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* Institute of General Surgery, Jinling Hospital, Nanjing 210002, China
National Chromatographic Research and Analysis Center, Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China
Published, JLR Papers in Press, June 1, 2005. DOI 10.1194/jlr.M500033-JLR200
1 To whom correspondence should be addressed. e-mail: liqiurong{at}yahoo.com
| ABSTRACT |
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These data demonstrate for the first time that DHA alters the lipid composition of membrane microdomains and suppresses IL-2 receptor signaling in T-cells. Thus, our data provide evidence for a functional modification in lipid rafts by DHA treatment and explain PUFA-mediated immunosuppressive effects.
Abbreviations: DHA, docosahexaenoic acid, 22:6n-3; EPA, eicosapentaenoic acid; IL, interleukin; JAK, Janus kinase; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin; STAT, signal transducer and activator of transcription
Supplementary key words polyunsaturated fatty acids lipid rafts fatty acid composition Janus kinase-signal transducer and activator of transcription signaling pathway
| INTRODUCTION |
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Lipid rafts are specialized in membrane microdomains, which are insoluble in nonionic detergents and can be isolated as detergent-resistant membrane domains. A large number of signaling proteins are targeted to lipid rafts. Therefore, lipid rafts serve as signaling platforms to facilitate efficient and specific signal transduction in living cells. Some findings have indicated that several proteins involved in the signaling pathway via the T-cell receptor, B-cell receptor, and IgE receptor are within lipid raft compartments (9, 10). Many of the critical signaling components involved in the T-cell receptor-mediated signal pathway are localized in rafts, including Fyn, Lck, and LAT Src family kinases (1012). G-proteins (13) and endothelial nitric oxide synthase (14) are also enriched in rafts. PUFA supplementation inhibited T-lymphocyte activation by modifying detergent-insoluble membrane domains and displacing signaling proteins from lipid rafts in T-cells (15).
The interleukin-2R (IL-2R) subunits complex (
, ß, and
c) is involved in both proliferative and activation-induced cell death signaling of T-cells. The high-affinity IL-2 receptors consist of the IL-2R
chain, a 55 kDa transmembrane glycoprotein with 251 amino acids, and IL-2Rß and IL-2R
c, which contribute to IL-2 binding and mediate signal transduction. Binding of IL-2 to the IL-2R results in the activation of Janus kinase-1 (JAK1) and JAK3 and their phosphorylation and activation (16). Subsequent phosphorylation of tyrosine residues in receptor chains results in the recruitment of the signal transducer and activator of transcription (STAT) (17). JAK-mediated phosphorylation of STAT proteins leads to their translocation to the nucleus, where STAT proteins regulate gene transcription. Because signals mediated through IL-2R are so important in the activation of T-cells, in our previous studies we revealed that IL-2R
, IL-2Rß, and IL-2R
c are associated with lipid rafts of T-cells and that eicosapentaenoic acid (EPA) could disrupt lipid raft integrity and alter the distribution of IL-2R
, IL-2Rß, and IL-2R
c in the plasma membrane and impair IL-2R signaling (our unpublished data).
In the present study, we examined the immunomodulatory effects of docosahexaenoic acid (DHA, 22:6n-3), the longest, most unsaturated fatty acid commonly found in membranes, by analyzing the raft lipid composition and the expression of key molecules of IL-2R signaling in both rafts and soluble fractions. Thus, our data provide evidence for a functional modification in lipid rafts by DHA treatment and explain PUFA-mediated immunosuppressive effects.
| EXPERIMENTAL PROCEDURES |
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antibody, IL-2Rß-specific goat anti-human IL-2Rß antibody, and goat anti-human common
chain (IL-2R
c) antibody from R&D Systems, Inc. (Minneapolis, MN), were used for Western blotting. Rabbit polyclonal antibodies of JAK1, JAK3, STAT5a, and STAT5b as well as anti-phosphotyrosine and phosphotyrosine STAT5 antibodies were purchased from Upstate, Inc. (Lake Placid, NY). Anti-caveolin-1 antibody was from BD Transduction Laboratories (Lexington, KY). The rabbit anti-goat IgG (heavy and light) horseradish peroxidase-conjugated antibody was obtained from Chemicon International, Inc. (Temecula, CA). Anti-rabbit IgG horseradish peroxidase-labeled secondary antibody was from Roche Diagnostics, Inc. PC5-conjugated mouse anti-human CD25 (IL-2R
) monoclonal antibodies and isotypic control were from Immunotech Coulter Co.
