Regulation Θ Kinase CB Activation

Ceramide Inhibits IL-2 Production by Preventing Protein Kinase C-Dependent NF- κB Activation: Possible Role in Protein Kinase C θ Regulation This information is current as of October 1, 2021. Nour Abboushi, Aimee El-Hed, Wissal El-Assaad, Lina Kozhaya, Marwan E. El-Sabban, Ali Bazarbachi, Rami Badreddine, Alicja Bielawska, Julnar Usta and Ghassan S. Dbaibo

J Immunol 2004; 173:3193-3200; ; Downloaded from doi: 10.4049/jimmunol.173.5.3193 http://www.jimmunol.org/content/173/5/3193

References This article cites 42 articles, 28 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/173/5/3193.full#ref-list-1

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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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ceramide Inhibits IL-2 Production by Preventing Protein Kinase C-Dependent NF-␬B Activation: Possible Role in Protein Kinase C␪ Regulation1

Nour Abboushi,2* Aimee El-Hed,2* Wissal El-Assaad,† Lina Kozhaya,* Marwan E. El-Sabban,‡ Ali Bazarbachi,* Rami Badreddine,* Alicja Bielawska,§ Julnar Usta,* and Ghassan S. Dbaibo3*†

The role of the sphingolipid ceramide in modulating the immune response has been controversial, in part because of conﬂicting data regarding its ability to regulate the transcription factor NF-␬B. To help clarify this role, we investigated the effects of ceramide on IL-2, a central NF-␬B target. We found that ceramide inhibited protein kinase C (PKC)-mediated activation of NF-␬B. Ceramide was found to signiﬁcantly reduce the kinase activity of PKC␪ as well as PKC␣, the critical PKC isozymes Downloaded from involved in TCR-induced NF-␬B activation. This was followed by strong inhibition of IL-2 production in both Jurkat T leukemia and primary T cells. Exogenous sphingomyelinase, which generates ceramide at the cell membrane, also inhibited IL-2 production. As expected, the repression of NF-␬B activation by ceramide led to the reduction of transcription of the IL-2 gene in a dose- dependent manner. Inhibition of IL-2 production by ceramide was partially overcome when NF-␬B nuclear translocation was reconstituted with activation of a PKC-independent pathway by TNF-␣ or when PKC␪ was overexpressed. Importantly, neither

the conversion of ceramide to complex glycosphingolipids, which are known to have immunosuppressive effects, nor its hydrolysis http://www.jimmunol.org/ to sphingosine, a known inhibitor of PKC, was necessary for its inhibitory activity. These results indicate that ceramide plays a negative regulatory role in the activation of NF-␬B and its targets as a result of inhibition of PKC. The Journal of Immunology, 2004, 173: 3193–3200.

he sphingolipid ceramide, the backbone of complex Among the targets of NF-␬B, IL-2 is a central regulator of the sphingolipids, has emerged as a regulator of the stress immune response that is produced by T cells upon antigenic or T response by virtue of its ability to induce growth suppres- mitogenic stimulation. Inhibition of IL-2 production achieves sig- sion, induction of cell differentiation, cell cycle arrest, apoptosis, nificant, clinically useful, immunosuppression. IL-2 is regulated and other biologic functions (1). Previous studies had established mostly at the level of transcription of its gene (16). Interaction of by guest on October 1, 2021 that the more complex glycosphingolipids derived from tumors or the TCR/CD3 complex with its specific Ag/MHC together with parasites exhibited immunosuppressive activity (2–6). A role for accessory signals provided by the APC initiate several signaling ceramide in regulating the immune response was suggested based pathways that result in the induction of the IL-2 gene. These in- on several observations, including its role in apoptosis. Addition- clude the activation of protein kinase C (PKC)4 and mobilization ally, in early studies ceramide was found to induce modest secre- of intracellular calcium. The downstream events that lead to the tion of IL-2 (7) and activation of the proinflammatory transcription expression of IL-2 include the activation of a number of specific factor NF-␬B (8–10). However, other groups showed that cer- transcription factors, including NF-␬B, NFAT, Oct-1, AP-1, Ets, amide did not activate NF-␬B (11–14). Because NF-␬B plays a and a CD28 responsive factor (17). These factors cooperate to critical role in the regulation of many immune and inflammatory responses by inducing several cytokines, cytokine receptors, form a multifactor complex that binds the enhancer region in a growth factors, and leukocyte adhesion molecules (15), clarifying stable manner and initiates transcription. Inhibition of one of these its regulation by ceramide became important for understanding the factors is sufficient to markedly depress IL-2 production. This is role of ceramide in the immune response. demonstrated by the ability of two widely used immunosuppres- sants, cyclosporin A and FK-506, to inhibit IL-2 production by blocking NF-AT activation. † ‡ Departments of *Biochemistry, Pediatrics, and Human Morphology, American Uni- Therefore, we decided to examine the effects of ceramide on the versity of Beirut, Faculty of Medicine, Beirut, Lebanon; and §Department of Bio- chemistry and Molecular Biology, Medical University of South Carolina, Charleston, pathways that lead to the activation of NF-␬B and its target, IL-2. SC 29425 The results indicate that ceramide is a potent inhibitor of IL-2 Received for publication July 14, 2003. Accepted for publication June 30, 2004. induction and that these effects appear to be mediated by the The costs of publication of this article were defrayed in part by the payment of page ability of ceramide to specifically inhibit PKC-mediated acti- charges. This article must therefore be hereby marked advertisement in accordance ␬ with 18 U.S.C. Section 1734 solely to indicate this fact. vation of NF- B. 1 This work was supported by Grant 0090859 from the National Science Foundation (to G.D.), The Diana Tamari-Sabbagh Foundation, and the Lebanese National Coun- cil for Scientific Research. 2 N.A. and A.E.-H. contributed equally to this work. 4 Abbreviations used in this paper: PKC, protein kinase C; C2-ceramide, N-acetyl- 3 Address correspondence and reprint requests to Dr. Ghassan Dbaibo, Department of sphingosine; C6-ceramide, N-hexanoylsphingosine; FB1, fumonisin B1; OPA, Pediatrics, American University of Beirut, P.O. Box 113/6044/B21, Beirut, Lebanon. O-phthalaldehyde; PDMP, D,L-threo-1-phenyl-2-decnoylamino-3-morpholino-1-pro- E-mail address: [email protected] panol; RPA, RNase protection assay.

