of June 25, 2018.This information is current as

Lipid RaftsActivation of a Neutral Sphingomyelinase in

T Cells by+and Proliferation of Human CD4Cholera Toxin B-Subunit Prevents Activation

Jean-Philippe Breittmayer and Claude AusselLaurence Lamy, Claudette Pelassy, Patricia Lagadec, Alexandre K. Rouquette-Jazdanian, Arnaud Foussat,

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

2005; 175:5637-5648; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2005 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Cholera Toxin B-Subunit Prevents Activation and Proliferationof Human CD4� T Cells by Activation of a NeutralSphingomyelinase 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-establishedphenomenon; nevertheless, the exact mechanism remained unclear. In the present study, we propose an explanation for therCTB-induced inhibition of CD4� T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipidcomposition of rafts. First, rCTB inhibits sphingomyelin synthesis; second, it enhances phosphatidylcholine synthesis; and third,it activates a raft-resident neutral sphingomyelinase resembling to neutral sphingomyelinase type 1, thus generating a transientceramide production. We demonstrated that these ceramides inhibit protein kinase C� phosphorylation and its translocation intothe 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, protein kinase C� inhibition, and NF-�B activation lead to CD4� T cellinhibition. The Journal of Immunology, 2005, 175: 5637–5648.

C holera toxin B-subunit (CTB)3, produced by the bacte-rium Vibrio cholerae, exerts profound immunomodula-tory effects on blood cell populations in vitro. CTB trig-

gers the polyclonal activation of B cells. This activation occurs inthe absence of significant proliferation and involves the up-regu-lation of a number of important molecules, namely MHC class II(Ia), B7, CD40, ICAM-1, and IL-2R� (CD25) (1, 2). CTB inducesselective apoptosis of T CD8� cells. This apoptosis is preceded byenhanced expression of CD25 and is independent of Fas (CD95) orTNF-�. It involves a NF-�B-dependent and caspase-3-dependentpathway (3). A similar effect is exerted by CTB in vivo where oraladministration of CTB leads to a demonstrable depletion of CD8�

T cells from both the Peyer’s patch and intraepithelial lymphocytecompartments (4). Heat-labile enterotoxin B-subunit (ETB) fromEscherichia coli, which is a close homologue of CTB, induces the

release of IL-10, IL-6, and TNF-� by human monocytes (5). CTBmodulates Ag processing and presentation by macrophages (6–8).CTB inhibits CD3- and PMA/ionomycin-induced murine T cellproliferation (9, 10). Such proliferation is inhibited even if CTB isadded hours after the start of culture (10). CTB also inhibits theproliferation of 2.10 cells (a human IL-2-dependent, CD4� T cellclone) in response to IL-2 produced endogenously on stimulationwith anti-TCR or provided exogenously (11). ETB induces nucleartranslocation of NF-�B in Jurkat cells (12). To conclude, CTBdisplays pleiotropic effects on human PBMCs (hPBMCs), it inhib-its CD4� T cell activation and proliferation, but the exact mech-anism remained unclear.

In this study, our results explain the inhibitory effect of rCTBboth on the activation and on the proliferation of human CD4� Tlymphocytes. rCTB specifically binds to the monosialogangliosideGM1, a raft marker, and strongly alters the lipid composition ofrafts of CD4� T lymphocytes. First, rCTB inhibits sphingomyelin(SM) synthesis, secondly it enhances phosphatidylcholine (Ptd-Cho) synthesis, and thirdly it activates a raft-resident neutral sphin-gomyelinase (SMase) resembling to NSM1, thus generating a tran-sient ceramide production. We demonstrated that these ceramidesinhibit protein kinase C� (PKC�) phosphorylation and its trans-location into the modified lipid rafts. We also demonstrated thatrCTB as ETB activates the NF-�B transcription factor. Further-more, we also show that rCTB induces NF-�B activation via theproduction of ceramides. Combined all together, raft modificationin terms of lipids, ceramide production, PKC� inhibition, andNF-�B activation lead to CD4� T cell inhibition.

Materials and MethodsCells

Citrate anticoagulated venous blood samples of healthy adult volunteersand buffy coats collected from normal donors by the Etablissement Fran-cais du Sang were obtained according to institutional guidelines. hPBMCswere isolated from either blood samples or buffy coats by centrifugation ona Ficoll-Hypaque density-gradient (1.077 g/ml). Interface PBMCs werepelleted, washed, and cultured in RPMI 1640 supplemented with 10% (v/v)heat-inactivated FCS, 50 U/ml penicillin G sodium, 50 �g/ml streptomycinsulfate, 2 mM L-glutamine, 1 mM sodium pyruvate, 20 mM HEPES, and

*Institut National de la Sante et de la Recherche Medicale (INSERM) Unit 576, IFR50, Hopital de l’Archet I, Nice Cedex 3, France; and †INSERM Unit 526, Activationdes Cellules Hematopoıetiques, Physiologie de la Survie et de la Mort Cellulaires etInfections Virales, IFR 50 Genetique et Signalisation Moleculaires, Faculte de Me-decine Pasteur, Nice Cedex 2, France

Received for publication July 29, 2004. Accepted for publication August 8, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Institut National de la Sante et de la Recherche Medi-cale. A.K.R.-J. is a recipient of a doctoral fellowship from the Ministere del’Enseignement Superieur et de la Recherche and from the Association pour la Re-cherche sur le Cancer.2 Address correspondence and reprint requests to Dr. Claude Aussel, Institut Nationalde la Sante et de la Recherche Medicale Unit 576, IFR 50, Hopital de l’Archet I, 151Route de Saint Antoine de Ginestiere, B.P. 79, 06202 Nice Cedex 3, France. E-mailaddress: aussel@unice.fr3 Abbreviations used in this paper: CTB, cholera toxin B-subunit; ETB, enterotoxinB-subunit; hPBMC, human PBMC; SM, sphingomyelin; PtdCho, phosphatidylcho-line; SMase, sphingomyelinase; PKC�, protein kinase C�; 7-AAD, 7-aminoactino-mycin D; m-�-CD, methyl-�-cyclodextrin; NAC, N-acetyl-L-cysteine; GSH, gluta-thione; PDMP, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol; FB1,fumonisin B1; PNS, postnuclear supernatant; PVDF, polyvinylidene difluoride; HSB,HEPES saline buffer; DAG, diacylglycerol; C-1-P, ceramide-1-phosphate; pCTB, pu-rified CTB; DRM, detergent-resistant membrane; FA, fatty acid; PtdEtn, phosphati-dylethanolamine; ASM, acidic SMase; SMS, SM synthase.

The Journal of Immunology

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1� minimal essential medium nonessential amino acid solution (InvitrogenLife Technologies). The human leukemic T cell line Jurkat was obtainedfrom American Type Culture Collection. Cells were cloned by limitingdilution. Clone D was selected on the basis of its IL-2 production whenactivated with PHA and the phorbolester 12-O-tetradecanoylphorbol-13-acetate. Jurkat clone D were grown in the same medium as PBMCs. Cellswere maintained at densities between 8 � 105 and 1 � 106/ml in a hu-midified incubator under 5% CO2 (Heraeus).

