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Nutritional Immunology

Dietary (n-3) Polyunsaturated Fatty Acids Remodel Mouse T-Cell Lipid Rafts

Yang-Yi Fan^*, David N. McMurray^*,,**, Lan H. Ly^* and Robert S. Chapkin^*,,2

^* Faculty of Nutrition, Center for Environmental and Rural Health, Texas A&M University and ^** Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, TX

²To whom correspondence should be addressed. E-mail: r-chapkin{at}tamu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In vitro evidence indicates that (n-3) polyunsaturated fatty acids (PUFA) suppress T-cell activation in part by displacing proteins from lipid rafts, specialized regions within the plasma membrane that play an important role in T-cell signal transduction. However, the ability of (n-3) PUFA to influence membrane microdomains in vivo has not been examined to date. Therefore, we compared the effect of dietary (n-3) PUFA on raft (liquid ordered) vs. soluble (liquid disordered) microdomain phospholipid composition in mouse T cells. Mice were fed diets containing either 5 g/100 g corn oil (control) or 4 g/100 g fish oil [contains (n-3) PUFA] + 1 g/100 g corn oil for 14 d. Splenic T-cell lipid rafts were isolated by density gradient centrifugation. Raft sphingomyelin content (mol/100 mol) was decreased (P < 0.05) in T cells isolated from (n-3) PUFA-fed mice. Dietary (n-3) PUFA were selectively incorporated into T-cell raft and soluble membrane phospholipids. Phosphatidylserine and glycerophosphoethanolamine, which are highly localized to the inner cytoplasmic leaflet, were enriched to a greater extent with unsaturated fatty acids compared with sphingomyelin, phosphatidylinositol and glycerophosphocholine. These data indicate for the first time that dietary (n-3) PUFA differentially modulate T-cell raft and soluble membrane phospholipid and fatty acyl composition.

KEY WORDS: • (n-3) fatty acids • docosahexaenoic acid • lipid rafts • T cell • sphingolipid

Within the T-cell plasma membrane, there are specific detergent-resistant domains in which key signal transduction proteins are localized. These regions are termed "lipid rafts" (1,2). Rafts are composed mainly of cholesterol and sphingolipids and therefore do not integrate well into the fluid phospholipid bilayers, causing them to form microdomains. Upon T-cell activation, rafts compartmentalize the ligated T-cell receptor (TcR) and associated signal-transducing molecules, thus providing an environment conducive to signal transduction (3). For example, the earliest mediators of T-cell proliferation [i.e., protein kinase C- (PKC), phospholipase C- (PLC), the linker for activation in T cells (LAT)], and T-cell apoptosis [i.e., Fas and Fas-ligand (Fas-L)], translocate to lipid rafts after stimulation (4–6). There is also an emerging paradigm that lipid rafts cluster at the T cell:antigen presenting cell interface, ultimately generating platforms specialized for processive and sustained TcR signaling (7). Although the essentiality of this immunological "synapse" with regard to T-cell activation has recently been challenged (8), there is overwhelming evidence that lipid raft integrity is a prerequisite for optimized TcR signal transduction and immune response (7,9–11). Interestingly, conditions that modify raft structure can disrupt these earliest steps of T-cell activation (1).

Many epidemiologic and clinical studies have demonstrated that (n-3) PUFA attenuate immune-mediated inflammatory diseases (12–14). The primary effector molecules are thought to be eicosapentaenoic acid [20:5(n-3), EPA] and docosahexaenoic acid [22:6(n-3), DHA]. We demonstrated recently that the anti-inflammatory properties of dietary (n-3) PUFA are the result of a coordinated direct effect on T-cell proliferation and activation-induced cell death (15,16). In addition, we demonstrated that dietary PUFA classes [(n-6) vs. (n-3)] are differentially incorporated into T-cell membranes (16–18).

Recent in vitro studies using a Jurkat T-cell line have shown that PUFA added in culture are capable of modifying lipid rafts and suppressing signal transduction (19,20). However, the ability of dietary (n-3) PUFA to influence plasma membrane subdomains, i.e., "liquid ordered" rafts or "liquid disordered" soluble membrane fractions has not been determined to date. Because the fidelity of such T-cell membrane remodeling may not be accurately represented using an in vitro manipulation (21), we investigated the in vivo effects of dietary (n-3) PUFA on splenic T-cell sphingolipid-rich plasma membrane microdomains (i.e., rafts) in mice.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Materials.