Materials and Methods Sphingosine measurement Cell culture Lipids were extracted by chloroform/methanol (2/1, v/v), and dihydrosphingosine (1 nmol) was added to the extract as an internal standard. Jurkat E6-1 leukemic T cells were obtained from American Type Culture The aqueous and cellular layers were re-extracted with chloroform/meth- Collection (Manassas, VA) and were grown in RPMI 1640 (Invitrogen Life anol (2/1, v/v), and chloroform layers from the two rounds of extraction Technologies, Paisley, U.K.) supplemented with 10% FCS (Invitrogen Life were combined and evaporated to dryness under N , then resuspended in Technologies). Jurkat cells were treated at a concentration of 1.5 ϫ 106 2 200 ␮l of chloroform and 800 ␮l of 0.125 M methanolic KOH and incu- cells/ml. PHA blasts were prepared according to standard protocols. All bated for 75 min at 37°C. The chloroform layer was combined with 1.4 ml treatments were performed in complete RPMI 1640 medium supplemented of chloroform and washed twice with 400 ␮lofHO, then dried under N with 10% serum. N-acetylsphingosine (C -ceramide), N-hexanoylsphin- 2 2 2 before O-phthalaldehyde (OPA) derivatization of sphingosine was per- gosine (C -ceramide), and dihydrosphingosine were prepared as described 6 formed as previously described (21). After incubation for 20 min at room previously (18). Cell viability studies were performed using the trypan blue ␮ method. Assessment of apoptosis was by annexin V labeling (Roche, temperature in the dark, 300 l of methanol/5 mM potassium phosphate Mannheim, Germany). buffer (90/10, v/v), pH 7.0, was added to the sample, and an aliquot of 100 ␮l was injected into the HPLC. HPLC analyses were conducted using an ␮ ϫ Quantification of IL-2 HP1100 model with C18 Ultrasphere ODS (5- m column, 4.6 mm 25 cm; Beckman Coulter, Fullerton, CA) with a 3-cm Hypersil ODS guard An IL-2 ELISA kit (R&D Systems, Minneapolis, MN) was used to quan- column. A linear gradient system, at a flow rate of 1 ml/min, was used, tify IL-2 production by treated cells, as indicated in the manufacturer’s starting from methanol/5 mM potassium phosphate buffer, pH 7.0 (90/10 instructions. (v/v) to 98/2 (v/v) at 30 min). Elution of the fluorescent OPA-sphingosine Total cellular RNA extraction peaks was detected using an HP1046A fluorescent detector with an exci- tation wavelength of 340 nm and an emission wavelength of 455 nm. The Total cellular RNA from treated or control cells was extracted using the retention times of sphingosine and dihydrosphingosine were 13 and 15.5