Abs, reagents, and radioactive products

rCTB was a kind gift of Dr. F. Anjuere and Prof. C. Czerkinsky (U721,Nice, France). rCTB was produced in a mutant strain of Vibrio cholerae 01deleted of its CT genes and transformed with a multicopy plasmid encod-ing CTB. The rCTB used was purified from the culture medium by acombination of salt precipitation and chromatographic methods, as previ-ously described (13), and has been already used in several studies (14–16).Rabbit polyclonal Ab anti-GM1, 1-phenyl-2-decanoylamino-3-morpho-lino-1-propanol, HCl (DL-threo-PDMP, hydrochloride/PDMP), and fumo-nisin B1 (FB1) from Fusarium moniliforme were purchased from Calbio-chem. mAb anti-PKC� (clone M4, IgG1), rabbit polyclonal Ab anti-phospho-PKC� (Ser657) (IgG), rabbit polyclonal Ab anti-p56Lck (IgG),and rabbit polyclonal Ab anti-LAT (IgG) were obtained from Upstate Bio-technology. Rabbit polyclonal Ab anti-PLC�1 (sc-81) was purchased fromSanta Cruz Biotechnology. Anti-CD3 mAb (clone X3, IgG2a) and anti-CD28 mAb (clone 28.2, IgG1) were produced in our laboratory. CD4-PerCP (clone SK3, IgG1), CD69-FITC (clone FN50, IgG1k), CD25-PE(clone M-A251, IgG1k), and apoptosis detection kit (annexin V-PE, 7-ami-noactinomycin D (7-AAD)) were purchased from BD Pharmingen. Perox-idase-labeled anti-rabbit IgG and rabbit anti-mouse IgG coupled to perox-idase were obtained from Rockland, and R-PE-Cy5-conjugatedstreptavidin was obtained from DakoCytomation. Methyl-�-cyclodextrin(m-�-CD), CTB, biotin-labeled CTB, monosialoganglioside-GM1,N-acetyl-L-cysteine (NAC), glutathione (GSH), cGMP, PMA, ionomycin,pepstatin, leupeptin, chymostatin, and anti-PMSF were purchased fromSigma-Aldrich. �2-macroglobulin was purchased from Roche. 1�,2�(n)-[3H]cholesterol (1.3–1.85 TBq/mmol), methyl-[3H]choline chloride (2.22–3.14 TBq/mmol), 9,10(n)-[3H]palmitic acid (37 MBq/mmol), [methyl-3H]thymidine (740 GBq/mmol), [�-32P]ATP, and [N-methyl-14C]SM (2.04Gbq/mmol, 55 mCi/mmol) were purchased from Amersham Biosciences.

In vitro T lymphocyte proliferative responses

Positive selection of CD4� T lymphocytes from freshly isolated hPBMCswas first performed using a fluorescence activated cell sorter (FACStar�;BD Biosciences). Reanalysis of the sorted population showed a purityhigher than 98%. Purified CD4� T lymphocytes were extensively washedthen resuspended in prewarmed culture medium at a cellular concentrationof 1 � 106/ml. Cell suspension was cultured in triplicate sets in flat-bottom96-well plates in the volume of 200 �l/well. Cells pretreated or not witheither rCTB (10 �g/ml) and/or others reagents (as detailed in the figurelegends) were stimulated or not by either PMA (10 ng/ml) plus ionomycin(100 nM) or soluble anti-CD3 mAb (5 �g/ml) plus anti-CD28 mAb (5�g/ml) for 72 h. The cultures were pulsed with 1 �Ci/well [3H]thymidineduring the last 16 h. Cells were harvested with a semiautomatic cell har-vester (Skatron Instruments) onto glass fiber filter paper. Filters were driedand counted by liquid scintillation in a Beckman Tricarb scintillation spec-trometer. Results are expressed as mean cpm � SEM of triplicate cultures.

Cell surface receptors staining

hPBMCs or Jurkat cells (1 � 106) were washed in cold PBS supplementedwith 0.1% BSA (pH 7.4) then incubated for 30 min in the dark at 4°C withthe appropriate fluorochrome-conjugated mAb according to the manufac-turer’s instructions. For GM1 indirect-staining, Jurkat cells were first in-cubated with biotin-labeled CTB (10 �g/ml). Then cells were washed andincubated (30 min at 4°C) in 100 �l of 1/25 dilution of RPE-Cy5-conju-gated streptavidin. Cells were washed again and fixed with 0.37%paraformaldehyde.

Cytometric analysis of T cell activation markers

Freshly isolated hPBMCs were washed then resuspended in prewarmedculture medium at a cellular concentration of 1 � 106/ml. Cell suspensionwas 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 (asdetailed in the figure legends) were stimulated or not by soluble anti-CD3mAb (5 �g/ml) plus anti-CD28 mAb (5 �g/ml) for 20 h. Then, cells werecostained with a PE-CD25 mAb, a FITC-CD69 mAb, and a PerCP-CD4mAb. hPBMCs were gated on lymphocytes according to their forward and

side angle light scatter. CD25 and CD69 surface expression on CD4� Tlymphocytes was determined flow cytometry after gating lymphocytes onthe basis of membrane expression of CD4. CD25 and CD69 up-regulationwas also examined on Jurkat cells pretreated or not with the indicated drugs(as detailed in the figure legends) then stimulated or not with PMA (10ng/ml) plus ionomycin (100 nM) for 20 h. The mean fluorescence intensityof 5000 cells was determined by flow cytometry (FACScan; BDBiosciences).

Viability measurement of treated cells

hPBMCs, treated or not with rCTB (10 �g/ml) for 72 h, were stained witha FITC-CD4 mAb. Then cells were incubated with both annexin-PE and7-AAD according to the manufacturer’s specifications. 7-AAD can be ex-cited by the 488-nm argon laser line and emits in the far red range of thespectrum; consequently, its spectral emission can be separated from theemissions of FITC and PE. The fluorescence parameters allow character-ization of necrotic cells (annexin-PE�/7-AAD�), apoptotic cells (annexin-PE�/7-AAD�), and viable cells (annexin-PE�/7-AAD�) in the chosensubset of FITC� cells. Moreover, viability of FB1- and PDMP-treatedJurkat cells was also compared with control Jurkat using the sametechnique.

Lipid rafts isolation

Raft isolation was accomplished using a combination of published proto-cols (17, 18). After radioactive labeling and/or pretreatment with rCTB (10�g/ml), Jurkat cells (80–100 � 106) were sonicated gently with a Vibracellsonicator (five bursts of 5 s, 5W; Bioblock Scientific) in 1 ml of ice-coldbuffer (25 mM HEPES, 150 mM NaCl, 5 mM EDTA, 10 mM sodiumpyrophosphate, 5 mM Na3VO4, and 10 mM NaF) supplemented with amixture of protease inhibitors (1 mM �-PMSF, 100 U/ml aprotinin,1mg/ml leupeptin, 1 mg/ml pepstatin, 2 mg/ml chymostatin, and 5 mg/ml�2-macroglobulin) and centrifuged at 800 gav at 2°C for 10 min to removenuclei and large debris. The resulting supernatant called postnuclear su-pernatant (PNS) was incubated with 0.5% Triton X-100 (PEG(9,10)-oc-tylphenyl ether) for 30 min at 2°C. The lysate was then adjusted to 1.33 Msucrose by the addition of 2 ml of 2 M sucrose and placed at the bottom ofan ultracentrifuge tube (Ultra-Clear; Beckman Instruments). A step sucrosegradient (0.2–0.9 M with 0.1 M steps, 1 ml each) was placed on top. Theweight percentage of sucrose was checked at room temperature using anAbbe-3L refractometer (Bioblock Scientific). The tubes were centrifuged at38 000,rpm (�250,000 � g, using the radial distance maximal (rmax: 158.8mm) for conversion with normogram) for 16–18 h (L8-70M Ultracentri-fuge; Beckman Instruments) in a SW41Ti rotor (Beckman Instruments) at2°C. One-milliliter fractions were harvested from the top. Rafts were re-covered from the low-density fractions 2 and 3 while the heavy/H fractions(soluble material) were recovered from the high-density fractions 8 and 9at the bottom of the ultracentrifuge tube.