Cell culture and fatty acid treatment
The human T-cell line Jurkat E6-1 (American Type Culture Collection, Manassas, VA) was grown under standard conditions in RPMI 1640 medium supplemented with 10% heat-inactivated bovine calf serum and penicillin/streptomycin (100 U/ml and 100 µg/ml, respectively) at 37°C with 5% CO2. For the modification of cellular lipids, the cells were cultured for 2 days in serum-free Iscove's modified Dulbecco's medium supplemented with 0.4% (w/v) BSA (fraction V, containing <3 µM total fatty acids), with the addition of 50 µM either DHA or stearic acid from stock solutions in ethanol (final concentration
0.5%). Stearic acid served as a control; it is considered the most suitable fatty acid to be added to control cultures because nonessential fatty acids are highly abundant in human serum, and this fatty acid was shown previously not to influence cellular PUFA content and the membrane subdomain distribution of proteins compared with controls treated with vehicle only (18). The other cells were treated with 1.5% methyl-ß-cyclodextrin for 1 h at 37°C to disrupt lipid rafts. After stimulation with 400 U/ml IL-2 for 30 min, cells were pelleted and lysed.
Isolation of lipid rafts
Lipid rafts were isolated from Jurkat T-cells as described (19) by discontinuous sucrose density gradient ultracentrifugation. The cells (2.5 x 107 cells/ml) were washed in Hanks' balanced salt solution three times and lysed in TKM buffer (50 mM Tris, pH 7.4, 25 mM KCl, 5 mM MgCl2, and 1 mM EDTA) containing 1% Brij-58 and protease inhibitor cocktail tablets (0.12 mg of antipain-HCl, 20 µg of bestatin, 40 µg of chymostatin, 0.12 mg of E-64, 20 µg of leupeptin, 20 µg of pepstatin, 0.12 mg of phosphoramidon, 0.8 mg of pefabloc, and 40 µg of aprotinin). Lysates were incubated on ice for 30 min, mixed with an equal volume of 80% sucrose in TKM buffer, and overlaid with 5.5 ml of 36% sucrose followed by 2.5 ml of 5% sucrose. The gradients were subjected to ultracentrifugation at 250,000 g at 4°C for 18 h with a 90 Ti rotor in an Optima L-80XP ultracentrifuge (Beckman Coulter, Inc., Fullerton, CA). Fractions of 1 ml were collected from the top of the gradients one by one.
Flow cytometry
The cells were washed twice with 10 mM PBS buffer (pH 7.2), suspended with 50 µl of PBS buffer, and incubated with Cy5-labeled CD25 (the IgG is conjugated to a tandem dye constituted of R-phycoerythrin covalently linked to cyanin 5.1 at 1 mol of PC5 per mole of IgG) in the dark at room temperature for 20 min.
Negative control cells were incubated with isotypic control antibody. Finally, the cells were washed twice in PBS and assessed by flow cytometry on FACScalibur (Becton Dickinson, Franklin Lakes, NJ). Data were then analyzed with CellQuest software (Becton Dickinson).