RNeasy Total RNA Mini kit (Qiagen, Hilden, Germany) as instructed by min, respectively. Downloaded from the manufacturer. PKC activity Quantification of mRNA by RNase protection assay (RPA) Cells were seeded in complete RPMI 1640 medium at a concentration of The RiboQuant Multiprobe RPA (BD Pharmingen, San Diego, CA) was ϫ 5 ␮ 7 10 cells/ml. The cells were preincubated with 15 MC6-ceramide used to detect and quantify IL-2 transcripts. The probe set consisted of a before stimulation with 15 ng/ml PMA for 20 min. Harvested cells were DNA template set of human cytokines. The templates were used for T7 washed once with 1ϫ cold PBS and suspended in 0.5 ml of cold homog- 32 polymerase-directed synthesis of a high specific activity, [␣- P]UTP-la- enization buffer (50 mM Tris-HCl, pH 7.5, containing 0.3% (w/v) 2-ME, 5 http://www.jimmunol.org/ beled, antisense RNA. Total RNA extracted from the cells was hybridized mM EDTA, 10 mM EGTA, and 50 ␮g/ml PMSF). Cells were lysed by with the labeled probe for 16 h in a heat block at 56°C according to the sonication on ice with four rounds of 15-s duration, vortexed, and then standard assay protocol. The RNA probe hybrid solution was then treated centrifuged at 500 ϫ g for 5 min at 4°C to remove unlysed cells and nuclei with RNase to digest ssRNA. The protected probe/mRNA hybrids were run (pellet). A fraction of the supernatant was taken as the total lysate, and the on a denaturing polyacrylamide gel and detected by autoradiography. The rest was centrifuged at 100,000 ϫ g for 40 min at 4°C to separate the quantity of each mRNA species was determined by the intensity of the cytosol from the membrane fractions. PKC isozyme activities were mea- band of the protected probe fragment. A lane was run on the gel with sured by a PKC enzyme assay system purchased from Amersham Bio- undigested probes that served as markers. From these markers, a standard sciences (Arlington Heights, IL) using isozyme-specific peptide substrates curve of migration distance vs log nucleotide length was plotted. This from Amersham Biosciences (PKC␣) and Calbiochem (PKC␪; La Jolla, curve was used to identify the RNase-protected bands on the gel corre- CA) as recommended by the manufacturer. The phosphorylated peptide sponding to different cytokines, including IL-2. was separated on binding paper, and 5% (v/v) acetic acid was used to wash by guest on October 1, 2021 32 EMSA the papers. P incorporated into the synthetic peptides was detected with a scintillation counter, and the counts were normalized to protein levels Nuclear extracts and EMSA were performed as described previously (11) determined using the Bradford protein assay. using NF consensus oligonucleotides (Santa Cruz Biotechnology, Santa For experiments using immunoprecipitated PKC␪, cells were lysed in 1 Cruz, CA). The specific bands were determined by competition experi- ml of lysis buffer containing 20 mM HEPES (pH 7.6), 250 mM NaCl, 1 ments using mutant oligonucleotides that did not bind to the specific tran- mM EDTA, 20 mM ␤-glycerophosphate, 1% Nonidet P-40, 20 mM p- scription factor (e.g., NF-␬B) in question or a 50-fold excess of unlabeled nitrophenyl phosphate, 0.1 mM Na3VO4, 1 mM DTT, 1 mM PMSF, and specific oligonucleotide. The specificity of the bands was also confirmed 1% protease inhibitor mixture (Roche) for1honice. The lysate was using specific Abs to the transcription factor components, e.g., anti-p50 or centrifuged at 16,000 ϫ g for 20 min at 4°C. A total of 2 mg of protein was anti-p65 for NF-␬B or anti-Fos and anti-Jun for AP-1 (Santa Cruz Bio- then incubated with 2 ␮gofPKC␪ Abfor1hat4°C, followed by overnight technology) in the incubation step, which resulted in a supershift of the incubation with 30 ␮l of protein G agarose. Samples were washed four specific band due to the bound Ab. times with lysis buffer and twice with kinase buffer. The beads were then incubated in kinase buffer containing [␥-32P]ATP and substrate to assess Luciferase assay activity. ␪ The pCEFL-PKC plasmid was a gift from Dr. S. Shaw (National Cancer Statistical analyses Institute, Bethesda, MD). The following luciferase reporter plasmids were used: NF-␬B (Stratagene, La Jolla, CA), AP-1 and NF-AT (gifts from Dr. Quantitative data are expressed as the mean Ϯ SD. Significant differences T. A. Chatila, Washington University, St. Louis, MO), and IL-2 (a gift among groups were examined by one-way ANOVA, followed by Bonfer- from Dr. A. Veillette, McGill University, Montreal, Canada). Cells were roni’s post-hoc test. A value of p Ͻ 0.05 was considered significant. seeded at 8 ϫ 105 cells in 0.5 ml of medium with 10% FBS and placed in a 24-well plate. Transfection with the respective plasmids was performed ␮ Results by adding 2 g of plasmid DNA dissolved with Lipofectamine (Invitrogen ␬ Life Technologies) in medium to each well in a dropwise fashion while Ceramide inhibits PMA-induced NF- B activation gently shaking the plate. The cells were incubated for 4 h before stimula- Jurkat cells costimulated with PMA and PHA were used to explore tion with PMA with or without C6-ceramide for 1.5 h. For IL-2 reporter experiments, cells were transfected by electroporation and treated simi- the effects of ceramide on IL-2 secretion. Whereas PMA functions larly, except PHA was included with PMA and incubation was performed mostly by activating several members of the PKC family, PHA for 24 h. At the end of the incubation, the Bright Glo luciferase assay provides complementary accessory signals, such as calcium mo- reagent (Promega, Madison, WI) was added, and luminescence was mea- bilization. Previously, we demonstrated that exogenous cell-per- sured using a luminometer. meable ceramides inhibit PMA-induced NF-␬B activation in these Ceramide measurement cells (22). To verify whether PHA cotreatment affected the inhib- ␬ Lipids were extracted from pelleted cells by the method of Bligh and Dyer itory effects of ceramide on PMA-induced NF- B, cells were stim- (19). Ceramide levels were measured by a modified diglycerol kinase assay ulated with PMA/PHA in the presence of cell-permeable synthetic ␬ as described previously (20) and normalized to lipid phosphate content. C6-ceramide, and specific NF- B-DNA binding after 1.5 h was The Journal of Immunology 3195