Immunoblot analysis

Aliquots (50 �l) of each sucrose density-gradient fraction were solubilizedin 50 �l of 2� Hoessli buffer (150 mM Tris-HCl (pH 8.5), 20% glycerol,5 mM EDTA, 5% SDS, and 10% 2-ME) and then resolved by 10% SDS-PAGE under reducing conditions, and proteins were transferred onto poly-vinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore).Membranes were blocked for 2 h at room temperature in a blocking buffercontaining 5% (w/v) nonfat dry milk in TBS (10 mM Tris-HCl and 140mM NaCl (pH 7.4)) and then incubated for 1 h with the appropriate Abdiluted 1000-fold in the same buffer. The membranes were washed exten-sively in TBS containing 0.4% (v/v) Tween 20. Detection was performedwith HRP-conjugated anti-rabbit or anti-mouse and ECL reagents (Amer-sham Biosciences) according to manufacturer’s instructions.

For phospho-PKC� immunoblotting, aliquots of fraction B (fraction2 � 3) solubilized in the same volume of 2� Hoessli buffer were loadedon 10% SDS-PAGE and then transferred to PVDF membranes as describedabove. Membranes were blocked for 2 h at room temperature in a blockingbuffer containing 5% (w/v) BSA in TBS and subsequently incubated withrabbit polyclonal Ab anti-phospho-PKC� (Ser657) for 2 h at room temper-ature. Phospho-PKC� signals were detected with HRP-conjugated goatanti-rabbit, followed by ECL. Membranes were then stripped in a buffer(pH 6.7) containing 62.5 mM Tris-HCl, 2% SDS, and 0.7% 2-ME andreblotted with Ab against PKC�.

Sphingolipid content manipulation

To reduce cellular sphingolipid content, Jurkat cells were cultured as con-trol cells in a medium supplemented with either FB1 (10 �M final con-centration) or PDMP (10 �M final concentration) for 4 days; FB1 and

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PDMP were included in each medium change. Inhibition of sphingolipidsynthesis was monitored by analyzing surface expression of GM1 by flowcytometry. GM1 replenishment of sphingolipid-depleted Jurkat cells wasconducted by incubating cells in serum-free RPMI 1640 containing GM1(0.5 mg/ml for 30 min) at 37°C.

Glycerophospholipids and SM content analysis

Jurkat cells were washed then incubated for 16–18 h in a HEPES salinebuffer (HSB) (pH 7.4), containing 137 mM NaCl, 2.7 mM KCl, 1 mMNa2HPO4, 12 H2O, 2.5 mM glucose, 20 mM HEPES, 5 mM MgCl2, 1 mMCaCl2, and 0.1% BSA at 37°C in the presence of 4 �Ci of either[3H]palmitic acid or [3H]choline chloride. Lipids were extracted and ana-lyzed from either whole cells (a) or fractions obtained afterultracentrifugation (b).

(a) rCTB-treated cells or control cells were rapidly sedimented, super-natants were discarded, and cell lipids were extracted with chloroform/methanol according to Bligh and Dyer (19) then separated by monodimen-sional thin-layer chromatography on plates LK6D Silica Gel 60 A(Whatman) in a solvent system composed of chloroform/methanol/aceticacid/water (75/45/12/3). Authentic phospholipid standards (Sigma-Aldrich) were run in parallel and detected with iodide vapors. Radioactivityin lipid spots was determined by using an automatic linear radiochromatog-raphy analyzer, Tracemaster 20 (Berthold), equipped with an 8-mm win-dow and the integration software supplied by the manufacturer.

(b) An aliquot (50 �l) of each different fraction obtained after ultracen-trifugation on sucrose density-gradient were extracted and analyzed as de-scribed above.

SM synthesis measurement

Jurkat cells (2 � 106) were maintained in 500 �l of HSB. At time 0, 4 �Ciof either [3H]palmitic acid or [3H]choline chloride were added, with orwithout rCTB, at the end of the treatment, and lipids were extracted andanalyzed as described above.

Cholesterol analysis

[3H]Cholesterol in toluene solution was first evaporated under N2 and dis-solved in ethanol just prior its use. Jurkat cells were washed then incubatedfor 16–18 h in HBS containing 4 �Ci of [3H]cholesterol. Raft purificationwas performed as detailed above. To determine the distribution of [3H]cho-lesterol, an aliquot (50 �l) of each different fraction obtained after ultra-centrifugation on sucrose density-gradient was mixed with Picofluor andcounted by liquid scintillation in a Beckman Tricarb scintillationspectrometer.

Assays for neutral- and acidic-SMase

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�M radioactive SM substrate, 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) wereplaced in a glass tube, and the organic solvent was removed under N2. Thedried [14C]SM was solubilized in 500 �l of 1% (w/v) �-octylglucoside bybrief sonication with a bath-type sonicator. Fifty-microliter aliquots of se-lected fractions (2 � 3 and 8 � 9) were assayed for the presence of dif-ferent SMase activities. Reactions were started by adding 50 �l of substratesolution. For the measurement of the neutral-SMase activity, this solutionconsisted in 10 �l of the stock solution of [14C]SM (0.5 nmol), 10 �l of abuffer consisting in 250 mM HEPES (pH 7.5), 50 mM MgCl2, and 0.5%(v/v) Triton X-100 and 30 �l of deionized water. After incubation at 37°Cfor 3 h, the reaction was stopped by adding 800 �l of chloroform/methanol(2/1, v/v) and 200 �l of deionized water. A 100-�l aliquot of the aqueousupper phase containing [14C]phosphorylcholine released from [N-methyl-14C]SM was collected and counted by liquid scintillation. The reaction waslinear within this frame, and the amount of [N-methyl-14C]SM hydrolyzedduring an assay did not exceed 10% of the total amount of radioactive SMadded. For calculation of the specific activities, values were corrected forvolume of the aqueous phase, volume of the sample, protein content, re-action time, and specific activity of the substrate. For the assay of acidic-SMase activity, the substrate solution consisted in 10 �l of the stock so-lution of [14C]SM (0.5 nmol) and 10 �l of 0.5 M sodium acetate buffer (pH4.8), consisting of 10 mM EDTA, 0.5% (v/v) Triton X-100, and 30 �l ofdeionized water. The initiation and termination of the reaction and thedetermination of the water-soluble radioactivity released from [N-methyl-14C]SM was proceed as described above.

Diacylglycerol (DAG) kinase assays

Total cellular ceramide levels were quantified by the DAG kinase assay as32P incorporated upon phosphorylation of ceramide to ceramide-1-phos-phate (C-1-P) by DAG kinase from Escherichia coli (23). After rCTBtreatment (10 �g/ml) for different period of time, Jurkat cells (5 � 106)were washed twice with ice-cold PBS. After centrifugation (1000 � g, 5min, 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(135 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.5 mMglucose, and 10 mM HEPES (pH 7.2)), and 30 �l of 100 mM EDTA. Thelipids of the organic phase were transferred to a new glass tube and driedunder a stream of N2. Lipid extracts were then subjected to mild alkalinehydrolysis (0.1 M KOH in methanol for 1 h at 37°C) to remove glycero-phospholipids. Five hundred microliters of chloroform, 270 �l of bufferedsaline solution, and 30 �l of 100 mM EDTA were added. After drying theorganic phase with N2, in vitro phosphorylation of extracted ceramides wasperformed as described by the manufacturer (RPN 200 kit; AmershamBiosciences). A total of 1 �Ci of [�-32P]ATP (4000 Ci/mmol) was used tostart the reaction. After 30 min at room temperature, the reaction wasstopped by extraction of lipids with 1 ml of chloroform/methanol/hydro-chloric acid (1 N) (100/100/1, v/v/v), 170 �l of buffered saline solution,and 30 �l of 100 mM EDTA. The lower organic phase was dried under N2.The samples were resuspended in 30 �l of chloroform/methanol (95/5, v/v)and spotted on plates LK6D Silica Gel 60 A. C-1-P was resolved by TLCusing chloroform/methanol/acetic acid (75/25/5, v/v/v) as solvent and mi-grated as a single spot at RF � 0.25. Linearity of the assay was establishedusing purified C16-ceramide (Sigma-Aldrich). Radioactivity in lipid spotswas determined by using an automatic linear radiochromatography ana-lyzer, Tracemaster 20 (Berthold), equipped with an 8-mm window and theintegration software supplied by the manufacturer.