Immunoblotting
The proteins in fractions isolated from sucrose density gradients were separated electrophoretically by SDS-PAGE on a Bio-Rad minigel apparatus (Bio-Rad, Inc., Hercules, CA) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Immunoblot analysis of IL-2R
, IL-2Rß, and IL-2R
c was performed using goat anti-human IL-2R
, IL-2Rß, and IL-2R
c antibodies and rabbit anti-goat IgG (H&L) horseradish peroxidase-conjugated antibody. Immunoblot analysis of JAK1, JAK3, STAT5a, STAT5b, and anti-phosphotyrosine was performed using rabbit anti-JAK1, anti-JAK3, anti-STAT5a, anti-STAT5b, anti-phosphotyrosine antibodies, phosphotyrosine STAT5, and anti-rabbit horseradish peroxidase-labeled antibodies. Membranes were developed according to standard immunoblotting procedures, detection was performed with the BM chemiluminescence Western blotting kit, and the membranes were exposed to films (Kodak BioMAX XAR; Eastman Kodak Co., Rochester, NY).
Fatty acid analysis
Detergent-resistant and detergent-soluble fractions from DHA-treated and control T-cells were freeze-dried before adding 1 ml of boron trifluoride-diethyl ether/methanol (1:3, v/v); 10 µg of heptadecanoic acid served as an internal standard. Methanolysis was performed by incubating at 75°C for 30 min. After cooling, 0.5 ml of water and 1.5 ml of n-hexane were added and stirred, then subjected to centrifugation at 2,000 g for 15 min. The organic phase was collected, dried by N2, and dissolved in 25 µl of chloroform, 2 µl of which was injected and analyzed by gas chromatography on a HP4890 system (Agilent Technologies, Palo Alto, CA). The system was equipped with a flame ionization detector, an HP3398A workstation, and a HP-FFAP quartz capillary column (30 m length, 0.32 mm inner diameter, 0.25 µm layer thickness). Gas chromatography was performed at 110°C, then increased at 8°C/min for 10 min, at 6°C/min to reach 230°C for 8 min, and at 6°C/min to 250°C for 3 min with a constant flow (10 p.s.i.) of high-purity N2. The fatty acid composition is expressed as mole percent. The significance of differences in fatty acid content between raft and soluble membrane fractions of DHA- and control-treated T-cells was calculated by paired Student's t-tests.
Mass spectrometry for raft phospholipid analysis
Total lipids in detergent-resistant and detergent-soluble fractions from DHA-treated and control T-cells were extracted. The samples were added with 1 ml of methanol containing 0.9% butylated hydroxytoluene and internal controls (1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine) and mixed. Four milliliters of chloroform was added and stirred with the addition of 1 ml of methanol. The final chloroform-methanol ratio was 2:1, and 1 ml of 50 mM KCl was added, mixed, and centrifuged at 2,000 g for 15 min. The organic phase was collected, filtered by organic filters, and evaporated-dried at 38°C.
An HP1100 series HPLC system (Agilent Technologies) was used. The LC separation was performed on a diol column (Nucleosil 100-5 OH) of 250 mm x 3.9 mm (inner diameter) x 5.0 µm (particle size). The total flow rate was 0.4 ml/min, and the column temperature was 35°C. Solvent mixture A consisted of hexane-1-propanol-formic acid-ammonia (79:20:0.6:0.07, v/v), and solvent mixture B consisted of 1-propanol-water-formic acid-ammonia (88:10:0.6:0.07, v/v). Mass spectrometric detection was performed on a QTRAP LC/MS/MS system from Applied Biosystems/MDS Sciex equipped with a turbo ion spray source. The split HPLC effluent entered the mass spectrometer through a steel electrospray ionization needle set at 5,500 V (in positive ion mode) or 4,500 V (in negative ion mode), and a heated capillary was set to 250°C. The ion source and ion optic parameters were optimized with respect to the positive or negative molecular related ions of the phospholipid standards. The flow rates of nitrogen drying gas and turbo gas were both 40 p.s.i. The declustering potential was set at 80 p.s.i. The other parameters were as follows: Enhanced Mass Scan as survey scan (mass range m/z 450950, scan speed 1,000 Dalton per second, trap time 20 ms) and Enhanced Product Ion Scan as dependent scan (scan speed 1,000 Da S1, trap time 150 ms, collision energy set at +35 eV in the positive ion mode and 40 eV in the negative ion mode).
| RESULTS |
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(CD25) expression on the surface of T-cells by FACS analysis. After IL-2 stimulation, 79.19% of the control cells (stearic acid) were CD25-positive. For Jurkat T-cells treated with DHA (25, 50, and 75 µM), the positive CD25 expression levels were 74.05, 59.75, and 10.98%, respectively (Fig. 1).