RPMI 1640 media and heat-inactivated fetal bovine serum were obtained from Irvine Scientific (Santa Ana, CA). Lymphocyte-M was purchased from Cedarlane (Toronto, Canada). T-cell purification columns were obtained from R&D Systems (Minneapolis, MN). Brij-58 was obtained from Fisher Scientific (Fair Lawn, NJ). Silica gel 60 G plates and all organic solvents were purchased from EM Science (Gibbstown, NJ). Fatty acid methyl ester (FAME) standards were purchased from Nu-Chek-Prep (Elysian, MN). Precast 4–20% Tris-glycine gels were obtained from Invitrogen (Carlsbad, CA). Peroxidase-conjugated cholera toxin B subunit was purchased from Sigma Chemical (St. Louis, MO). Mouse monoclonal anti-Lck was obtained from Transduction Laboratories (Los Angeles, CA). Rabbit polyclonal anti-LAT was obtained from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal anti-CD3 was purchased from BD Pharmingen (Los Angeles, CA). Peroxidase-labeled goat anti-mouse and anti-rabbit immunoglobulin (Ig)G were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Corn oil was obtained from Degussa BioActives (Champaign, IL) and menhaden fish oil was provided by the NIH (Fish Oil Test Material Program, Washington DC).

Animals and diets.

All experimental procedures using laboratory animals were approved by the University Laboratory Animal Care Committee of Texas A&M University. Pathogen-free female C57BL/6 mice (n = 180; Frederick Research Facility, Frederick, MD), weighing 16–18 g, were randomly divided into two groups of 90 mice. For 2 wk, mice had free access to one of the two semipurified diets, which were adequate in all nutrients (22). Diets varied only in the oil composition, i.e., either corn oil (CO) or an (n-3) PUFA-enriched fish-corn oil (FO) mixture (4:1, w/w) at 5 g/100 g diet. The basic diet composition, expressed as g/100 g was: casein, 20; sucrose, 42; cornstarch, 22; cellulose, 6; AIN-76 mineral mix, 3.5; AIN-76 vitamin mix, 1 (23), DL-methionine, 0.3; choline chloride, 0.2; Tenox 20A (containing 32% glycerol, 30% corn oil, 20% tert-butylhydroquinone, 15% propylene glycol, 3% citric acid) 0.1; and oil, 5 (24). The fatty acid composition of the diets, as determined by gas chromatography, is shown in Table 1.

Mice were killed by CO₂ asphyxiation. T cells were isolated from spleens as described previously (24). Briefly, spleens were homogenized in complete RPMI medium (RPMI 1640 with 25 mmol/L HEPES supplemented with 10% heat-inactivated fetal bovine serum, 1 x 10⁵ U/L penicillin, 100 mg/L streptomycin, 2 mmol/L L-glutamine, and 10 µmol/L 2-mercaptoethanol), followed by passage through a 149-µm wire mesh filter to create single-cell suspensions. Erythrocytes were removed by density gradient centrifugation over Lymphocyte-M. Total lymphocytes were loaded onto a negative-selection mouse T-cell purification column to purify the T-cell population (>90% CD3 positive) (21). T cells isolated from 30 mice (3 x 10⁸ cells) were pooled for raft isolation.

Density gradient centrifugation and isolation of lipid rafts.

Raft microdomains were isolated from mouse T cells as described by Tamir et al. (25), with slight modification. T cells were lysed in lysis buffer [100 mmol/L NaCl, 2 mmol/L EDTA, 4.1 mmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 0.2 mmol/L Na₃VO₄, 50 µmol/L NaF, 25 mmol/L HEPES, 3.2 µmol/L aprotinin, 88 µmol/L leupeptin, 160 µmol/L bestain, 60 µmol/L pepstain A and 56 µmol/L E-64, pH 6.9] supplemented with 1% Brij-58. Cell lysates were passed through a 27G needle once, followed by a 30-min incubation on ice. A solution containing 850 g/L sucrose (in lysis buffer) was added to the lysate and mixed by pipetting to generate a 450 g/L sucrose lysate. Cell lysates were transferred to the bottom of a 2-mL polyallomer ultracentrifuge tube, which was subsequently overlaid with 350 and 50 g/L sucrose, respectively. After centrifugation at 200,000 x g (Beckman Coulter Optima Max-E Ultracentrifuge, TLS 55 rotor) for 16 h at 4°C, aliquots from the top (low density detergent insoluble glycolipid enriched raft fraction), and from the bottom (cytosol-high density membrane detergent soluble fraction) of the tube were collected for lipid analysis. In addition, for the purpose of examining protein distribution patterns, aliquots consisting of 200-µL fractions (5 fractions total), followed by 500-µL fractions (2 fractions total) were collected sequentially from the top of the gradient for immunoblot analysis.

