were GM1 binding by specific Abs is not able to inhibit CD4ϩ T cells, obtained in Jurkat cells (Fig. 2B). whereas GM1-rCTB interaction is necessary to exert inhibitory ϩ effect. Furthermore, we questioned whether Ac anti-GM1 would SM is necessary for rCTB-induced CD4 T cell inhibition potentiate the effects of rCTB or would block its activity. CD4ϩ T Because cholesterol is not required for rCTB-induced inhibition of lymphocytes incubated with a combination of rCTB plus Ac anti- CD4ϩ T lymphocytes, we investigated a role for SM in rCTB- GM1 were stimulated by PMA/ionomycin and [3H]thymidine in- induced CD4ϩ T inhibition. To this end, we used Jurkat cells in- corporation was measured. As shown in Fig. 1A, Ac anti-GM1 stead of hPBMCs because these cells are more suitable for study- neither potentiates nor blocks the effect of rCTB on PMA/iono- ing lipid metabolism because their global metabolism is greatly by guest on October 1, 2021

FIGURE 1. Effect of rCTB and rabbit polyclonal Abs anti-GM1 on PMA plus ionomycin-induced CD4ϩ T lymphocyte proliferation. A, FACS-sorted CD4ϩ T lymphocytes were pretreated or not in 96- well flat-bottom plates with either pCTB, rCTB, rab- bit polyclonal Abs anti-GM1, pCTB ϩ cGMP, or rCTB ϩ Abs anti-GM1 for 30 min at the indicated concentrations, then CD4ϩ T lymphocytes were stimulated or not with 10 ng/ml PMA plus 100 nM ionomycin. Proliferation was monitored by [3H]thy- midine incorporation during the last 16 h of culture. CD4ϩ T lymphocytes were harvested for beta scin- tillation counting. Data represent one of three simi- lar experiments. The values represent means Ϯ SEM. B, Jurkat cells (1 ϫ 106) were first incubated with Abs anti-GM1 (30 min at 4°C), then GM1 re- ceptors were stained with pCTB-biotin ϩ streptavi- din-RPE-Cy5 (30 min at 4°C) as indicated in Mate- rials and Methods. GM1 staining was measured by cytometric analysis. The Journal of Immunology 5641