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was detected in low density, detergent-resistant membrane fractions of T-cells after solubilization with the nonionic detergent Brij-58 (Fig. 2). The IL-2Rß and IL-2R
c chains were also detected consistently in the lipid raft fractions but not in the soluble membrane fractions (Fig. 2). IL-2R chains were enriched in lipid rafts and localized within lipid raft microdomains. Lipid rafts play an important role in the regulation of the IL-2R signaling pathway. After DHA treatment, IL-2R
, IL-2Rß, and IL-2R
c were detected not only in raft fractions but also in soluble membrane fractions (Fig. 2), which indicated that IL-2R was partly displaced from lipid rafts after this treatment. Our results revealed that DHA could disrupt lipid raft integrity and alter the distribution of IL-2R
, IL-2Rß, and IL-2R
c in the plasma membrane and impair IL-2R signaling.
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| DISCUSSION |
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PUFAs displace proteins from rafts and affect cytoplasmic and transmembrane proteins in rafts by posttranslational regulation with fatty acyl moieties (15). The alteration in membrane fatty acyl composition should affect membrane lipids residing in the exoplasmic leaflet and also has indirect effects on the membrane subdomain distribution of acylated proteins (10). Lipid alteration of phospholipids, including PC, SM, PE, PS, and PI, in lipid rafts would have particular effects on displacing proteins from the functional lipid rafts. EPA treatment in T-cells resulted in the enrichment of PUFAs in the cytoplasmic leaflet as well as the exoplasmic leaflet of rafts (10). Therefore, we analyzed the incorporation of fatty acids in raft lipids not only in the exoplasmic lipid leaflet (PC and SM) (Tables 2, 3) but also in the cytoplasmic membrane lipid leaflet (PE, PI, and PS) (Tables 4 6) after DHA treatment. DHA treatment significantly increased the concentrations of PC species substituted with 36:3 (16:0/20:3), 38:6 (18:1/20:5, 16:0/22:6, 18:2/20:4), 38:5 (18:1/20:4, 18:2/20:3, 16:0/22:5), and 40:6 (18:1/22:5, 18:0/22:6, 20:3/20:3, 20:2/20:4) acyl chains (Table 2). SM consists of choline, ceramide, and fatty acids and is very difficult to split by electrospray ionization MS. Table 3 shows the total amounts of carbon and unsaturated bonds of fatty acids and ceramide. Substitution of SM with 38:3 acyl chains was increased significantly in rafts of DHA-treated cells compared with control cells (Table 3). Alteration of the fatty acyl composition of exoplasmic leaflet lipids (PC and SM) could indirectly affect the displacement of proteins from lipid rafts (10). PE species substituted with 36:5 (16:0/20:5), 36:5 (16:1/20:4), 38:6 (16:0/22:6, 18:2/20:4), 38:5 (18:0/20:5, 16:0/22:5, 18:1/20:4), 40:7 (18:1/22:6), 40:6 (20:2/20:4, 18:2/22:4, 18:0/22:6, 18:1/22:5), and 40:5 (18:0/22:5, 18:1/22:4, 18:2/22:3, 20:1/20:4) acyl chains (Table 4); PS species substituted with 38:4 (18:0/20:4), 38:6 (18:0/20:6), and 40:5 (18:0/22:5) acyl chains (Table 5); and PI species substituted with 36:4 (16:0/20:4), 40:6 (18:0/22:6), and 40:5 (18:0/22:5) acyl chains in rafts in DHA-treated cells compared with control cells (Table 6). DHA treatment changes the fatty acyl composition of lipids from the cytoplasmic leaflet in rafts. Thus, the alterations of fatty acyl chains in the cytoplasmic and exoplasmic lipid leaflet of lipid rafts from DHA-treated cells play an important role in raft structure and its lipid environment.