Semiquantitative RT-PCR

After cell treatment, total RNA was isolated from Jurkat cells using TRIzolReagent (Invitrogen Life Technologies) based on method derived byChomczynski and Sacchi (24). RNA (150 ng) was then reverse transcribedusing the SuperScript II RNAase H� reverse transcriptase (Invitrogen LifeTechnologies) following the manufacturer’s instructions and resuspendedin 150 �l final volume. cDNAs (5 �l) or water as control were amplifiedby PCR in a final volume of 25 �l using the Platinum TaqDNA Polymerase(Invitrogen Life Technologies) and 300 nM of forward and reverse prim-ers. RT-PCR was typically performed for 35 cycles (denaturation at 95°Cfor 20 s, annealing at 68°C for 1 min, extension at 72°C for 1 min). Primerswere designed using the PRIMER Express Software 1.5 (Applied Biosys-tems). The following 5� and 3� primers were as follows: human CD69, 5�primer (5�-CGTAGCAGAGAACAGCTCTTTGC-3�) and 3� primer (5�-ACAGGACAGGAACTTGGAAGGA-3�), and human CD25, 5� primer(5�-GGGACTGCTCACGTTCATCA-3�) and 3� primer (5�-TTCAACATGGTTCCTTCCTTGTAG-3�). �-actin was used as loading control.

EMSAs

Total cellular extracts were prepared in Totex lysis buffer (20 mM HEPES(pH 7.9), 350 mM NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM MgCl2,0.5 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 10 �g/mlaprotinin). Supernatants from a 15,000 � g, 10 min centrifugation at 4°Cwere collected. The NF-�B probe used for mobility shift assay was con-stituted of a synthetic double-stranded oligonucleotide containing theNF-�B site of the Ig� promoter (5�-GATCCAAGGGACTTTCCATG-3�).The 32P-end-labeled probe (T4 kinase) was incubated with Totex extractsamples (20 �g) for 25 min at room temperature. Complexes were thenseparated by electrophoresis on a 5% nondenaturating polyacrylamide gelin 0.5 � Tris-borate EDTA. Dried gels were subjected to autoradiography.

Luciferase assays

Jurkat cells were transiently transfected by electroporation (320 V, 960 �F)with 10 �g of a luciferase reporter gene controlled by a minimal thymidinekinase promoter and six reiterated �B sites (�Bx6 thymidine kinase luc). At36 h after transfection, cells were stimulated as indicated. Cells werewashed twice in PBS (pH 7.2) and lysed in 100 �l of reporter lysis buffer(Promega). Luciferase activity was assayed by luminometry (Lumat;EG&G Berthold) using the Promega luciferase assay system. Normaliza-tion of transfection efficiency was done using a cotransfected �-galactosi-dase expression vector. Luciferase activity was determined in triplicate andexpressed as fold increase relative to basal activity seen in untreated un-stimulated mock-transfected cells.

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ResultsGM1-rCTB interaction but not GM1-anti-GM1 Abs interactioninhibits PMA/ionomycin-induced CD4� T proliferation

Previous works have demonstrated that CTB is able to inhibit Tcell activation and proliferation induced by either polyclonal mi-togens or by specific Ags (9–11). To see whether GM1 bindingalone is sufficient or not to inhibit PMA/ionomycin-induced CD4�

T proliferation, CD4� T lymphocytes were pretreated with variousconcentrations of either purified CTB (pCTB), rCTB, or rabbitpolyclonal anti-GM1 Abs, then cells were stimulated with PMA/ionomycin, and proliferation was measured. Fig. 1A unambigu-ously demonstrates that PMA/ionomycin-induced CD4� T cellproliferation is inhibited by both pCTB and rCTB in a dose-de-pendent manner. Our result unambiguously demonstrates that theinhibition of the proliferation of CD4� T lymphocytes is not dueto cAMP produced by contaminant cholera toxin A-subunit be-cause rCTB gives the same results as pCTB. Furthermore, we in-vestigated whether cGMP inhibit the effects of pCTB. Indeed, ifpCTB is contaminated by cholera toxin A-subunit, it will producecAMP, and it is well known that cGMP and cAMP have antago-nistic action on proliferation of T lymphocytes (25). As shown inFig. 1A, cGMP does not prevent the pCTB-induced inhibition ofthe PMA/ionomycin-induced proliferation of CD4� T lympho-cytes, thus indicating that pCTB does not exert its effect via AMPc.In contrast with rCTB, anti-GM1 Abs have no effects on PMA/ionomycin-induced CD4� T cell proliferation. In conclusion,GM1 binding by specific Abs is not able to inhibit CD4� T cells,whereas GM1-rCTB interaction is necessary to exert inhibitoryeffect. Furthermore, we questioned whether Ac anti-GM1 wouldpotentiate the effects of rCTB or would block its activity. CD4� Tlymphocytes incubated with a combination of rCTB plus Ac anti-GM1 were stimulated by PMA/ionomycin and [3H]thymidine in-corporation was measured. As shown in Fig. 1A, Ac anti-GM1neither potentiates nor blocks the effect of rCTB on PMA/iono-

mycin-induced proliferation. Fig. 1B clearly shows that theepitopes recognized by either rCTB or Ac anti-GM1 are different.There is no competition between rCTB and Ac anti-GM1 for thebinding of GM1. This result may explain why Ac anti-GM1 doesnot block the action of rCTB. The epitope recognized by Ac anti-GM1 may be not involved in the inhibition of CD4� T lympho-cytes because they do not potentiate the effects of rCTB.

The integrity of cholesterol-rich raft is not required for rCTB-induced inhibition of CD4� T lymphocytes

The monosialoganglioside GM1 is certainly the most com-monly used lipid raft marker. Cholesterol extraction by m-�-CDdisrupts cholesterol-rich rafts and raft-resident molecules leaverafts. However, a recent study (26) showed that depletion of73% of cell cholesterol with m-�-CD significantly affects therecovery in detergent-resistant membranes (DRMs) of GM1acetylated or acylated with C8 or unsaturated fatty acids (FAs)but not of GM1 acylated with C18, C22, or C24 FAs. To seewhether cholesterol-rich raft integrity is required for rCTB-induced CD4� T cell inhibition, we first treated hPBMCs withm-�-CD to disrupt cholesterol-rich rafts. Then cholesterol-de-pleted hPBMCs were pretreated with rCTB or pCTB and cellswere stimulated with PMA/ionomycin. As shown in Fig. 2A,cholesterol depletion of CD4� T lymphocytes with m-�-CDdoes not prevent rCTB and pCTB to inhibit PMA/ionomycin-induced CD69 and CD25 up-regulation. The same results wereobtained in Jurkat cells (Fig. 2B).

SM is necessary for rCTB-induced CD4� T cell inhibition

Because cholesterol is not required for rCTB-induced inhibition ofCD4� T lymphocytes, we investigated a role for SM in rCTB-induced CD4� T inhibition. To this end, we used Jurkat cells in-stead of hPBMCs because these cells are more suitable for study-ing lipid metabolism because their global metabolism is greatly

FIGURE 1. Effect of rCTB and rabbit polyclonalAbs anti-GM1 on PMA plus ionomycin-inducedCD4� T lymphocyte proliferation. A, FACS-sortedCD4� T lymphocytes were pretreated or not in 96-well flat-bottom plates with either pCTB, rCTB, rab-bit polyclonal Abs anti-GM1, pCTB � cGMP, orrCTB � Abs anti-GM1 for 30 min at the indicatedconcentrations, then CD4� T lymphocytes werestimulated or not with 10 ng/ml PMA plus 100 nMionomycin. Proliferation was monitored by [3H]thy-midine incorporation during the last 16 h of culture.CD4� T lymphocytes were harvested for beta scin-tillation counting. Data represent one of three simi-lar experiments. The values represent means �SEM. B, Jurkat cells (1 � 106) were first incubatedwith Abs anti-GM1 (30 min at 4°C), then GM1 re-ceptors were stained with pCTB-biotin � streptavi-din-RPE-Cy5 (30 min at 4°C) as indicated in Mate-rials and Methods. GM1 staining was measured bycytometric analysis.