PDMP inhibits GM1 synthesis whereas FB1 inhibits both SM and GM1 synthesis. As shown in Fig. 3C, rCTB largely inhibits PMA/ionomycin-induced CD69 and CD25 up-regulation (bar 4 vs bar 2). As expected, rCTB has little effect on CD69 and CD25 expression on PDMP-treated Jurkat cells (bar 7 vs bar 6), but rCTB re-exerts its important inhibitory effect on GM1-restored Jurkat cells (bar 8 vs bars 7 and 4). It indicates that rCTB can inhibit PMA/ionomycin-induced CD69 and CD25 up-regulation either via endogenous or exogenous GM1. It means that exogenous GM1 is as active as endogenous one. Furthermore, as expected, rCTB has little effect on CD69 and CD25 expression on FB1-treated Jurkat cells (bar 11 vs bar 10). In sharp contrast with PDMP treatment, GM1 restoration of FB1-treated Jurkat cells does not allow rCTB to exert its inhibitory effect on PMA/ionomycin-induced CD69 and CD25 up-regulation (bar 12 vs bar 4). This result clearly indicates that SM is necessary to rCTB to exert its inhibitory effect on PMA/ ionomycin-induced CD69 and CD25 up-regulation. To determine whether mRNA correlates with the cell surface expression of CD69 and CD25, RT-PCRs were performed (Fig. 3D). In accordance with the flow cytometry, CTB inhibits mRNA Downloaded from synthesis of CD69 and CD25. FB1 and PDMP treatment prevent the inhibitory effect of CTB on PMA/ionomycin-induced CD69 and CD25 up-regulation. Exogenous GM1-addition in PDMP- treated Jurkat cells allows CTB to inhibit T cell activation. In ␤ contrast, exogenous GM1-addition in FB1-treated Jurkat cells does FIGURE 2. Cholesterol rich-raft disruption by m- -CD does not pre- http://www.jimmunol.org/ not allow CTB to inhibit T cell activation. It unambiguously dem- vent rCTB to inhibit PMA/ionomycin-induced CD69 and CD25 up-regu- lation. A, Freshly isolated hPBMCs were treated or not with m-␤-CD (10 onstrates that if cholesterol is not required, SM is necessary to mM, 10 min at 37°C). Then cells pretreated or not with rCTB (10 ␮g/ml) GM1 signaling via the binding of CTB. or pCTB (10 ␮g/ml) for 30 min were stimulated or not by PMA (10 ng/ml) rCTB inhibits SM synthesis and enhances PtdCho synthesis plus ionomycin (100 nM) for 20 h. Then cells were costained with a PE- CD25 mAb, a FITC-CD69 mAb, and a PerCP-CD4 mAb. hPBMCs were Because we demonstrated that SM is required for GM1 signaling gated on lymphocytes according to their forward and side angle light scat- via the binding of rCTB (Fig. 3), we analyzed the synthesis of SM ϩ ter. CD25 and CD69 surface expression on CD4 T lymphocytes was in control and in rCTB-treated Jurkat cells. We also analyzed the determined flow cytometry after gating lymphocytes on the basis of mem- synthesis of PtdCho and phosphatidylethanolamine (PtdEtn) (Fig. by guest on October 1, 2021 brane expression of CD4. Data represent one of three similar experiments. 4). The uptake of [3H]palmitic acid and [3H]choline chloride is not B, CD25 and CD69 up-regulation was also examined on Jurkat cells pre- ␤ affected by rCTB treatment (data not shown). We show that treated or not with m- -CD, pretreated or not with either rCTB or pCTB, 3 3 and stimulated or not with PMA/ionomycin as in A. Data represent one of [ H]palmitic acid-labeled SM synthesis and [ H]choline-labeled three similar experiments. The values represent means Ϯ SEM. SM synthesis are both inhibited in rCTB-treated cells compared with control cells (Ϫ54 and Ϫ56%, respectively). In contrast, [3H]palmitic acid-labeled PtdCho synthesis and [3H]choline-la- beled Ptcho synthesis are both enhanced in rCTB-treated Jurkat more rapid than those of hPBMCs. Indeed, Jurkat cells do spon- cells (ϩ122 and ϩ 84% respectively). [3H]Palmitic acid-labeled taneously proliferate whereas hPBMCs do not. Incorporation of PtdEtn is not affected by rCTB treatment (data not shown). [3H]palmitic acid or [3H]choline is quite equal in Jurkat cells and in hPBMCs. But the kinetic of SM synthesis is completely differ- rCTB induces SM hydrolysis ent. After a 4 h-incubation with tritiated precursors, it is already Jurkat cells prelabeled with either [3H]palmitic acid (Fig. 5A, up- possible to easily detect SM synthesis in Jurkat cells, whereas it is per graph)or[3H]choline (Fig. 5A, lower graph) were left un- impossible in hPBMCs. We used two drugs to modulate the level treated or incubated with rCTB for different period of time vary- of SM and/or gangliosides: FB1 and PDMP. FB1 prevents the ing from 0 to 30 min. The analysis of lipids extracted from production of both SM and gangliosides (27). PDMP specifically whole cells indicates that rCTB treatment results in a time- diminishes levels of endogenous gangliosides (28, 29). Note that dependent decrease of SM (Fig. 5A). The decrease of SM is PDMP treatment also results in a significant accumulation of maximal at time 30 min and reaches 47% for [3H]palmitic acid- SM (30). labeled SM and 46% for [3H]choline-labeled SM. We studied the surface expression of GM1 both in FB1- and To study SM hydrolysis in lipid rafts, Jurkat cells, prelabeled PDMP-treated Jurkat cells. As shown in Fig. 3B, FB1 and PDMP with either [3H]palmitic acid (Fig. 5B)or[3H]choline (Fig. 5C), considerably diminish the level of the ganglioside GM1 after a were treated or not with rCTB for 30 min and rafts were purified 4-day treatment. Reduction of GM1 is time dependent, but treat- The analysis of the fractions was first performed by liquid scintil- ments cannot be longer extended because beyond a 4-day incuba- lation counting (data not shown). The distribution of the tritiated tion, cell viability starts to decline. After a 4-day treatment, FB1- FA clearly indicates that this saturated FA is preferentially incor- treated cells exhibits only 34% of control GM1 and cell viability is porated into fractions 2 and 3 corresponding to membrane rafts greater than 93%. PDMP-treated Jurkat only possess 29% of GM1, compared with fractions 8 and 9 corresponding to the detergent- and cell viability is not affected at all. Incubation of GM1-depleted soluble material. A further analysis by TLC of the lipid composi- Jurkat with exogenous GM1 (0.5 mg/ml for 30 min) approximately tion of the sucrose density-fractions unambiguously indicates that multiplies by 3.5 the basal content. the raft fraction is highly enriched in [3H]palmitic acid-labeled SM 5642 rCTB-INDUCED SMase ACTIVATION CAUSES CD4ϩ T CELL INHIBITION Downloaded from

