Elevated serum free fatty acid concentrations inhibit T lymphocyte signaling THOMAS M. STULNIG,1 MARKUS BERGER, MICHAEL RODEN, HARALD STINGL, ¨ USL DANIEL RAEDERSTORFF,* AND WERNER WALDHA Department of Internal Medicine III, University of Vienna, Vienna, Austria; and *Hoffmann-La Roche Ltd., Basel, Switzerland ABSTRACT Unbound cis-unsaturated free (i.e., nonesterified) fatty acids (FFA) inhibit T lymphocyte activation in vitro and therefore may exert immunosuppressive effects. However, in blood serum the major proportion of FFA is tightly bound to albumin, whereas unbound FFA are hardly detectable. Since serum FFA elevation occurs under pathological conditions like insulin resistance or cancer, which are often associated with a disturbed immune response, we addressed the question of whether increased serum FFA concentrations could affect T lymphocyte activation under in vivo conditions. Our studies revealed that 1) addition of pure long-chain cisunsaturated FFA in the absence of albumin inhibited calcium response in cultured Jurkat T cells. 2) In healthy volunteers, serum FFA elevation by a lipid/ heparin infusion, including predominantly unsaturated fatty acids, decreased calcium response of cultured T cells in contrast to studies without heparin. 3) Most notably, stepwise increasing serum FFA by lipid/heparin infusion also inhibited calcium response of simultaneously isolated autologous peripheral blood T lymphocytes as well as their CD4ⴙ and CD8ⴙ subsets. In conclusion, our data emphasize that serum FFA elevation is able to exert immunosuppressive effects in vivo.—Stulnig, T. M., Berger, M., Roden, M., Stingl, H., Raederstorff, D., Waldha¨usl, W. Elevated serum free fatty acid concentrations inhibit T lymphocyte signaling. FASEB J. 14, 939 –947 (2000)
Key Words: signal transduction 䡠 calcium signaling 䡠 lipids 䡠 antigen receptors 䡠 glycosylphosphatidylinositols
Free, i.e., nonesterified, fatty acids (FFA) are major substrates for energy metabolism, and consequently large quantities of FFA have to be supplied to peripheral cells. Since FFA are highly insoluble in the aqueous phase, their major proportion is transported in blood bound to albumin, thereby keeping the concentration of unbound FFA extremely low (1). However, the efficient binding of FFA to albumin not only mediates their solubility, but also prevents direct effects of unbound FFA on various 0892-6638/00/0014-0939/$02.25 © FASEB
cellular functions. Activation, degranulation, and cytolytic functions of lymphocytes in particular are inhibited in vitro by the naturally prevailing cisunsaturated species of unbound FFA (2– 6). Accordingly, diseases with increased serum FFA concentrations such as diabetes mellitus (7), cancer (8, 9), and states of ischemia (10, 11) are often associated with disturbances in immune function (12, 13). Since activation of T lymphocytes plays a pivotal role for initiating immune response and cell-mediated cytotoxic activity, inhibition of lymphocyte activation by elevated serum FFA levels could evoke a clinically relevant immunosuppressive effect. Only a minor proportion of serum FFA occurs in an unbound (and thus thermodynamically active) form due to several FFA binding sites on albumin (14, 15). Former attempts to measure the concentration of unbound FFA in serum revealed a huge variety of results (16 –19), but the latest and methodologically most reliable determinations estimate the normal serum concentration of unbound FFA to be below 10 nmol/l (1). Thus, the measurable serum concentration of unbound FFA appears ⬃100 times less than the minimal concentration required to elicit a functional effect in lymphocytes in the absence of albumin (2, 5), thereby arguing against a pathophysiological relevance of FFA for lymphocyte function. However, from the physiological and clinical point of view the exact concentration of unbound FFA is much less relevant than their effect on cell function. The cellular effects of unbound cis-unsaturated FFA are due to partition of FFA into cellular membranes, hence perturbing their biophysical properties (20). In the biological situation, an equilibrium in the distribution of FFA between FFA binding proteins in serum and cytosol as well as target cell membranes is formed, and the resulting membrane concentration of cis-unsaturated FFA seems to determine their subsequent action (20). Thus, in order to provide 1 Correspondence: Department of Internal Medicine III, Division of Endocrinology and Metabolism, University of Vienna, Wa¨hringer Gu¨rtel 18 –20, A-1090 Vienna, Austria. E-mail:
[email protected]
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reliable information on potential immunosuppressive effects of FFA in vivo, equilibration of FFA between serum proteins and cells must be allowed to occur in an experimental setting on lymphocyte function. Therefore we addressed the question of whether serum FFA elevation could alter T cell signaling. The rise in cytoplasmic calcium concentration, which is a major event in T cell signal transduction and required for T cell activation (21–23), was evaluated in three different settings systematically approximating the in vivo situation. 1) Pure FFA were added to Jurkat T cells in the absence of albumin to determine effects of various unbound FFA in cultured T cells. 2) Serum FFA were elevated in healthy volunteers by simultaneous infusion of predominantly unsaturated lipids and heparin during hyperinsulinemic-euglycemic clamp studies, and sera collected during these metabolic studies were added to cultured T cells prior to stimulation. 3) Finally, sera and peripheral blood lymphocytes were simultaneously collected during a similar metabolic study, and the activation of autologous T cell subsets was studied in the presence of corresponding sera.