evaluated. Fig. 1A shows that there was signiﬁcant dose-dependent ␬ inhibition of NF- BbyC6-ceramide. Treatment of cells with PHA alone did not activate NF-␬B, whereas PMA alone potently

activated it and this was strongly inhibited by C6-ceramide (Fig. 1B). These results suggested that PHA cotreatment did not modulate the inhibitory effects of ceramide on the PMA-induced pathway of NF-␬B. Speciﬁc DNA binding of NFAT, AP-1, and Ets after stimulation with PMA was also assayed by EMSA. Comparison of PMA-

treated extracts in the presence or the absence of C6-ceramide showed no difference in binding of any of these transcription factors to their speciﬁc cognate sequences at 90 min (Fig. 1C). Thus, the effects of ceramide at this early time point (1.5 h) appeared to be limited to the inhibition of PMA-induced NF-␬B. This was veriﬁed using luciferase reporter plasmid constructs driven by NF-␬B, AP-1, or NF-AT. Transient transfection of Jurkat cells with these constructs showed that PMA treatment alone for 1.5 h activated NF-␬B-driven luciferase expression, but not that driven

by AP-1 or NF-AT (Fig. 1D). Cotreatment with C6-ceramide (20 ␮M) resulted in signiﬁcant inhibition of NF-␬B-driven activity. Downloaded from Ceramide inhibits PKC␪ and PKC␣ activities Several PKC isozymes are involved in TCR-induced stimulation. PKC␪ recently emerged as a critical regulator of NF-␬B activation after TCR ligation, where it is rapidly recruited to the site of TCR clustering (23). Its loss abrogates TCR-induced NF-␬B activation http://www.jimmunol.org/ in mature T cells (24). Ceramide differentially modulates various PKC isozymes (14, 25–29), but its effects on PKC␪ are unknown. This was explored in Jurkat cells, where PMA-induced PKC␪ and PKC␣ activities were measured in the presence or the absence of ceramide. It was found that ceramide potently inhibited the membrane-associated activation of both isozymes after stimulation with PMA for 20 min (Fig. 2, A and B). These ﬁndings indicated that ceramide was able to inhibit the key PKC isozyme involved in by guest on October 1, 2021 early T cell activation in addition to its previously reported ability to inhibit classical PKC isozymes.

Fas-induced ceramide results in PKC␪ inhibition Fas is known to play a central role in activation-induced T cell death. Previous studies implicated ceramide as one of the signaling pathways stimulated by Fas during apoptosis (30). To determine whether the observed effects with exogenous ceramide were rele- vant to normal T cell regulation, ceramide levels and PKC␪ activity were measured after T cell activation in the context of Fas stimulation (Fig. 2, C and D). In Fas-stimulated cells, it was found that ceramide levels increased significantly and that treatment with FIGURE 1. Ceramide inhibits PMA-induced NF-␬B activation. A, Cer- PMA/PHA at 8 or 14 h after Fas stimulation resulted in a slight amide inhibits PMA/PHA-induced activation of NF-␬B in a dose-depen- further increase in ceramide. As shown previously (30), when fu- dent manner. Jurkat cells were treated with vehicle, PMA (10 ng/ml) and PHA (10 ␮g/ml; P/P), or TNF-␣ (5 nM) with the indicated concentrations monisin B1 (FB1), an inhibitor of de novo ceramide synthesis was of C6-ceramide for 90 min. EMSA was performed on nuclear proteins added to cells before Fas stimulation, ceramide accumulation was using specific NF-␬B-labeled oligonucleotide probe. The specific NF-␬B ␪ significantly inhibited (Fig. 2C). When PKC activity was exam- band is indicated as well as a nonspecific band (n.s.) and free unbound ined, it was found that PMA/PHA treatment could not induce probe. TNF-␣ was used as a positive control for NF-␬B activation. B, PKC␪ activity 8 and 14 h after Fas stimulation (Fig. 2D). When PMA, but not PHA, induced NF-␬B activation that was blocked by cer- ceramide synthesis was inhibited by FB1, Fas stimulation no amide. Jurkat cells were treated as described in A, except that PMA and ␮ longer inhibited PKC␪ activity. These results suggested that Fas- PHA treatments were separate, and C6-ceramide was used at 20 M. C, induced ceramide synthesis was necessary for Fas-induced inhibi- Ceramide does not affect DNA binding of AP1, NF-AT, or ETS. Jurkat tion of PKC␪. cells were treated with vehicle or PMA (10 ng/ml) and the indicated concentrations of C6-ceramide for 90 min. EMSAs were performed on equal Synthetic or natural ceramides inhibit IL-2 production amounts of nuclear protein extracts using transcription factor-specific oli- ␬ ␪ ␬ gonucleotide probes. D, Ceramide inhibits PMA-induced NF- B luciferase The inhibition of PKC and NF- B by ceramide led us to deter- reporter activity. Jurkat cells were transiently transfected with reporter con- mine the effects of this inhibition on IL-2 secretion. When co- structs, as indicated, before treatment with PMA with (^) or without (Ⅺ) stimulated with PMA and PHA, Jurkat cells produced large ceramide for 1.5 h. Luciferase activity in transfected cells was measured as amounts of IL-2 in a time-dependent manner, reaching a plateau of described in Materials and Methods and is presented as relative to activity 3300 Ϯ 417 pg/ml by 24 h (data not shown). Jurkat cells were in control untreated cells. 3196 CERAMIDE INHIBITS IL-2 PRODUCTION