FIGURE 3. SM is necessary for rCTB-induced CD4ϩ T inhibition. A, FB1 inhibits both SM and gangliosides. PDMP specifically prevents the formation of gangliosides while enhancing SM production. B, Jurkat cells (1 ϫ 106) were cultured with or without FB1 or PDMP (in both case, 10 ␮M final http://www.jimmunol.org/ concentration) for 24, 48, 72, and 96 h. At 96 h, half the culture was supplemented with GM1 (0.5 mg/ml for 30 min). Surface expression of GM1 was measured by flow cytometry. Data represent one of three similar experiments. The values represent means Ϯ SEM. C, Jurkat cells (1 ϫ 106) pretreated or not with either FB1 (10 ␮M) or PDMP (10 ␮M) for 4 days were incubated or not with exogenous GM1 (0.5 mg/ml) for 30 min. Then cells pretreated or not with rCTB (10 ␮g/ml) for 30 min were stimulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for 20 h. Surface expression of CD69 and CD25 was measured by flow cytometry as in Fig. 2B. Data represent one of three similar experiments. The values represent means Ϯ SEM. D, Jurkat cells were treated as in C, except that they were stimulated by PMA/ionomycin for 6 h. RNA was extracted, and RT-PCR was performed using primers amplifying either CD69, CD25, or ␤-actin. Data represent one of three similar experiments.

(Fig. 5B). Comparison between fractions 2 and 9 shows that by guest on October 1, 2021 [3H]choline-labeled SM is less predominant than [3H]palmitic ac- id-labeled SM in raft fractions (Fig. 5C). rCTB treatment results in an important loss of SM from raft fractions while soluble fractions remain unchanged (Fig. 5, B and C). Fig. 5, B and C, also show that rCTB enhances PtdCho synthesis. When it is labeled with palmitic acid, PtdCho accumulation only occurs in raft fractions; by contrast, when it is labeled with choline, PtdCho is found in rafts as well as in soluble fractions. Because saturated FA carbon chains are known to interact with cholesterol, we investigated the effect of SM degradation on cel- lular [3H]cholesterol content. In control cells, a clear enrichment of [3H]cholesterol is observed in raft fractions compared with deter- gent-soluble fractions (Fig. 6). rCTB treatment results in a de- crease of cholesterol in agreement with previous reports (31, 32), which demonstrated that the hydrolysis of plasma membrane SM alters cellular cholesterol homeostasis. We observed that this cho- lesterol decrease specifically occurs in rafts.

rCTB activates a NSM1-like in lipid rafts that produces ceramides Because we observed an important decrease of raft-SM in rCTB- treated Jurkat, we were interested in characterizing the rCTB-in- duced SMase activity. For that purpose, Jurkat cells were treated or FIGURE 4. rCTB inhibits SM synthesis and enhances PtdCho synthe- not with rCTB and raft isolation was performed. Raft fractions, sis. Jurkat cells (2 ϫ 106) were maintained in 500 ␮l of HSB. At time 0, 4 ␮Ci of either [3H]palmitic acid (A)or[3H]choline chloride (B) were and fractions containing the Triton X-100-soluble material were added, with (10 ␮g/ml, f) or without rCTB (Ⅺ), at the end of the treatment assayed for either neutral or acidic SMase (ASM) activity. As (120 min); lipids were extracted and analyzed as described in Materials shown in Fig. 7A, a neutral pH optimum SMase activity was found and Methods. Data represent one of three similar experiments. The values in raft of rCTB-treated Jurkat cells. This result indicates that a represent means Ϯ SEM. raft-resident neutral SMase is involved in GM1 signaling via the The Journal of Immunology 5643 Downloaded from http://www.jimmunol.org/