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more rapid than those of hPBMCs. Indeed, Jurkat cells do spon-taneously proliferate whereas hPBMCs do not. Incorporation of[3H]palmitic acid or [3H]choline is quite equal in Jurkat cells andin hPBMCs. But the kinetic of SM synthesis is completely differ-ent. After a 4 h-incubation with tritiated precursors, it is alreadypossible to easily detect SM synthesis in Jurkat cells, whereas it isimpossible in hPBMCs. We used two drugs to modulate the levelof SM and/or gangliosides: FB1 and PDMP. FB1 prevents theproduction of both SM and gangliosides (27). PDMP specificallydiminishes levels of endogenous gangliosides (28, 29). Note thatPDMP treatment also results in a significant accumulation ofSM (30).

We studied the surface expression of GM1 both in FB1- andPDMP-treated Jurkat cells. As shown in Fig. 3B, FB1 and PDMPconsiderably diminish the level of the ganglioside GM1 after a4-day treatment. Reduction of GM1 is time dependent, but treat-ments cannot be longer extended because beyond a 4-day incuba-tion, cell viability starts to decline. After a 4-day treatment, FB1-treated cells exhibits only 34% of control GM1 and cell viability isgreater than 93%. PDMP-treated Jurkat only possess 29% of GM1,and cell viability is not affected at all. Incubation of GM1-depletedJurkat with exogenous GM1 (0.5 mg/ml for 30 min) approximatelymultiplies by 3.5 the basal content.

PDMP inhibits GM1 synthesis whereas FB1 inhibits both SMand GM1 synthesis. As shown in Fig. 3C, rCTB largely inhibitsPMA/ionomycin-induced CD69 and CD25 up-regulation (bar 4 vsbar 2). As expected, rCTB has little effect on CD69 and CD25expression on PDMP-treated Jurkat cells (bar 7 vs bar 6), but rCTBre-exerts its important inhibitory effect on GM1-restored Jurkatcells (bar 8 vs bars 7 and 4). It indicates that rCTB can inhibitPMA/ionomycin-induced CD69 and CD25 up-regulation either viaendogenous or exogenous GM1. It means that exogenous GM1 isas active as endogenous one. Furthermore, as expected, rCTB haslittle effect on CD69 and CD25 expression on FB1-treated Jurkatcells (bar 11 vs bar 10). In sharp contrast with PDMP treatment,GM1 restoration of FB1-treated Jurkat cells does not allow rCTBto exert its inhibitory effect on PMA/ionomycin-induced CD69 andCD25 up-regulation (bar 12 vs bar 4). This result clearly indicatesthat 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 surfaceexpression of CD69 and CD25, RT-PCRs were performed (Fig.3D). In accordance with the flow cytometry, CTB inhibits mRNAsynthesis of CD69 and CD25. FB1 and PDMP treatment preventthe inhibitory effect of CTB on PMA/ionomycin-induced CD69and CD25 up-regulation. Exogenous GM1-addition in PDMP-treated Jurkat cells allows CTB to inhibit T cell activation. Incontrast, exogenous GM1-addition in FB1-treated Jurkat cells doesnot allow CTB to inhibit T cell activation. It unambiguously dem-onstrates that if cholesterol is not required, SM is necessary toGM1 signaling via the binding of CTB.

rCTB inhibits SM synthesis and enhances PtdCho synthesis

Because we demonstrated that SM is required for GM1 signalingvia the binding of rCTB (Fig. 3), we analyzed the synthesis of SMin control and in rCTB-treated Jurkat cells. We also analyzed thesynthesis of PtdCho and phosphatidylethanolamine (PtdEtn) (Fig.4). The uptake of [3H]palmitic acid and [3H]choline chloride is notaffected by rCTB treatment (data not shown). We show that[3H]palmitic acid-labeled SM synthesis and [3H]choline-labeledSM synthesis are both inhibited in rCTB-treated cells comparedwith control cells (�54 and �56%, respectively). In contrast,[3H]palmitic acid-labeled PtdCho synthesis and [3H]choline-la-beled Ptcho synthesis are both enhanced in rCTB-treated Jurkatcells (�122 and � 84% respectively). [3H]Palmitic acid-labeledPtdEtn is not affected by rCTB treatment (data not shown).

rCTB induces SM hydrolysis

Jurkat cells prelabeled with either [3H]palmitic acid (Fig. 5A, up-per graph) or [3H]choline (Fig. 5A, lower graph) were left un-treated or incubated with rCTB for different period of time vary-ing from 0 to 30 min. The analysis of lipids extracted fromwhole cells indicates that rCTB treatment results in a time-dependent decrease of SM (Fig. 5A). The decrease of SM ismaximal at time 30 min and reaches 47% for [3H]palmitic acid-labeled SM and 46% for [3H]choline-labeled SM.

To study SM hydrolysis in lipid rafts, Jurkat cells, prelabeledwith either [3H]palmitic acid (Fig. 5B) or [3H]choline (Fig. 5C),were treated or not with rCTB for 30 min and rafts were purifiedThe analysis of the fractions was first performed by liquid scintil-lation counting (data not shown). The distribution of the tritiatedFA clearly indicates that this saturated FA is preferentially incor-porated into fractions 2 and 3 corresponding to membrane raftscompared with fractions 8 and 9 corresponding to the detergent-soluble material. A further analysis by TLC of the lipid composi-tion of the sucrose density-fractions unambiguously indicates thatthe raft fraction is highly enriched in [3H]palmitic acid-labeled SM

FIGURE 2. Cholesterol rich-raft disruption by m-�-CD does not pre-vent rCTB to inhibit PMA/ionomycin-induced CD69 and CD25 up-regu-lation. A, Freshly isolated hPBMCs were treated or not with m-�-CD (10mM, 10 min at 37°C). Then cells pretreated or not with rCTB (10 �g/ml)or pCTB (10 �g/ml) for 30 min were stimulated or not by PMA (10 ng/ml)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 weregated on lymphocytes according to their forward and side angle light scat-ter. CD25 and CD69 surface expression on CD4� T lymphocytes wasdetermined flow cytometry after gating lymphocytes on the basis of mem-brane expression of CD4. Data represent one of three similar experiments.B, CD25 and CD69 up-regulation was also examined on Jurkat cells pre-treated or not with m-�-CD, pretreated or not with either rCTB or pCTB,and stimulated or not with PMA/ionomycin as in A. Data represent one ofthree similar experiments. The values represent means � SEM.

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(Fig. 5B). Comparison between fractions 2 and 9 shows that[3H]choline-labeled SM is less predominant than [3H]palmitic ac-id-labeled SM in raft fractions (Fig. 5C). rCTB treatment results inan important loss of SM from raft fractions while soluble fractionsremain unchanged (Fig. 5, B and C). Fig. 5, B and C, also showthat rCTB enhances PtdCho synthesis. When it is labeled withpalmitic acid, PtdCho accumulation only occurs in raft fractions;by contrast, when it is labeled with choline, PtdCho is found inrafts as well as in soluble fractions.