pretreated with C6-ceramide for 2 h before stimulation with PMA/ PHA. Signiﬁcant, dose-dependent inhibition of IL-2 production was observed (Fig. 3A). Notably, this effect was observed in the absence of ceramide-induced cell death, as assayed at 24 h by both trypan blue uptake and annexin V labeling (data not shown). To determine whether our observations in Jurkat cells could be extended to normal T cells, PHA blasts were isolated from human PBMCs and subjected to similar treatments. When additionally treated with PMA, PHA blasts produced IL-2 up to a level of 2200 pg/ml within 3–6 h. When PHA blasts were pretreated for 2 h with

increasing concentrations of C6-ceramide before stimulation with PMA, profound inhibition of IL-2 production was observed (Fig. 3B). Additionally, there was no ceramide-induced cell death observed in these cells (data not shown). Similar results were ob-

tained when C2-ceramide was used instead of C6-ceramide (data not shown). These ﬁndings suggested that our results were not Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 2. Exogenous or Fas-generated ceramide inhibits PKC. A and B, Ceramide inhibits PMA-induced PKC␪ (A) and PKC␣ (B) activation. ␮ Jurkat cells were preincubated with 15 MC6-ceramide before stimulation with 15 ng/ml PMA for 20 min. Specific PKC activity was determined in FIGURE 3. Inhibition of IL-2 production by C6-ceramide in Jurkat the total lysate as well as in the cytoplasmic and membrane fractions as cells. A, Ceramide inhibits IL-2 production. Cells were pretreated with the indicated. C, Fas-generated ceramide is inhibited by FB1. Jurkat cells were indicated concentrations of C6-ceramide or ethanol vehicle for 2 h before stimulated with Fas Ab for 8 or 14 h before treatment with PMA/PHA for stimulation with PMA/PHA. IL-2 levels were measured in the supernatant 30 min as indicated. FB1 (100 ␮M) was added with Fas Ab treatment as at 24 h and presented as a percentage of PMA/PHA-treated cells. B, Inhi- indicated. Ceramide levels in cell lipid extracts were measured as described bition of IL-2 production by C6-ceramide. PHA blasts from healthy donors ϫ 6 in Materials and Methods and normalized to lipid phosphate. D, Fas-gen- were seeded at 1.5 10 cells/ml in 10% FCS and treated with PMA (10 erated ceramide inhibits PKC␪ activity, and this is reversed by FB1. Cells ng/ml) in the presence of vehicle or C6-ceramide at the indicated concen- were treated as described in C, and specific PKC␪ activity was measured trations. IL-2 was measured at 16 h. C, Inhibition of IL-2 production by as described in A. Data shown are representative of two or three identical bacterial sphingomyelinase. Jurkat cells were treated with PMA/PHA in experiments with similar results. the presence of vehicle or the indicated concentrations of bacterial sphingomyelinase. IL-2 levels were measured and are presented as a percentage ,ء .of the PMA/PHA-treated control value. Error bars represent the SD Statistically significant difference compared with the control. The Journal of Immunology 3197

unique to the Jurkat cell line, but could also be produced in pri- vested 24 h after treatment, lysed with hypotonic buffer to release mary T cells. intracellular IL-2, and combined with their respective medium. The ability of natural ceramide, which is hydrophobic and dif- Measurement of IL-2 revealed that there was no evidence of in- ﬁcult to deliver to cells, to produce the same effects as synthetic tracellular trapping of IL-2 after ceramide treatment (data not ceramide was examined next. Exogenous bacterial sphingomyeli- shown). This indicated that ceramide did not inhibit the secretion nase, which hydrolyzes membrane sphingomyelin, was used to of formed IL-2. generate natural ceramide at the cell membrane. PMA/PHA-stim- In addition to the early signals that are responsible for initiating ulated Jurkat cells were cotreated with a range of concentrations of IL-2 production, late signals are required for its sustained produc- sphingomyelinase, as shown in Fig. 3C. Inhibition of IL-2 production, e.g., ERK activation, which also activates NF-␬B (32). There- tion occurred at very low concentrations of sphingomyelinase. fore, it became important to examine whether ceramide was inter- These studies indicated that the observed effects with synthetic fering with these late signals. This was important because ceramide and Fas-generated, de novo synthesized ceramide could ceramide was shown to modulate ERK activity in some systems also be produced by natural ceramide generated at the cell (33). The effect of delaying the addition of ceramide to stimulated