Immunoblotting.

The seven gradient fractions (described above) isolated from mouse T cells were concentrated by SpeedVac (Savant Instruments, Holbrook, NY). Protein concentrations were measured by the bicinchoninic acid assay (26). Concentrated samples (0.5–2 µg) were immunoblotted with GM1, LAT, lymphoid-specific Src family kinase (Lck) or CD3 antibodies using the method of Davidson et al. (27) to evaluate the membrane domain localization of select markers. Briefly, samples were treated with SDS sample buffer and subjected to electrophoresis in a 4–20% precast Tris-glycine gel. After electrophoresis, proteins were electroblotted onto a polyvinylidene fluoride membrane using a Hoefer Mighty Small Transphor Unit (Pharmacia, Piscataway, NJ) at 400 mA for 100 min. After transfer, the membrane was incubated with specific primary antibodies. For GM1 detection, samples were incubated with peroxidase conjugated cholera toxin B subunit. For LAT detection, samples were incubated with rabbit anti-LAT antibody overnight at 4°C, followed by peroxidase-labeled goat anti-rabbit IgG incubation for 1 h at room temperature. For Lck and CD3 detection, samples were incubated with mouse anti-Lck or anti-CD3 antibodies, followed by peroxidase labeled goat anti-mouse IgG incubation. Bands were developed using Super Signal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL), and the blots were scanned and quantified using a BioRad Fluor-S Max MultiImager System (Hercules, CA).

Measurement of phospholipid fatty acid composition.

Total lipids in liquid ordered membrane raft and liquid disordered soluble fractions from CO- and FO-fed mice were extracted by the method of Folch et al. (28). Total lipid phosphorus was measured as described by Duck-Chong (29). Individual phospholipid classes were separated by one-dimensional TLC on silica gel 60 G plates using chloroform/methanol/acetic acid/water (50:37.5:3.5:2, v/v/v/v) as the developing solvent. Isolated individual phospholipid classes were spiked with 50 ng heptadecanoic acid (17:0) as internal standard and transesterified in the presence of 6% methanolic HCl. FAME were subsequently analyzed by capillary gas chromatography as previously described (30).

Statistical analysis.

Data were analyzed using one-way ANOVA. Differences of P < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Characterization of T-cell raft and soluble membrane fractions.

To determine whether T-cell raft and soluble membrane properties were influenced by different levels of (n-3) PUFA in the diet, we optimized the methodology to detect the effect of diet on T-cell membrane raft composition (Fig. 1). Similar to previous reports using Brij 58 as the primary detergent to lyse T-cell membranes, the protein distribution patterns confirmed the well-documented structural features of lipid rafts, i.e., the significant enrichment of the ganglioside GM-1, a raft positive marker, and the exclusion of CD3, a raft negative marker (19,20,25,31,32). In addition, the LAT and Lck were distributed in both raft and soluble membrane fractions under nonstimulated conditions (33).

View larger version (49K): [in this window] [in a new window]   FIGURE 1 Characterization of mouse splenic T-cell plasma membrane microdomains. Membranes were isolated from mice fed a semipurified diet. GM-1, raft positive marker; CD3, raft negative marker; LAT and Lck, two signaling markers with a mixed localization. Upper panel (A): Western immunoblots of 1-µg protein aliquots. Numbers represent fraction number. Lower panel (B): Summary of immunoblot densitometry plots. Fractions 1–3 represent liquid ordered rafts; 4 and 5 represent intermediate density membrane fractions; and on the far right of the x-axis, soluble-liquid disordered fractions 6 and 7. Jurkat T cell standard was used as a positive control.