Because saturated FA carbon chains are known to interact withcholesterol, 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 SMalters cellular cholesterol homeostasis. We observed that this cho-lesterol decrease specifically occurs in rafts.

rCTB activates a NSM1-like enzyme in lipid rafts that producesceramides

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 ornot with rCTB and raft isolation was performed. Raft fractions,and fractions containing the Triton X-100-soluble material wereassayed for either neutral or acidic SMase (ASM) activity. Asshown in Fig. 7A, a neutral pH optimum SMase activity was foundin raft of rCTB-treated Jurkat cells. This result indicates that araft-resident neutral SMase is involved in GM1 signaling via the

FIGURE 4. rCTB inhibits SM synthesis and enhances PtdCho synthe-sis. Jurkat cells (2 � 106) were maintained in 500 �l of HSB. At time 0,4 �Ci of either [3H]palmitic acid (A) or [3H]choline chloride (B) wereadded, with (10 �g/ml, f) or without rCTB (�), at the end of the treatment(120 min); lipids were extracted and analyzed as described in Materialsand Methods. Data represent one of three similar experiments. The valuesrepresent means � SEM.

FIGURE 3. SM is necessary for rCTB-induced CD4� T inhibition. A, FB1 inhibits both SM and gangliosides. PDMP specifically prevents the formationof gangliosides while enhancing SM production. B, Jurkat cells (1 � 106) were cultured with or without FB1 or PDMP (in both case, 10 �M finalconcentration) 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 wasmeasured by flow cytometry. Data represent one of three similar experiments. The values represent means � SEM. C, Jurkat cells (1 � 106) pretreatedor 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 pretreatedor 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 andCD25 was measured by flow cytometry as in Fig. 2B. Data represent one of three similar experiments. The values represent means � SEM. D, Jurkat cellswere treated as in C, except that they were stimulated by PMA/ionomycin for 6 h. RNA was extracted, and RT-PCR was performed using primersamplifying either CD69, CD25, or �-actin. Data represent one of three similar experiments.

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rCTB. Furthermore, this neutral SMase activity is almost entirelyinhibited by GSH. It indicates that the involved enzyme is prob-ably a NSM1-like one. ASM has not been found implicated in thatprocess (data not shown).

SM hydrolysis results in the formation of ceramides and phos-phocholine. The topology of ceramide formation determines itsfunction (33). When SM from the outer leaflet is hydrolyzed, cer-amides generated in this outer leaflet form ceramides-rich do-mains, while ceramides generated from the small SM pool in theplasma membrane inner leaflet serve as second messengers in sig-nal transduction. Using the DAG kinase assay, we dosed the cer-amide production upon the binding of GM1 by rCTB. As shown inFig. 7B, rCTB induces a rapid and transient production of cer-amides. The ceramide production reaches its maximal at 30 minand returns near to the basal level at 45 min. This result suggeststhat ceramides produced by rCTB are rapidly metabolized and re-enter into the SM cycle.

GSH and NAC pretreatment inhibits the effects of rCTB

On one hand, we demonstrated that rCTB-GM1 association inhib-its CD4� T lymphocyte activation (Figs. 2, A and B, 3, D and E)and proliferation (Fig. 1). In the other hand, we demonstrated that

1) rCTB inhibits SM synthesis (Fig. 4) and 2) activates a NSM1-like enzyme in lipid rafts that produces transient ceramides (Fig.7). To demonstrate that rCTB-induced CD4� T lymphocyte inhi-bition is due in part to SM level modifications, we pretreated Jurkatcells with either the antioxidant GSH or NAC. NAC acts as aprecursor of reduced GSH biosynthesis and consequently it inhib-its neutral SMase. As shown in Fig. 8A, pretreatment with GSH orNAC inhibits the inhibitory effect of rCTB on Jurkat cells. rCTB-treated Jurkat do not up-regulate CD69 and CD25 when they arestimulated by PMA/ionomycin. By contrast, rCTB-treated cellsthat have been pretreated before with NAC or GSH are able toup-regulate the activation markers CD69 and CD25.

To determine whether mRNA correlates with the cell surfaceexpression of CD69 and CD25, RT-PCRs were performed (Fig.8B). In total accordance with the flow cytometry, the inhibitoryeffect of CTB on Jurkat cells is abolished when cells are pretreatedwith NAC.

To link the absence of proliferation of rCTB-treated CD4� Tlymphocytes and the neutral SMase activity (i.e., the transient ac-cumulation of ceramides), purified CD4� T lymphocytes were pre-treated or not with GSH or NAC before being treated or not withrCTB (10 �g/ml, 30 min). Then cells were stimulated either by

FIGURE 5. rCTB induces SM hydrolysis. A, Jurkat cells (2 � 106) were first prelabeled until isotopic equilibrium with either [3H]palmitic acid (uppergraph) or [3H]choline chloride (lower graph), then washed and treated (f) or not (�) with rCTB (10 �g/ml) for different periods of time varying from0 to 30 min. Lipids were extracted with chloroform/methanol then analyzed by TLC on silicagel plates. These graphs are representative of three differentand independent experiments. The values represent means � SEM. B, A PNS preparation of Jurkat cells (80–100 � 106) metabolically prelabeled for 16 huntil 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 fractionatedon a sucrose density-gradient as described in Materials and Methods. After ultracentrifugation, 1-ml fractions were harvested from the top. Lipids fromeach fraction were extracted with chloroform/methanol then analyzed by TLC on silicagel plates. The distribution of [3H]palmitic acid-labeled SM,[3H]palmitic acid-labeled PtdCho, and [3H]palmitic acid-labeled PtdEtn is shown for control (�) 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 centrifugetube to fractions 8 and 9. These graphs are representative of three different and independent experiments. The values represent means � SEM. C, Jurkatcells (80–100 � 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 distributionof [3H]choline-labeled SM and [3H]choline-labeled PtdCho is shown for control (�) 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 � SEM.

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CD3/CD28 or by PMA/ionomycin. As expected, rCTB inhibitsless the proliferation of GSH- or NAC-pretreated CD4� T lym-phocytes than the proliferation of control lymphocytes.

Thus, these results demonstrate that the rCTB-induced SMaseactivation is responsible for the observed inhibition (activation andproliferation) of CD4� T lymphocytes when they are preincubatedwith rCTB.

SM hydrolysis is required for rCTB-induced NF-�B activation

Heat-labile ETB from Escherichia coli, a close homologue ofCTB, is known to activate nuclear translocation of NF-�B in Jurkatcells (12). Activation of NF-�B by rCTB has never been investi-gated before. Translocation of NF-�B was visualized after 1 h ofrCTB (10 �g/ml) stimulation by its binding to a radioactive probecontaining �B sites from the Ig� promoter (Fig. 9A, lane 2 com-pared with unstimulated cells, lane 1). In PDMP-treated Jurkatcells, rCTB fails to translocate NF-�B. In addition, in FB1-treatedcells, rCTB also fails to translocate NF-�B (Fig. 9A, lane 11 vslane 2). Furthermore, rCTB is able to translocate NF-�B in GM1-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 isnecessary to NF-�B translocation by rCTB. Preincubation withGSH leads to an inhibition of NF-�B DNA-binding activity (Fig.9A, lane 4 vs lane 2), indicating that SM hydrolysis is required forrCTB-induced NF-�B translocation.

NF-�B activation was measured in Jurkat cells transfected witha reporter luciferase gene under the control of NF-�B (Fig. 9B). Intotal accordance with EMSAs, NF-�B activation was inhibitedboth in PDMP- and in FB1-treated cells and was restored only inGM1-recompleted, PDMP-treated cells, indicating that SM is re-quired for NF-�B activation by rCTB. rCTB stimulation leads toan 8-fold increase in luciferase activity that is strongly decreasedby �50% by GSH-or NAC-pretreatment, indicating that SM hy-drolysis is involved in rCTB-induced NF-�B activation. GSH andNAC has no effect on baseline luciferase activity.

rCTB treatment prevents PKC� phosphorylation andtranslocation into modified lipid rafts

Because rCTB treatment strongly modifies the lipid compositionof rafts (1) rCTB diminishes SM synthesis (Fig. 4), 2) hydrolyzesSM, and 3) enhances PtdCho synthesis (Fig. 5)), we were inter-ested in studying the distribution of raft-resident proteins highlyinvolved in T cell activation. As shown by Fig. 10A, rCTB treat-

ment modifies neither the distribution of linker for activation of Tcells nor Lck. Then, we studied the effect of rCTB on PMA-in-duced recruitment of PKC� into lipid rafts (Fig. 10B, upper panel).As shown in Fig. 10B, upper panel, PMA induces a partial trans-location of PKC� into lipid rafts. rCTB treatment (i.e., raft mod-ifications in terms of lipids) importantly prevents PKC� translo-cation into lipid rafts. Interestingly, GSH and NAC pretreatment,which prevents rCTB to inhibit Jurkat activation, allows PKC� toredistribute itself after rCTB pretreatment and PMA stimulation.