membrane. cells was examined. The addition of C6-ceramide to PMA/PHA- treated cells was made several hours before, simultaneously, or Ceramide inhibits IL-2 mRNA transcription delayed for 1, 2, 4, or 6 h after stimulation. As shown in Fig. 5, The ability of ceramide to inhibit PKC-induced NF-␬B activation inhibition of IL-2 production was maximal when cells were pre- suggested that ceramide might be interfering with transcription of treated with ceramide, but also occurred when ceramide was added ␬ the IL-2 gene, which is NF- B dependent. The effects of C6-cer- at the same time as PMA/PHA or when its addition was delayed Downloaded from amide on the level of IL-2 mRNA were examined after3hof for up to 2 h. Further delay in the addition of ceramide resulted in

stimulation. As shown in Fig. 4A,C6-ceramide inhibited the pro- the gradual loss of inhibition. These experiments indicated that duction of IL-2 mRNA transcripts in a dose-dependent manner ceramide was inhibiting an early event (within the ﬁrst 2 h) im- without affecting the housekeeping gene GAPDH. Additionally, portant in IL-2 regulation. the transcript of the NF-␬B-inducible IL-2R␣ gene was potently inhibited (Fig. 4A). Also, the time-dependent production of IL-2 Ceramide inhibition of PMA-induced NF␬B is bypassed mRNA was inhibited by ceramide at all times examined (Fig. 4B). by TNF-␣ http://www.jimmunol.org/ These experiments indicated that ceramide was inhibiting IL-2 at NF-␬B is activated by many pathways. Some are PKC dependent, the level of transcription. whereas others, such as TNF-␣, are PKC independent (34, 35). ␣ Ceramide inhibits an early event in IL-2 production Previously, it was shown that ceramide does not inhibit TNF- - induced NF-␬B activation (22). The ability of TNF-␣ treatment to Ceramide has been reported to perturb secretory functions in other overcome the inhibitory effects of ceramide on PMA-induced models (31). Therefore, it was important to determine whether NF-␬B activation was examined. When Jurkat cells treated with ceramide inhibited the secretion of formed IL-2 in addition to in- PMA and C6-ceramide for 1.5 h were additionally treated with hibiting IL-2 transcription. Jurkat cells were stimulated with PMA/ TNF-␣ at 2 nM, NF-␬B activation was reconstituted, and the ce- by guest on October 1, 2021 PHA in the presence or the absence of C6-ceramide and were har- ramide-induced inhibition was bypassed (Fig. 6A). To determine whether the reconstitution of NF-␬B activation by TNF-␣ allowed the production of IL-2 in the presence of ceramide, Jurkat cells

were treated with PMA/PHA and C6-ceramide, with or without TNF-␣ for 14 h. As shown in Fig. 6B, IL-2 production was partially restored by the addition of TNF-␣. These results suggested that ceramide modulated its inhibitory effect on NF-␬B mobilization by inhibition of the PKC pathway of NF-␬B activation and that TNF-␣ could partially overcome this inhibition.

FIGURE 4. Ceramide inhibits IL-2 mRNA synthesis. A, Dose-depen- ␣ dent inhibition of IL-2 and IL-2R mRNA synthesis by C6-ceramide. Ju- rkat cells were treated with PMA/PHA and C6-ceramide at the indicated concentrations. RNA was collected at 3 h, and the speciﬁc mRNA species FIGURE 5. Ceramide inhibits an early event in IL-2 production. Jurkat ␮ were detected as described in Materials and Methods. B, Time-dependent cells were prepared as described in Fig. 1. C6-ceramide (20 M) was added inhibition of IL-2 mRNA by ceramide. Cells were treated with PMA/PHA at the indicated time points relative to PMA/PHA treatment, and IL-2 lev- ␮ and C6-ceramide (20 M). Total RNA was extracted at the indicated time els were measured at 16 h. Data are representative of three different ex- Statistically different values ,ء .points. GAPDH is a housekeeping gene that serves as a control for tech- periments. Error bars represent the SD nical variations. compared with the control. 3198 CERAMIDE INHIBITS IL-2 PRODUCTION

IL-2 luciferase activity was measured at 24 h, it was found that overexpression of PKC␪ overcame the inhibition that was induced by ceramide and partially restored IL-2 production.