FIGURE 5. rCTB induces SM hydrolysis. A, Jurkat cells (2 ϫ 106) were first prelabeled until isotopic equilibrium with either [3H]palmitic acid (upper graph)or[3H]choline chloride (lower graph), then washed and treated (f) or not (Ⅺ) with rCTB (10 ␮g/ml) for different periods of time varying from 0 to 30 min. Lipids were extracted with chloroform/methanol then analyzed by TLC on silicagel plates. These graphs are representative of three different and independent experiments. The values represent means Ϯ SEM. B, A PNS preparation of Jurkat cells (80–100 ϫ 106) metabolically prelabeled for 16 h until isotopic equilibrium with [3H]palmitic acid and stimulated or not by rCTB (10 ␮g/ml) for 30 min was treated with Triton X-100 at 4°C and fractionated on a sucrose density-gradient as described in Materials and Methods. After ultracentrifugation, 1-ml fractions were harvested from the top. Lipids from 3 each fraction were extracted with chloroform/methanol then analyzed by TLC on silicagel plates. The distribution of [ H]palmitic acid-labeled SM, by guest on October 1, 2021 [3H]palmitic acid-labeled PtdCho, and [3H]palmitic acid-labeled PtdEtn is shown for control (Ⅺ) and rCTB-treated cells (f) from the top to the bottom, respectively. The buoyant fractions (rafts) at the top of the gradient correspond to fractions 2 and 3, while the soluble material at the bottom of the centrifuge tube to fractions 8 and 9. These graphs are representative of three different and independent experiments. The values represent means Ϯ SEM. C, Jurkat cells (80–100 ϫ 106) labeled with [3H]choline chloride as in B were treated by rCTB as in B, then raft purification was performed as in B. The distribution of [3H]choline-labeled SM and [3H]choline-labeled PtdCho is shown for control (Ⅺ) and rCTB-treated cells (f) from the top to the bottom, respectively. These graphs are representative of three different and independent experiments. The values represent means Ϯ SEM. rCTB. Furthermore, this neutral SMase activity is almost entirely 1) rCTB inhibits SM synthesis (Fig. 4) and 2) activates a NSM1- inhibited by GSH. It indicates that the involved enzyme is prob- like enzyme in lipid rafts that produces transient ceramides (Fig. ably a NSM1-like one. ASM has not been found implicated in that 7). To demonstrate that rCTB-induced CD4ϩ T lymphocyte inhi- process (data not shown). bition is due in part to SM level modifications, we pretreated Jurkat SM hydrolysis results in the formation of ceramides and phos- cells with either the antioxidant GSH or NAC. NAC acts as a phocholine. The topology of ceramide formation determines its precursor of reduced GSH biosynthesis and consequently it inhib- function (33). When SM from the outer leaflet is hydrolyzed, cer- its neutral SMase. As shown in Fig. 8A, pretreatment with GSH or amides generated in this outer leaflet form ceramides-rich do- NAC inhibits the inhibitory effect of rCTB on Jurkat cells. rCTB- mains, while ceramides generated from the small SM pool in the treated Jurkat do not up-regulate CD69 and CD25 when they are plasma membrane inner leaflet serve as second messengers in sig- stimulated by PMA/ionomycin. By contrast, rCTB-treated cells nal transduction. Using the DAG kinase assay, we dosed the cer- that have been pretreated before with NAC or GSH are able to amide production upon the binding of GM1 by rCTB. As shown in up-regulate the activation markers CD69 and CD25. Fig. 7B, rCTB induces a rapid and transient production of cer- To determine whether mRNA correlates with the cell surface amides. The ceramide production reaches its maximal at 30 min expression of CD69 and CD25, RT-PCRs were performed (Fig. and returns near to the basal level at 45 min. This result suggests 8B). In total accordance with the flow cytometry, the inhibitory that ceramides produced by rCTB are rapidly metabolized and re- effect of CTB on Jurkat cells is abolished when cells are pretreated enter into the SM cycle. with NAC. To link the absence of proliferation of rCTB-treated CD4ϩ T GSH and NAC pretreatment inhibits the effects of rCTB lymphocytes and the neutral SMase activity (i.e., the transient ac- On one hand, we demonstrated that rCTB-GM1 association inhib- cumulation of ceramides), purified CD4ϩ T lymphocytes were pre- its CD4ϩ T lymphocyte activation (Figs. 2, A and B,3,D and E) treated or not with GSH or NAC before being treated or not with and proliferation (Fig. 1). In the other hand, we demonstrated that rCTB (10 ␮g/ml, 30 min). Then cells were stimulated either by 5644 rCTB-INDUCED SMase ACTIVATION CAUSES CD4ϩ T CELL INHIBITION

FIGURE 6. rCTB lowers cholesterol level in rafts. A PNS preparation of Jurkat cells (80–100 ϫ 106) labeled for 16 h until isotopic equilibrium with [3H]cholesterol and stimulated or not by rCTB (10 ␮g/ml) for 30 min was treated with Triton X-100 at 4°C and fractionated on a sucrose density- gradient as described in Materials and Methods. The nine different frac- tions obtained were assayed for radioactivity by liquid scintillation. The distribution of [3H]cholesterol in the gradient is shown for control (Ⅺ) and rCTB-treated cells (f). This graph is representative of three different and independent experiments. The values represent means Ϯ SEM. Downloaded from CD3/CD28 or by PMA/ionomycin. As expected, rCTB inhibits less the proliferation of GSH- or NAC-pretreated CD4ϩ T lym- phocytes than the proliferation of control lymphocytes. Thus, these results demonstrate that the rCTB-induced SMase FIGURE 7. rCTB activates a neutral SMase. A, Jurkat cells (80–100 ϫ activation is responsible for the observed inhibition (activation and 106) were pretreated or not with rCTB (10 ␮g/ml) or pCTB (10 ␮g/ml) for ϩ proliferation) of CD4 T lymphocytes when they are preincubated 30 min. Raft isolation was performed as in Fig. 5B, except that RPMI 1640 http://www.jimmunol.org/ with rCTB. supplemented with a mixture of protease inhibitors was used in place of the initial buffer. Fifty-microliter aliquots of selected fractions (2 ϩ 3 and 8 ϩ SM hydrolysis is required for rCTB-induced NF-␬B activation 9) were assayed with or without GSH (3 mM) for the presence of either Heat-labile ETB from Escherichia coli, a close homologue of neutral SMase activity or ASM activity as described in Materials and ␬ Methods. This graph is representative of three different and independent CTB, is known to activate nuclear translocation of NF- B in Jurkat Ϯ ϫ 6 ␬ experiments. The values represent means SEM. B, Jurkat cells (5 10 ) cells (12). Activation of NF- B by rCTB has never been investi- were treated (f)ornot(Ⅺ) with rCTB (10 ␮g/ml) for the indicated ␬ gated before. Translocation of NF- B was visualized after1hof times. Lipids were extracted and, after mild alkaline hydrolysis, sub- rCTB (10 ␮g/ml) stimulation by its binding to a radioactive probe