The data indicate that dietary (n-3) PUFA are capable of profoundly altering the composition of phospholipids that constitute lipid rafts in T-cell plasma membrane. Raft sphingomyelin (ChoCer) content (mol/100 mol) was decreased (P < 0.05) 44% in T cells isolated from (n-3) PUFA fed mice (Table 2). There was also a modest increase (P = 0.062) in the other membrane phospholipids in the relative abundance of glycerophosphoethanolamine (EtnGpl) in (n-3) PUFA-enriched lipid rafts.

Because unsaturated fatty acids in model membranes greatly reduce raft formation (34) and can displace signaling proteins (20), we also examined the changes in the fatty acid composition of T-cell rafts and liquid disordered soluble membrane domains in response to dietary (n-3) PUFA. The fatty acid composition (nmol/µg phosphorus and mol/100 mol) of individual phospholipid classes are described in Tables 34567. Because the inclusion of (n-3) PUFA in the diet reduced ChoCer mass in lipid rafts (Table 2), we initially examined the fatty acyl composition of this phospholipid class. The mean number of monounsaturated fatty acids was higher (P < 0.05) in T-cell rafts of FO-fed mice compared with CO-fed mice (Table 3). In contrast, the unsaturation index was not altered in the liquid disordered soluble membrane fraction (Table 3). For raft glycerophosphocholine (ChoGpl), the total PUFA and unsaturation indices (P < 0.05) in FO-fed mice were less (P < 0.05) than in CO-fed mice. Interestingly, despite the significant decrease of (n-6) PUFA, no (n-3) PUFA were detected in the FO raft fraction. In contrast, FO-fed mice had elevated (P < 0.05) (n-3) PUFA in the soluble ChoGpl membrane fraction (Table 4). Because there was a tendency for dietary (n-3) PUFA to increase EtnGpl mass in lipid rafts (Table 2), we also examined the fatty acyl composition of this phospholipid class. Dietary EPA and DHA were highly enriched in both raft and soluble EtnGpl, largely at the expense of (n-6) PUFA, e.g., 22:4(n-6) (Table 5). However, the unsaturation index was elevated only in the FO-soluble fraction. Phosphatidylserine (PtdSer) in raft and soluble membrane fractions responded similarly with respect to (n-3) PUFA incorporation (Table 6). In contrast, phosphatidylinositol (PtdIns) in rafts was highly resistant to (n-3) PUFA enrichment after FO feeding (Table 7).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The significant effects of diets rich in (n-3) PUFA on T-cell function have been firmly established in both humans and experimental animals (12,13,17,36–39). This is important because the typical Western diet contains 10–20 times more (n-6) than (n-3) PUFA (40). Unfortunately, no cogent, unifying hypothesis exists at present to address the mechanisms by which (n-3) PUFA selectively suppress T-cell immune function compared with (n-6) PUFA (the major dietary form of PUFA in the U.S. diet). Because lipid rafts are important signaling platforms for T cells (41,42), we determined the ability of dietary fish oil, containing EPA and DHA, to influence plasma membrane microdomain phospholipid composition. Initially we optimized the methodology to detect the effect of fish oil feeding on T-cell membrane raft composition. The observed protein distribution patterns (Fig. 1) confirm the well-documented structural features of lipid rafts, i.e., the significant enrichment of the ganglioside GM-1, a raft positive marker and the exclusion of CD3, a raft negative marker (19,31,32). In addition, the elevated sphingolipid content and persistence of a saturated fatty acyl environment in lipid rafts is consistent with the presence of liquid ordered microdomains (20,42).

Our data indicate that dietary (n-3) PUFA are capable of profoundly altering the composition of phospholipids that constitute lipid rafts in the murine T-cell plasma membrane. Raft sphingomyelin content (mol/100 mol) was significantly decreased in T cells isolated from (n-3) PUFA–fed mice (Table 2). This novel and unexpected observation is noteworthy because sphingolipids, which are largely restricted to the outer (exoplasmic) leaflet of the plasma membrane bilayer, are required to facilitate raft formation and T-cell activation (32,35). Because rafts can be disrupted by depletion of sphingolipids (35), dietary (n-3) PUFA may alter surface receptor protein function and T-cell responsiveness by altering raft phospholipid composition. This hypothesis is supported by recent in vitro studies using a Jurkat T-cell line in which PUFA enrichment selectively modified lipid rafts and suppressed signal transduction (19,20). In addition, it has been demonstrated that CD28 engagement triggers cytoskeletal and intracellular kinase-rich lipid raft microdomain rearrangements that result in stabilization of the immunological synapse, i.e., the junction between the T-cell and the antigen-presenting cell (2). Therefore, any dietary factors that alter lipid raft properties, e.g., (n-3) PUFA, might be expected to modulate CD28 costimulatory function. This is consistent with recent in vivo reports indicating that dietary DHA alters CD28 function in primary murine T-cells (15,24).