Effects of ceramide are not due to its metabolism to gangliosides or sphingosine The previously described immunosuppressive effects of sphingolipids were attributed mostly to complex glycosphingolipids. Therefore, it became important to determine whether the observed IL-2 inhibition by ceramide was due to its metabolism to gangliosides that, in turn, were producing the inhibitory effects. The UDP- glucosyl:ceramide transferase inhibitor, D,L-threo-1-phenyl-2-de- canoylamino-3-morpholino-1-propanol (PDMP), was used. PDMP prevents the synthesis of cerebrosides and gangliosides from ceramide and leads to the accumulation of cellular ceramide (36). Jurkat cells were treated with PDMP (50 ␮M), resulting in an increase in cellular ceramide levels by 4-fold within 6 h (data not shown). Jurkat cells were pretreated with PDMP, either alone or in

combination with C6-ceramide, for 2 h before stimulation. PDMP treatment alone prevented IL-2 production (Fig. 7A). This indi- Downloaded from cated that the elevation of natural cellular ceramide induced by

PDMP was sufficient to produce inhibition. When C6-ceramide treatment was combined with PDMP, complete inhibition of IL-2 production was still observed. Indeed, PDMP potentiated the inhibitory effects of the lower concentrations of ceramide. These findings indicated that conversion of exogenously added ceramide http://www.jimmunol.org/ to gangliosides was not necessary to produce IL-2 suppression and that the natural ceramide that accumulated in response to treatment with PDMP cooperated with the exogenously added synthetic ceramide to produce IL-2 inhibition. Sphingosine is another sphingolipid that has been demonstrated to inhibit PKC, albeit by a different mechanism (37). Because sphingosine is structurally related to ceramide (it lacks the fatty acyl chain), and both can be converted into one another by single- by guest on October 1, 2021 step biochemical reactions, it became important to determine whether ceramide produced its effects by hydrolysis to sphingosine. First, the ability of sphingosine itself to produce effects on NF-␬B and IL-2 comparable to those of C -ceramide was studied. FIGURE 6. Ceramide-mediated inhibition of NF-␬B and IL-2 activa- 6 Jurkat cells were activated with PMA and cotreated with sphin- tion is bypassed by TNF-␣ or PKC␪ overexpression. A, TNF-␣ restores NF-␬B binding in the presence of ceramide. Jurkat cells were treated with gosine. Sphingosine, which is more cell permeable than ceramide, ␣ ␮ produced inhibition of NF-␬B at high concentrations (Fig. 7B), vehicle, PMA (10 ng/ml), TNF- (2 nM), or C6-ceramide (20 M) as indicated for 90 min. EMSA was performed on nuclear extracts, and the followed by inhibition of IL-2 at a later time point. Sphingosine specific NF-␬B band is indicated. The nonspecific band (n.s.) and free cellular levels after C6-ceramide treatment of Jurkat cells were probe are indicated. B, TNF-␣ partially reconstitutes IL-2 secretion in the measured. Because the first 6 h after ceramide addition were crit- presence of ceramide. Jurkat cells were treated with PMA/PHA as de- ical for its inhibitory effects, sphingosine levels were measured scribed in Fig. 1 with the indicated concentrations of C6-ceramide and during that time frame and were found not to change in response ␣ TNF- for 14 h. The IL-2 concentration was measured from supernatant. to C -ceramide treatment (Fig. 7C). Thus, although sphingosine ␪ 6 C, PKC overexpression partially overcomes ceramide-mediated inhibition was capable of producing effects on IL-2 production similar to of IL-2 luciferase reporter activity. Jurkat cells were transiently cotrans- those of ceramide by virtue of its ability to inhibit PKC, the effects fected with IL-2 luciferase reporter construct with either vector (Ϫ)or ␪ ϩ of ceramide did not appear to be mediated by sphingosine. PKC ( ) constructs and were treated with PMA/PHA and C6-ceramide as indicated. Luciferase activity was measured as described in Fig. 1D. Discussion The aim of this study was to clarify the role of ceramide in the regulation of NF-␬B and NF-␬B-dependent IL-2 production. Our ␪ Overexpression of PKC partially overcomes the inhibitory findings are in agreement with previous observations showing that effects of ceramide ceramide does not activate NF-␬B (11–13). They are also in agree- ␪ To determine whether PKC inhibition by ceramide contributed to ment with a recent study showing that C2-ceramide inhibits the the observed effects on IL-2 production, Jurkat cells were tran- production of IL-2 in rat splenocytes, although comparatively siently transfected with PKC␪ or vector in addition to either higher concentrations of ceramide were used, and significant ap- NF-␬B or IL-2 luciferase reporter constructs and were subse- optosis was observed (38). Based on the current study, the inhib- quently treated with PMA/PHA in the presence or the absence of itory effects of ceramide on IL-2 and NF-␬B appear to be mediated ceramide (Fig. 6C). PKC␪ overexpression resulted in a 2-fold in- by inhibition of PKC, particularly PKC␪. Additionally, and in the crease in NF-␬B-driven luciferase activity in response to PMA at context of Fas stimulation, this inhibition might have a role during 3 h compared with vector-transfected cells (data not shown). When Fas-induced apoptosis by neutralizing a survival signal for T cells. The Journal of Immunology 3199