In this study, we found that after IL-2 stimulation in Jurkat T-cells, IL-2R
, IL-2Rß, and IL-2R
c chain proteins were detected in the fractions of membrane lipid rafts (Fig. 2), consistent with the data of Matkó, Bodnár, and Vereb (26). It was also confirmed using fluorescence resonance energy transfer and confocal microscopy that IL-2R
, IL-15R
, IL-2/15Rß, and IL-2R
c subunits formed supramolecular receptor clusters in lipid rafts (27). These findings were different from those of a previous study (28), which suggested that IL-2R
was enriched in lipid rafts and that the IL-2Rß and IL-2R
c chains were found only in detergent-soluble membranes. Our results indicate that DHA treatment caused IL-2R
, IL-2Rß, and IL-2R
c subunit displacement from membrane lipid rafts. The partial IL-2R chains removing from lipid rafts to soluble membrane fractions were very important for the DHA-induced suppression of the IL-2R signaling pathway.
The phosphorylated Janus family kinases (JAK1 and JAK3 kinases and phosphotyrosine) in response to IL-2 were not detected in lipid rafts but in soluble membrane fractions (Fig. 3), consistent with a previous report (26). To date, very little data are available for the influence of PUFAs on the signaling properties of cytokine receptors and JAKs in rafts (27, 29). Some results have indicated that JAKs were detected in rafts, whereas other reports showed JAKs enriched in detergent-soluble membranes (27). In addition, according to the standard model of STAT signaling upon IL-2-induced receptor activation, monomeric cytosolic STAT5s are recruited to the cytoplasmic tail of the respective plasma membrane receptor and phosphorylated by JAK family kinases, then dimerize and translocate to the nucleus to modulate gene expression. Most of reports showed that the association of STATs with plasma membrane rafts was always excluded from cellular membranes (3035). However, in 1997, Koshelnick and colleagues (36) first found STAT1, one of the STAT families, in a low density, detergent-resistant fraction of human kidney tumor epithelial cells. Later, STAT3, activated PY-STAT3, and PY-STAT1 were also detectable in this special domain of cell membrane (35), which initiated the study of "raft-STAT signaling transduction" in many cell types (37). Furthermore, in this study, we detected the presence of STAT5a and STAT5b not only in detergent-soluble fractions but also in detergent-resistant rafts. In addition, after DHA treatment, STAT5a and STAT5b were not detected in lipid raft fractions but were located in detergent-soluble fractions. In addition, the results in Fig. 5 showed that phosphorylated STAT5 was associated with detergent-soluble fractions and decreased with DHA treatment. We suggest that STATs in membrane rafts may be a transient early stage in cytokine signaling and that membrane rafts containing STATs may contain physical sites for integrating the combinatorial effects of different cytokines and different activation pathways. Our discovery of the alteration of STATs in rafts was an extension of work characterizing the subcellular distribution of STAT proteins.
In summary, our data strongly demonstrate that altered raft lipid environment affects the membrane subdomain distribution of proteins in the IL-2R signaling pathway in DHA-treated T-cells. Such partial disorganization in lipid rafts induced by DHA treatment mediates the immunodepressive function and its immunomodulatory effect. However, the exact mechanism underlying these alterations after PUFA treatment remains obscure in most instances, whether in vivo or in a given clinical situation. This awaits further investigation.
| ACKNOWLEDGMENTS |
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Manuscript received January 25, 2005 and in revised form April 26, 2005 and in re-revised form May 23, 2005.
| REFERENCES |
|---|
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|
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í, I. J. Ansotegui, W. Knapp, and H. Stockinger. 1991. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science. 254: 10161019.
RI and lipid raft components are regulated by the actin cytoskeleton. J. Cell Sci. 113: 10091019.[Abstract]
í, W. Waldhäusl, and T. M. Stulnig. 2002. LAT displacement from lipid rafts as a molecular mechanism for the inhibition of T cell signaling by polyunsaturated fatty acids. J. Biol. Chem. 277: 2841828423.This article has been cited by other articles:
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