Then, we investigated the phosphorylation status of PKC�within rafts (Fig. 10B, lower panel). The same amount of PKC� inrafts for control and rCTB-treated cells was subjected to SDS-PAGE, and the membrane was immunoblotted with anti-phospho-PKC� mAb. Fig. 10B, lower panel, clearly shows that rCTB in-hibits PKC� phosphorylation on Ser657.

DiscussionIn the present article, we propose a mechanism for the inhibitoryeffect of rCTB both on the activation and on the proliferation ofhuman CD4� T lymphocytes. rCTB specifically binds to GM1, araft marker, and strongly modifies the lipid composition of rafts.First, rCTB inhibits SM synthesis; second, it enhances PtdChosynthesis; and third, it activates a raft-resident neutral SMase, thus

FIGURE 6. rCTB lowers cholesterol level in rafts. A PNS preparationof Jurkat cells (80–100 � 106) labeled for 16 h until isotopic equilibriumwith [3H]cholesterol and stimulated or not by rCTB (10 �g/ml) for 30 minwas 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. Thedistribution of [3H]cholesterol in the gradient is shown for control (�) andrCTB-treated cells (f). This graph is representative of three different andindependent experiments. The values represent means � SEM.

FIGURE 7. rCTB activates a neutral SMase. A, Jurkat cells (80–100 �106) were pretreated or not with rCTB (10 �g/ml) or pCTB (10 �g/ml) for30 min. Raft isolation was performed as in Fig. 5B, except that RPMI 1640supplemented with a mixture of protease inhibitors was used in place of theinitial buffer. Fifty-microliter aliquots of selected fractions (2 � 3 and 8 �9) were assayed with or without GSH (3 mM) for the presence of eitherneutral SMase activity or ASM activity as described in Materials andMethods. This graph is representative of three different and independentexperiments. The values represent means � SEM. B, Jurkat cells (5 � 106)were treated (f) or not (�) with rCTB (10 �g/ml) for the indicatedtimes. Lipids were extracted and, after mild alkaline hydrolysis, sub-jected to phosphorylation by the DAG kinase in the presence of[�-32P]ATP as described in Materials and Methods. The resulting C-1-Pwas separated by TLC. Radioactivity in lipid spots was determined byusing an automatic linear radiochromatography analyzer. This graph isrepresentative of three different and independent experiments. The val-ues represent means � SEM.

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generating a transient ceramide production. These ceramides in-hibit PKC� phosphorylation and its translocation into the modifiedlipid rafts. Furthermore, ceramides activate NF-�B. We hypothe-size that combined all together, raft modification in terms of lipids,ceramide production, PKC� inhibition, and NF-�B activation leadto T cell inhibition.

Gangliosides and glycosylphosphatidylinositol-anchored pro-teins are frequently used as positive controls for raft purification.GM1 is certainly the most commonly used raft marker. It is to notethat gangliosides and glycosylphosphatidylinositol-anchored pro-teins are almost ever used indiscriminately as if they were equal.Nevertheless, Vyas et al. (34) demonstrated that GM1 does notcocluster with GD3 on intact neurons. Gomez-Mouton et al. (35)show that the acquisition of a motile phenotype in T lymphocytesresults in the asymmetric redistribution of GM3- and GM1-en-riched raft domains to the leading edge and to the uropod, respec-tively. Furthermore, Millan et al. (36) have demonstrated that

CD59 and GM1 cluster in different membrane subdomains of Ju-rkat cells. Furthermore, m-�-CD extracts cholesterol from TritonX-100-resistant membranes without affecting the buoyant proper-ties of Thy-1 and GM1 (37, 38). The occurrence of GM1 in DRMsdepends on its ceramide moiety. Depletion of 73% of cellular cho-lesterol with m-�-CD does not affect the recovery in DRMs ofGM1 acylated with C18-, C22-, or C24-saturated FAs (26). Com-bined all together, these data suggest that GM1 resides in a subsetof lipid raft that is insensitive to cholesterol depletion by m-�-CD.Our data support and extend this earlier observation because weshow that m-�-CD-treatment does not affect the inhibitory effect ofrCTB via GM1.

CTB and ETB are known to modulate leukocyte function. Thisproperty is attributed to the ability of the B-subunit to bind GM1.Nevertheless, it has been demonstrated that GM1 binding alone,contrary to expectations, is not sufficient to initiate toxin action.Fraser et al. (12) described the properties of ETB (H57S), a mutantB-subunit with a His3Ser substitution, at position 57. The mutantstill binds to GM1 but is found to be severely defective in inducing

FIGURE 8. NAC and GSH reverse the inhibitory effect of rCTB. A,Jurkat cells (1 � 106) pretreated or not with NAC (10 mM) or GSH (3 mM)for 1 h were treated or not with rCTB (10 �g/ml) for 30 min. Then cellswere stimulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for20 h. Surface expression of CD69 and CD25 was measured by flow cy-tometry as in Fig. 2B. This graph is representative of three different andindependent experiments. The values represent means � SEM. B, Jurkatcells were treated as in A, except that they were stimulated by PMA/iono-mycin for 6 h. RNA was extracted, and RT-PCR was performed usingprimers amplifying either CD69, CD25, or �-actin. Data represent one ofthree similar experiments. C, FACS-sorted CD4� T lymphocytes werepretreated or not in 96-well flat-bottom plates with either NAC (10 mM) orGSH (3 mM) for 30 min, then CD4� T lymphocytes treated or not withrCTB (10 �g/ml) for 30 min were stimulated or not with either 10 ng/mlPMA plus 100 nM ionomycin or soluble anti-CD3 mAb plus anti-CD28mAb (5 �g/ml each). Proliferation was monitored by [3H]thymidineincorporation during the last 16 h of culture. CD4� T lymphocytes wereharvested for beta scintillation counting. This graph is representative ofthree different and independent experiments. The values representmeans � SEM.

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, 4days), or FB1 (10 �M, 4 days), or GSH (3 mM for 1 h). Sphingolipid-depleted cells were preincubated or not with GM1 (0.5 mg/ml) for 30 min.Then cells were treated or not with rCTB (10 �g/ml) for 1 h. The total cellextracts were then incubated with a radioactive double-stranded oligonu-cleotide encompassing the �B site of the Ig� promoter. Complexes werethen separated by nondenaturing electrophoresis followed by autoradiog-raphy. Data represent one of three similar experiments. B, For sphingolipiddepletion experiments, Jurkat cells were pretreated or not with eitherPDMP (10 �M, 3 days) or FB1 (10 �M, 3 days), then cells were trans-fected with 10 �g of luciferase reporter gene. At 36 h after transfection(Jurkat cells were maintained for 36 h in a medium containing eitherPDMP or FB1), cells preincubated or not with GM1 (0.5 mg/ml) for 30 minwere treated or not with rCTB (10 �g/ml) for 30 min. For NAC and GSHexperiments, 36 h after transfection, Jurkat cells were pretreated or not withNAC (25 mM, 1 h) or GSH (3 mM, 1 h), then cells were treated or not withrCTB (10 �g/ml) for 30 min. Luciferase activity was measured as detailedin Materials and Methods. Data represent one of three similar experiments.The values represent means � SEM.