jected to phosphorylation by the DAG kinase in the presence of by guest on October 1, 2021 containing ␬B sites from the Ig␬ promoter (Fig. 9A, lane 2 com- [␥-32P]ATP as described in Materials and Methods. The resulting C-1-P pared with unstimulated cells, lane 1). In PDMP-treated Jurkat was separated by TLC. Radioactivity in lipid spots was determined by cells, rCTB fails to translocate NF-␬B. In addition, in FB1-treated using an automatic linear radiochromatography analyzer. This graph is cells, rCTB also fails to translocate NF-␬B (Fig. 9A, lane 11 vs representative of three different and independent experiments. The val- Ϯ lane 2). Furthermore, rCTB is able to translocate NF-␬B in GM1- ues represent means SEM. restored PDMP-treated cells (Fig. 9A, lane 8 vs lane 2); by con- trast, rCTB cannot translocate NF-␬B in GM1-restored, FB1- treated cells (Fig. 9A, lane 12 vs lane 2), indicating that SM is ment modifies neither the distribution of linker for activation of T necessary to NF-␬B translocation by rCTB. Preincubation with cells nor Lck. Then, we studied the effect of rCTB on PMA-in- GSH leads to an inhibition of NF-␬B DNA-binding activity (Fig. duced recruitment of PKC␣ into lipid rafts (Fig. 10B, upper panel). 9A, lane 4 vs lane 2), indicating that SM hydrolysis is required for As shown in Fig. 10B, upper panel, PMA induces a partial trans- rCTB-induced NF-␬B translocation. location of PKC␣ into lipid rafts. rCTB treatment (i.e., raft mod- NF-␬B activation was measured in Jurkat cells transfected with ifications in terms of lipids) importantly prevents PKC␣ translo- a reporter luciferase gene under the control of NF-␬B (Fig. 9B). In cation into lipid rafts. Interestingly, GSH and NAC pretreatment, total accordance with EMSAs, NF-␬B activation was inhibited which prevents rCTB to inhibit Jurkat activation, allows PKC␣ to both in PDMP- and in FB1-treated cells and was restored only in redistribute itself after rCTB pretreatment and PMA stimulation. GM1-recompleted, PDMP-treated cells, indicating that SM is re- Then, we investigated the phosphorylation status of PKC␣ quired for NF-␬B activation by rCTB. rCTB stimulation leads to within rafts (Fig. 10B, lower panel). The same amount of PKC␣ in an 8-fold increase in luciferase activity that is strongly decreased rafts for control and rCTB-treated cells was subjected to SDS- by ϳ50% by GSH-or NAC-pretreatment, indicating that SM hy- PAGE, and the membrane was immunoblotted with anti-phospho- drolysis is involved in rCTB-induced NF-␬B activation. GSH and PKC␣ mAb. Fig. 10B, lower panel, clearly shows that rCTB in- NAC has no effect on baseline luciferase activity. hibits PKC␣ phosphorylation on Ser657.

rCTB treatment prevents PKC␣ phosphorylation and Discussion translocation into modified lipid rafts In the present article, we propose a mechanism for the inhibitory Because rCTB treatment strongly modifies the lipid composition effect of rCTB both on the activation and on the proliferation of of rafts (1) rCTB diminishes SM synthesis (Fig. 4), 2) hydrolyzes human CD4ϩ T lymphocytes. rCTB specifically binds to GM1, a SM, and 3) enhances PtdCho synthesis (Fig. 5)), we were inter- raft marker, and strongly modifies the lipid composition of rafts. ested in studying the distribution of raft-resident proteins highly First, rCTB inhibits SM synthesis; second, it enhances PtdCho involved in T cell activation. As shown by Fig. 10A, rCTB treat- synthesis; and third, it activates a raft-resident neutral SMase, thus The Journal of Immunology 5645 Downloaded from