APCs (40). Also, conventional PKC isoforms as well as PKC␪ were demonstrated to synergize with calcineurin to activate NF-␬B and the IL-2 promoter (41, 42). The mechanisms by which PKC activates NF-␬B are not clear, but direct or indirect activation of I␬B kinases are possibilities. Our results show that ceramide specifically inhibits PKC-mediated, but not TNF-␣-induced, NF-␬B activation. Indeed, this inhibition can be bypassed by the overexpression of PKC␪ or the addition of TNF-␣. This indicates that ceramide does not inhibit the TNF-␣-induced I␬B kinases and that it has no direct effect on NF-␬B, such as modulating its DNA- binding activity or inducing its proteolysis. Whether ceramide inactivates other kinases that are downstream of PKC remains to be determined. Ceramide appears to differentially modulate various PKC isoforms. A recent study showed that ceramide inactivates PKC␣ in Molt-4 and Jurkat cells (26). This inactivation appears to be mediated by dephosphorylation of PKC␣ and without interfering with its ability to translocate from the cytosol to the membrane that normally occurs after its activation. This is in contrast to the direct inhibitory effects of sphingosine on PKC that result from compet- Downloaded from itive, but reversible, inhibition of the regulatory domain (37). Also, ␤ inhibition of PKC II by ceramide was demonstrated in Molt-4 cells (26). In contrast, ceramide has been shown to activate PKC␨ (25). Our findings in this study show that ceramide, either exogenously added or generated by Fas ligation, inhibits PKC␪ activation. This is the first evidence that ceramide regulates this PKC http://www.jimmunol.org/ isoform. Additional studies evaluating the effects of ceramide on PKC␪ regulation are ongoing in our laboratory. Our results suggest that the inhibition of PKC-induced NF-␬B activation by ceramide is sufficient to significantly disable IL-2 transcription and that this is partially overcome by overexpression of PKC␪. However, other mechanisms, such as the negative regulation of other necessary transcription factors, cannot be com- pletely ruled out based on our experiments. As mentioned above, by guest on October 1, 2021 optimal IL-2 transcription occurs when all the necessary transcription factors are present to form a complex that interacts with the promoter (17). If one of these factors is inhibited, the induction of the gene is down-regulated. FIGURE 7. The effects of ceramide are not due to its metabolism to Previous studies have shown that glycosphingolipids cause in- gangliosides or sphingosine. A, Effects of PDMP treatment on IL-2 pro- hibition of the proliferation of activated T cells. Additionally, sev- duction and inhibition by ceramide. Jurkat cells were pretreated with the eral parasites with a rich and complex array of glycosphingolipids

indicated concentrations of C6-ceramide alone, PDMP alone, or their com- in their membranes may evade the immune response not only by bination for 2 h before stimulation with PMA/PHA. IL-2 levels were mea- immune suppression mediated by the glycosphingolipids, but also sured in the supernatants at 24 h and are shown as a percentage of the by the different ceramides present that may specifically down-reg- PMA/PHA-treated control value. Data represent at least three separate ex- ulate T cell activation (5, 6, 43). Our studies suggest that bacterial Statistically significant difference ,ء .periments. Error bars show the SD sphingomyelinases secreted by pathogenic bacteria may induce the compared with the control. B, Sphingosine inhibits PMA-mediated NF-␬B activation. Jurkat cells were treated with PMA (10 ng/ml) or vehicle with generation of endogenous ceramide by host cells, leading to local or without the indicated concentrations of sphingosine. Nuclear extracts immunosuppression. were prepared at 90 min to evaluate NF-␬B activation as described above. These findings support a new role for ceramide as a putative

C, Lack of sphingosine generation after C6-ceramide treatment. Jurkat cells immunosuppressor lipid that is independent of, but complementary were seeded at 0.5 ϫ 106 cells/ml in complete medium containing 10% to, its role in the stress response. Additional studies will be needed ␮ FCS and rested overnight. They were treated with C6-ceramide (20 M), to explore this role. The emergence of PKC␪ as the central PKC and lipids were harvested at the indicated time points. The cell pellet was isoform involved in TCR signaling and our discovery that cer- washed carefully before measurements of C6-ceramide and sphingosine amide can inhibit its activity provide an opportunity for better were performed as described in Materials and Methods. The results rep- understanding the role of ceramide in regulating the immune resent three different experiments. The lipid content is shown on the y-axis. response. Acknowledgments Until recently, the relative importance of the different PKC iso- Part of this work was performed at the Research Core Facility of American forms in IL-2 production was not known, although both classic (␣, University of Beirut. We thank Dr. George Nemer for helpful discussions. ␤, and ␥) and novel (␪ and ⑀) isoforms were implicated (24, 39). Recent evidence has shown that PKC␪ is critically important for References ␬ 1. Hannun, Y. A., and L. M. Obeid. 2002. The ceramide-centric universe of lipid- TCR-mediated NF- B activation, and that it is the only isoform of mediated cell regulation: stress encounters of the lipid kind. J. Biol. Chem. PKC that is translocated to the site of contact between T cells and 277:25847. 3200 CERAMIDE INHIBITS IL-2 PRODUCTION

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