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leukocyte signaling. For example, it fails to trigger caspase-3-me-diated T CD8� lymphocyte apoptosis. It does not activate NF-�Bin Jurkat cells. It fails to induce a potent anti-B-subunit response inmice, and it also fails to serve as mucosal adjuvant. In the samemanner, CTB (H57A) binds GM1 but lacks immunomodulatory ortoxic activity (39). In the present study, we demonstrate that GM1ligation alone, via rabbit polyclonal Abs anti-GM1, is not able toinhibit CD4� T lymphocyte activation and proliferation. We dem-onstrate that GM1 ligation by Abs does not lead to SMase activa-tion and SM hydrolysis (data not shown). Aman et al. (39) hy-pothesize that CT may require interaction, not only with GM1, butalso with another molecule to exert its biological activity. For

Aman et al. (39), it is conceivable that rCTB binding to GM1 inlipid rafts would position it to interact with signaling moleculesthat participate in toxin-mediated immune cell modulation. In re-spect with our experimental data, it is quite conceivable that theintermediary molecule involved in the toxin action would be araft-resident neutral SMase.

The isoform of SMase (acidic or neutral), thus the topology ofceramide formation, dictates its outcome (33). The activation ofASM is believed to theoretically allow the hydrolysis of the mainpool of SM from the outer plasma membrane leaflet, thus resultingin the formation of ceramide-rich domains (40), while the activa-tion of neutral SMase would rather allow the hydrolysis of thesmall pool of SM from the inner leaflet resulting, this time, in thegeneration of ceramides that will act as second messengers. In thisstudy, 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 byrCTB reaches 47% for [3H]palmitic acid-labeled SM and 46% for[3H]choline-labeled SM. It probably represents more than the mi-nor pool of SM located in the inner leaflet. Thus, what remainsunclear is how neutral SMase gains access to the major pool ofSM. It has been hypothesized that neutral SMase would hydrolyzeSM 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 bythe externalization of phosphatidylserine (data not shown).

In this study, we demonstrated that rCTB regulates SM at twolevels. First, rCTB inhibits SM synthesis, and second, rCTB acti-vates SM hydrolysis. These two complementary actions both con-tribute to cell cycle arrest of CD4� T lymphocytes. Indeed, theinhibition of SM synthesis 1) causes the inhibition of DAG syn-thesis and 2) increases the level of ceramides; furthermore, SMhydrolysis generates ceramides. Consequently, rCTB enhancesceramides content by two different manners. Ceramides:DAG ratioplays an important role in cell proliferation, and numerous targetsof ceramides are known to negatively regulate cell proliferation.

The biochemical synthesis of SM occurs via the action of aPtdCho:ceramide choline phosphotransferase (SM synthase(SMS)), which transfers the phosphorylcholine (phosphocholine)moiety from PtdCho onto the primary hydroxyl of ceramide, thusproducing SM and DAG (41, 42). Thus, the inhibition of SMSreduces the intracellular level of DAG. It has been published thatDAG generated during SM synthesis is involved in PKC activationand cell proliferation (43). It constitutes the first way by whichrCTB inhibits PKC� in CD4� T lymphocytes. Furthermore, theinhibition of SMS also increases the level of ceramides (44, 45). Itmay constitute a supplementary way, in addition to SM hydrolysis,to elevate ceramide levels. Flores et al. (46) studied the changes inthe balance between DAG and ceramides during cell proliferation,cell arrest, and apoptosis in T lymphocytes. Accordingly, augmen-tation of ceramides and diminution of DAG favor cell arrest.

Ceramides also target specific proteins inducing cell cycle ar-rest. Ceramides induce a Go-G1 cell cycle arrest, and this wasmechanistically shown to be due to the induction of dephosphor-ylation of the retinoblastoma gene product (Rb) (47). Furthermore,it has been also reported that the treatment of NIH 3T3 cells witha specific inhibitor of glucosylceramide synthase, which inducesceramide accumulation, causes a G2-M cell cycle arrest, possiblymediated by ceramide-induced inhibition of the cyclin-dependentp34cdc2 and Cdk2 kinases (48). Another study has shown that cer-amides specifically inactivates the cyclin-dependent kinase Cdk2through activation of PP1 and PP2A phosphatases (49). Ceramidesalso inactivates protein kinase B/Akt (50). In addition, it has been

FIGURE 10. rCTB inhibits the phosphorylation and the translocation ofPKC� into rafts. A, Jurkat cells (80–100 � 106) were left untreated orincubated with CTB (10 �g/ml) for 30 min. Raft purification was achievedas in Fig. 5B. The nine fractions, usually harvested from the top of theultracentrifuge tube, were pooled as follows: lane E, (9 � 8); lane D, (7 �6); lane C, (5 � 4); lane B, (3 � 2); and lane A, 1. The pooled fractions(lanes A–E) were subjected to SDS-PAGE, transferred onto PVDF mem-branes, and immunoblotted with the indicated antibodies. Data representone of three similar experiments. B, Jurkat cells (80–100 � 106) pretreatedor not with NAC (25 mM) for 1 h or GSH (3 mM) for 1 h were pretreatedor not with pCTB or rCTB (10 �g/ml) for 30 min. Then cells were stim-ulated or not by PMA (10 ng/ml) plus ionomycin (100 nM) for 1 h. Raftisolation was achieved as in Fig. 5B. Upper panel, Fractions B and E weresubjected to SDS-PAGE, transferred onto PVDF membranes, and immu-noblotted with anti-PKC� mAb. Lower panel, Twenty-five microliters offraction B “control” and 65 �l of fraction B “rCTB-treated cells,” to havethe same quantity of PKC�, were subjected to SDS-PAGE, transferredonto PVDF membranes, and immunoblotted with anti-phospho-PKC�(Ser657) Ab. Then membrane was stripped and reblotted with anti-PKC�mAb. Data represent one of three similar experiments.

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demonstrated that endogenous ceramides, produced either by over-expression of bacterial SMase or by daunorubicin treatment, in-hibit mRNA synthesis of telomerase RT and telomerase activityvia inactivation of c-Myc transcription factor (51, 52).

In this study, we observed that rCTB inhibits PKC� phosphor-ylation and prevents its translocation into rafts. PKC� is a pro-growth cellular regulator, and its inactivation can explain cell cyclearrest. PKC� inhibition by rCTB can be easily explained by threeways. First, PKC� is activated by DAG, and because of the inhi-bition of SM synthesis, DAG level is diminished in rCTB-treatedlymphocytes. Consequently, PKC� is less activated in rCTB-treated cells. Second, ceramides are known to inactivate PKC� viaa phosphatase (53, 54). Third, rafts are markedly modified in termsof lipids. Indeed, rCTB-treated lymphocytes possess rafts with lessSM, less cholesterol, but more PtdCho than control cells. It is quiteconceivable that these lipid modifications strongly alter the an-choring of PKC� into these modified rafts because ceramides donot inhibit PMA-induced translocation of PKC� by themselves(53). Collectively, all of these evidences can explain the inhibitionof PKC� by rCTB.

Finally, we observed that rCTB induces the activation of NF-�B. rCTB-induced NF-�B activation is inhibited in FB1-treatedcells, suggesting that SM is required. Furthermore, NAC and GSHprevent rCTB-induced NF-�B activation, it means that SM hydro-lysis i.e., ceramide production is responsible for the rCTB-inducedNF-�B activation. Ceramides are well known to activate NF-�B(55), but the genes involved remain to be elucidated.

AcknowledgmentsWe thank Dr. Laurence Lamy, Dr. Isabelle Foucault, Romain Gallais, andFrank Leporati. We also thank Prof. Cecil Czerkinsky and Dr. FabienneAnjuere for providing us the rCTB. We are also grateful to Alexis de laDaudaf for helpful discussion.

DisclosuresThe authors have no financial conflict of interest.

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