FIGURE 9. SM hydrolysis is required for rCTB-induced NF-␬B acti-

vation. A, Jurkat cells were pretreated or not with either PDMP (10 ␮M, 4 http://www.jimmunol.org/ days), or FB1 (10 ␮M, 4 days), or GSH (3 mM for 1 h). Sphingolipid- FIGURE 8. NAC and GSH reverse the inhibitory effect of rCTB. A, depleted cells were preincubated or not with GM1 (0.5 mg/ml) for 30 min. 6 Jurkat cells (1 ϫ 10 ) pretreated or not with NAC (10 mM) or GSH (3 mM) Then cells were treated or not with rCTB (10 ␮g/ml) for 1 h. The total cell for 1 h were treated or not with rCTB (10 ␮g/ml) for 30 min. Then cells extracts were then incubated with a radioactive double-stranded oligonu- were stimulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for cleotide encompassing the ␬B site of the Ig␬ promoter. Complexes were 20 h. Surface expression of CD69 and CD25 was measured by flow cy- then separated by nondenaturing electrophoresis followed by autoradiog- tometry as in Fig. 2B. This graph is representative of three different and raphy. Data represent one of three similar experiments. B, For sphingolipid independent experiments. The values represent means Ϯ SEM. B, Jurkat depletion experiments, Jurkat cells were pretreated or not with either cells were treated as in A, except that they were stimulated by PMA/iono- PDMP (10 ␮M, 3 days) or FB1 (10 ␮M, 3 days), then cells were trans- by guest on October 1, 2021 mycin for 6 h. RNA was extracted, and RT-PCR was performed using fected with 10 ␮g of luciferase reporter gene. At 36 h after transfection primers amplifying either CD69, CD25, or ␤-actin. Data represent one of (Jurkat cells were maintained for 36 h in a medium containing either ϩ three similar experiments. C, FACS-sorted CD4 T lymphocytes were PDMP or FB1), cells preincubated or not with GM1 (0.5 mg/ml) for 30 min pretreated or not in 96-well flat-bottom plates with either NAC (10 mM) or were treated or not with rCTB (10 ␮g/ml) for 30 min. For NAC and GSH ϩ GSH (3 mM) for 30 min, then CD4 T lymphocytes treated or not with experiments, 36 h after transfection, Jurkat cells were pretreated or not with rCTB (10 ␮g/ml) for 30 min were stimulated or not with either 10 ng/ml NAC (25 mM, 1 h) or GSH (3 mM, 1 h), then cells were treated or not with PMA plus 100 nM ionomycin or soluble anti-CD3 mAb plus anti-CD28 rCTB (10 ␮g/ml) for 30 min. Luciferase activity was measured as detailed 3 mAb (5 ␮g/ml each). Proliferation was monitored by [ H]thymidine in Materials and Methods. Data represent one of three similar experiments. ϩ incorporation during the last 16 h of culture. CD4 T lymphocytes were The values represent means Ϯ SEM. harvested for beta scintillation counting. This graph is representative of three different and independent experiments. The values represent means Ϯ SEM. CD59 and GM1 cluster in different membrane subdomains of Ju- rkat cells. Furthermore, m-␤-CD extracts cholesterol from Triton generating a transient ceramide production. These ceramides in- X-100-resistant membranes without affecting the buoyant proper- hibit PKC␣ phosphorylation and its translocation into the modified ties of Thy-1 and GM1 (37, 38). The occurrence of GM1 in DRMs lipid rafts. Furthermore, ceramides activate NF-␬B. We hypothe- depends on its ceramide moiety. Depletion of 73% of cellular cho- size that combined all together, raft modification in terms of lipids, lesterol with m-␤-CD does not affect the recovery in DRMs of ceramide production, PKC␣ inhibition, and NF-␬B activation lead GM1 acylated with C18-, C22-, or C24-saturated FAs (26). Com- to T cell inhibition. bined all together, these data suggest that GM1 resides in a subset Gangliosides and glycosylphosphatidylinositol-anchored pro- of lipid raft that is insensitive to cholesterol depletion by m-␤-CD. teins are frequently used as positive controls for raft purification. Our data support and extend this earlier observation because we GM1 is certainly the most commonly used raft marker. It is to note show that m-␤-CD-treatment does not affect the inhibitory effect of that gangliosides and glycosylphosphatidylinositol-anchored pro- rCTB via GM1. teins are almost ever used indiscriminately as if they were equal. CTB and ETB are known to modulate leukocyte function. This Nevertheless, Vyas et al. (34) demonstrated that GM1 does not property is attributed to the ability of the B-subunit to bind GM1. cocluster with GD3 on intact neurons. Gomez-Mouton et al. (35) Nevertheless, it has been demonstrated that GM1 binding alone, show that the acquisition of a motile phenotype in T lymphocytes contrary to expectations, is not sufficient to initiate toxin action. results in the asymmetric redistribution of GM3- and GM1-en- Fraser et al. (12) described the properties of ETB (H57S), a mutant riched raft domains to the leading edge and to the uropod, respec- B-subunit with a His3Ser substitution, at position 57. The mutant tively. Furthermore, Milla´n et al. (36) have demonstrated that still binds to GM1 but is found to be severely defective in inducing 5646 rCTB-INDUCED SMase ACTIVATION CAUSES CD4ϩ T CELL INHIBITION