Because unsaturated fatty acids in model membranes greatly reduce raft formation (34) and can displace signaling proteins (20), we also examined the changes in the fatty acid composition of T-cell rafts in response to dietary (n-3) PUFA. Interestingly, only PtdSer and EtnGpl in the raft and liquid disordered soluble membrane fractions incorporated dietary EPA and DHA, largely at the expense of (n-6) PUFA content. In general, although changes in all phospholipid classes induced by dietary fish oil containing EPA and DHA were markedly mitigated in rafts compared with the soluble membrane fraction, ChoGpl and PtdIns in T-cell rafts were entirely devoid of any (n-3) PUFA. Therefore, the notion that PUFA chains are excluded from rafts (43) is not entirely accurate. It would appear that certain classes of raft phospholipids do incorporate significant levels of highly unsaturated (n-3) PUFA, which may lead to adaptive changes in ChoCer content in an attempt by the T cell to maintain a constant bilayer configuration, e.g., tight acyl chain packing. Further studies are warranted to elucidate the mechanisms that regulate diet-induced membrane microdomain alterations and the resultant modulation of cell function.

Dietary fish oil, containing (n-3) PUFA, has been shown to suppress human T-cell function via reductions in the secretion of interleukin-2, the primary autocrine and paracrine T-cell growth factor, and subsequent proliferation (44). In addition to their ability to alter membrane function/dynamics, dietary EPA and DHA suppress arachidonic acid–derived prostaglandin (PG)E₂ production (45,46). However, PGE₂ is antiproliferative for T cells (12,45,47). Therefore, this putative mechanism is not consistent with the suppressed T-cell proliferation that was observed after dietary EPA and DHA supplementation (15,48). It is now generally accepted that the inhibitory effects of (n-3) PUFA on T-cell proliferation are not mediated by eicosanoids or lipid peroxidation (12). Another mechanism by which (n-3) PUFA could alter T-cell function might involve peroxisome proliferator-activated receptors (PPAR). In addition to their ability to alter membrane function/dynamics, dietary PUFA are also ligands for certain nuclear receptors. Although some ligands for PPAR (, ) are known to modulate T-cell function (49,50), this class of nuclear receptor binds (n-3) and (n-6) PUFA with equal affinity and lacks fatty acid class [(n-3) vs. (n-6)] specificity (51–53). Therefore, the unique effects of (n-3) PUFA are likely not mediated via PPAR.

In conclusion, we have shown for the first time that dietary (n-3) PUFA differentially modulate T-cell raft (liquid ordered) and soluble (liquid disordered) membrane phospholipid and fatty acyl composition. Because the wide variety of lipids found in membranes, and particularly membrane rafts, actively participate in signal transduction pathways, these results support the hypothesis that dietary (n-3) PUFA alter T-cell membrane microdomain composition and may therefore influence signaling complexes and modulate T-cell activation in vivo.

	ACKNOWLEDGMENTS

 
The authors thank Kirsten Switzer for her valuable technical assistance in the completion of these studies. We also acknowledge Degussa BioActives for the generous provision of corn oil.

	FOOTNOTES

 
¹ Supported by NIH grants DK53055 and P30-ES09106.

³ Abbreviations used: ChoCer, sphingomyelin; ChoGpl, glycerophosphocholine; CO, corn oil; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; EtnGpl, glycerophosphoethanolamine; Fas-L, Fas-ligand; FO, fish oil; LAT, linker for activation in T cells; Lck, lymphoid-specific Src family kinase; PG, prostaglandin; PKC, protein kinase C-; PLC, phospholipase C-; PPAR, peroxisome proliferator-activated receptors; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; TcR, T-cell receptor.

Manuscript received 3 January 2002. Initial review completed 5 February 2003. Revision accepted 21 February 2003.

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