Aman et al. (39), it is conceivable that rCTB binding to GM1 in lipid rafts would position it to interact with signaling molecules that participate in toxin-mediated immune cell modulation. In re- spect with our experimental data, it is quite conceivable that the intermediary molecule involved in the toxin action would be a raft-resident neutral SMase. The isoform of SMase (acidic or neutral), thus the topology of ceramide formation, dictates its outcome (33). The activation of ASM is believed to theoretically allow the hydrolysis of the main pool of SM from the outer plasma membrane leaflet, thus resulting in the formation of ceramide-rich domains (40), while the activa- tion of neutral SMase would rather allow the hydrolysis of the small pool of SM from the inner leaflet resulting, this time, in the generation of ceramides that will act as second messengers. In this study, we demonstrated that rCTB hydrolyzes SM in a time-de- pendent manner via the activation of a neutral SMase that is in- hibited by GSH. Thus, this enzyme involved in that process re- sembles to NSM1. However, the quantity of hydrolyzed SM by 3

rCTB reaches 47% for [ H]palmitic acid-labeled SM and 46% for Downloaded from [3H]choline-labeled SM. It probably represents more than the mi- nor pool of SM located in the inner leaflet. Thus, what remains unclear is how neutral SMase gains access to the major pool of SM. It has been hypothesized that neutral SMase would hydrolyze SM after its flip-flop from the outer leaflet to the inner leaflet (33),

but we did not observe any flip-flop of phospholipids as studied by http://www.jimmunol.org/ the externalization of phosphatidylserine (data not shown). In this study, we demonstrated that rCTB regulates SM at two levels. First, rCTB inhibits SM synthesis, and second, rCTB acti- vates SM hydrolysis. These two complementary actions both con- tribute to cell cycle arrest of CD4ϩ T lymphocytes. Indeed, the inhibition of SM synthesis 1) causes the inhibition of DAG syn- thesis and 2) increases the level of ceramides; furthermore, SM FIGURE 10. rCTB inhibits the phosphorylation and the translocation of hydrolysis generates ceramides. Consequently, rCTB enhances ␣ ϫ 6

PKC into rafts. A, Jurkat cells (80–100 10 ) were left untreated or by guest on October 1, 2021 ceramides content by two different manners. Ceramides:DAG ratio incubated with CTB (10 ␮g/ml) for 30 min. Raft purification was achieved as in Fig. 5B. The nine fractions, usually harvested from the top of the plays an important role in cell proliferation, and numerous targets ultracentrifuge tube, were pooled as follows: lane E,(9ϩ 8); lane D,(7ϩ of ceramides are known to negatively regulate cell proliferation. 6); lane C,(5ϩ 4); lane B,(3ϩ 2); and lane A, 1. The pooled fractions The biochemical synthesis of SM occurs via the action of a (lanes A–E) were subjected to SDS-PAGE, transferred onto PVDF mem- PtdCho:ceramide choline (SM synthase branes, and immunoblotted with the indicated antibodies. Data represent (SMS)), which transfers the phosphorylcholine (phosphocholine) one of three similar experiments. B, Jurkat cells (80–100 ϫ 106) pretreated moiety from PtdCho onto the primary hydroxyl of ceramide, thus or not with NAC (25 mM) for1horGSH(3mM)for1hwere pretreated producing SM and DAG (41, 42). Thus, the inhibition of SMS ␮ or not with pCTB or rCTB (10 g/ml) for 30 min. Then cells were stim- reduces the intracellular level of DAG. It has been published that ulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for 1 h. Raft isolation was achieved as in Fig. 5B. Upper panel, Fractions B and E were DAG generated during SM synthesis is involved in PKC activation and cell proliferation (43). It constitutes the first way by which subjected to SDS-PAGE, transferred onto PVDF membranes, and immu- ϩ noblotted with anti-PKC␣ mAb. Lower panel, Twenty-five microliters of rCTB inhibits PKC␣ in CD4 T lymphocytes. Furthermore, the fraction B “control” and 65 ␮l of fraction B “rCTB-treated cells,” to have inhibition of SMS also increases the level of ceramides (44, 45). It the same quantity of PKC␣, were subjected to SDS-PAGE, transferred may constitute a supplementary way, in addition to SM hydrolysis, onto PVDF membranes, and immunoblotted with anti-phospho-PKC␣ to elevate ceramide levels. Flores et al. (46) studied the changes in 657 (Ser ) Ab. Then membrane was stripped and reblotted with anti-PKC␣ the balance between DAG and ceramides during cell proliferation, mAb. Data represent one of three similar experiments. cell arrest, and apoptosis in T lymphocytes. Accordingly, augmen- tation of ceramides and diminution of DAG favor cell arrest. leukocyte signaling. For example, it fails to trigger caspase-3-me- Ceramides also target specific proteins inducing cell cycle ar- ϩ diated T CD8 lymphocyte apoptosis. It does not activate NF-␬B rest. Ceramides induce a Go-G1 cell cycle arrest, and this was in Jurkat cells. It fails to induce a potent anti-B-subunit response in mechanistically shown to be due to the induction of dephosphor- mice, and it also fails to serve as mucosal adjuvant. In the same ylation of the retinoblastoma gene (Rb) (47). Furthermore, manner, CTB (H57A) binds GM1 but lacks immunomodulatory or it has been also reported that the treatment of NIH 3T3 cells with toxic activity (39). In the present study, we demonstrate that GM1 a specific inhibitor of glucosylceramide synthase, which induces

ligation alone, via rabbit polyclonal Abs anti-GM1, is not able to ceramide accumulation, causes a G2-M cell cycle arrest, possibly inhibit CD4ϩ T lymphocyte activation and proliferation. We dem- mediated by ceramide-induced inhibition of the cyclin-dependent onstrate that GM1 ligation by Abs does not lead to SMase activa- p34cdc2 and Cdk2 (48). Another study has shown that cer- tion and SM hydrolysis (data not shown). Aman et al. (39) hy- amides specifically inactivates the cyclin-dependent kinase Cdk2 pothesize that CT may require interaction, not only with GM1, but through activation of PP1 and PP2A phosphatases (49). Ceramides also with another molecule to exert its biological activity. For also inactivates protein kinase B/Akt (50). In addition, it has been The Journal of Immunology 5647 demonstrated that endogenous ceramides, produced either by over- 11. Marmor, M. D., and M. Julius. 2001. Role for lipid rafts in regulating interleu- expression of bacterial SMase or by daunorubicin treatment, in- kin-2 receptor signaling. Blood 98: 1489–1497. 12. Fraser, S. A., L. de Haan, A. R. Hearn, H. K. Bone, R. J. Salmond, A. J. Rivett, hibit mRNA synthesis of RT and telomerase activity N. A. Williams, and T. R. Hirst. 2003. Mutant Escherichia coli heat-labile toxin via inactivation of c-Myc transcription factor (51, 52). B subunit that separates toxoid-mediated signaling and immunomodulatory ac- ␣ tion from trafficking and delivery functions. Infect. Immun. 71: 1527–1537. In this study, we observed that rCTB inhibits PKC phosphor- 13. Lebens, M., S. Johansson, J. Osek, M. Lindblad, and J. Holmgren. 1993. Large- ylation and prevents its translocation into rafts. PKC␣ is a pro- scale production of Vibrio cholerae toxin B subunit for use in oral vaccines. growth cellular regulator, and its inactivation can explain cell cycle Biotechnology 11: 1574–1578. ␣ 14. George-Chandy, A., K. Eriksson, M. Lebens, I. Nordstrom, E. Schon, and arrest. PKC inhibition by rCTB can be easily explained by three J. Holmgren. 2001. Cholera toxin B subunit as a carrier molecule promotes an- ways. First, PKC␣ is activated by DAG, and because of the inhi- tigen presentation and increases CD40 and CD86 expression on antigen-present- bition of SM synthesis, DAG level is diminished in rCTB-treated ing cells. Infect. Immun. 69: 5716–5725. ␣ 15. Anjuere, F., A. George-Chandy, F. Audant, D. Rousseau, J. Holmgren, and lymphocytes. Consequently, PKC is less activated in rCTB- C. Czerkinsky. 2003. Transcutaneous immunization with cholera toxin B subunit treated cells. Second, ceramides are known to inactivate PKC␣ via adjuvant suppresses IgE antibody responses via selective induction of Th1 im- a phosphatase (53, 54). Third, rafts are markedly modified in terms mune responses. J. Immunol. 170: 1586–1592. 16. Anjuere, F., C. Luci, M. Lebens, D. Rousseau, C. Hervouet, G. Milon, of lipids. Indeed, rCTB-treated lymphocytes possess rafts with less J. Holmgren, C. Ardavin, and C. Czerkinsky. 2004. In vivo adjuvant-induced SM, less cholesterol, but more PtdCho than control cells. It is quite mobilization and maturation of gut dendritic cells after oral administration of cholera toxin. J. Immunol. 173: 5103–5111. conceivable that these lipid modifications strongly alter the an- 17. Montixi, C., C. Langlet, A. M. Bernard, J. Thimonier, C. Dubois, M. A. Wurbel, choring of PKC␣ into these modified rafts because ceramides do J. P. Chauvin, M. Pierres, and H. T. He. 1998. Engagement of T cell receptor not inhibit PMA-induced translocation of PKC␣ by themselves triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17: 5334–5348. (53). Collectively, all of these evidences can explain the inhibition

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