MATERIALS AND METHODS Reagents and antibodies Lymphocytes were stimulated with mouse monoclonal antibodies (mAbs) directed against CD3ε (OKT3, IgG2a; Ortho Pharmaceuticals, Raritan, N.J.) and CD59 (MEM-43, IgG2a), kindly provided by Dr. V. Horˇejsˇ´ı, Institute of Molecular Genetics (Prague, Czech Republic) and partly obtained from Monosan (Uden, The Netherlands). Antibodies were crosslinked by F(ab⬘)2 fragments of goat anti-mouse (GAM)-IgG from Sigma (St. Louis, Mo.) or Jackson (West Grove, Pa.). Phycoerythrin-labeled antibodies against CD4 (Leu-3a), CD8 (Leu-2a), CD20 (Leu-16), CD33 (Leu-M9), and CD56 (Leu19; all IgG1) were purchased from Becton Dickinson (San Jose, Calif.).
Metabolic studies A To induce production of free fatty acids under controlled conditions resembling insulin resistance as it occurs in diabetes mellitus, hyperinsulinemic (⬃450 pmol/l)-euglycemic (⬃5 mmol/l) clamp studies were performed for 180 min as detailed previously (24). Briefly, at 7:30 a.m. catheters were placed in one antecubital vein of the left and right arm for blood sampling and infusions, respectively. Regular human insulin (Actrapid; Novo Nordisc, Denmark) was administered as a primed continuous intravenous (i.v.) infusion [7.1 pmol/ (kg 䡠 min)]. Constant fasting plasma glucose concentrations were achieved by a variable i.v. glucose infusion (1.1 M). Subjects were studied under two experimental conditions: 1) elevation of serum FFA concentrations (‘high FFA’) induced by i.v. infusion of a triglyceride emulsion (1.5 ml/min; Intralipid 20%, Kabi Pharmacia, Stockholm, Sweden; for fatty acid composition, see Table 1) combined with a bolus (250 IU)-continuous i.v. infusion of heparin [0.2 IU/(kg 䡠 min)] (25, 26); and 2) fasting plasma FFA concentrations (‘low FFA’) during i.v. triglyceride infusion (1.5 ml/min) only. Heparin was used to stimulate lipoprotein lipase, which catalyzes hydrolysis of triglycerides (27). Blood was drawn at 0, 15, 60, 120, and 180 min for use in stimulation experiments and chemical analyses. Metabolic studies B Experiments for stepwise increasing serum FFA concentrations were begun at 5:30 a.m. (⫽ ⫺390 min) with insertion of venous catheters as described above. The i.v. infusion of triglyceride emulsion (Intralipid 20%) was raised in two steps (0 –180 min, 0.5 ml/min; 180 –360 min, 1.0 ml/min) during simultaneous administration of heparin [bolus: 200 IU; continuous infusion 0.2 IU/(kg 䡠 min)]. Somatostatin (0.1 g/kg 䡠 min) was infused from ⫺10 min until ⫹360 min to inhibit endogenous hormone secretion. From 0 to 360 min, insulin TABLE 1. Fatty acid composition of lipid infusion Fatty acid
C14:0b C16:0 C16:1 (n⫺7) C18:0 C18:1 (n⫺7) C18:1 (n⫺9) C18:2 (n⫺6) C18:3 (n⫺3) C20:0 C20:1 (n⫺9) C20:4 (n⫺6) C22:0 C22:4 (n⫺6) C22:5 (n⫺6) C22:5 (n⫺3) C22:6 (n⫺3) C24:0 Total saturated Total unsaturated Total monoenes Total polyenes
Subjects Metabolic studies A included eight healthy male subjects [age 25.3⫾2.2 years (mean⫾sd); body mass index 23.15 ⫾ 2.93 kg/m2, serum albumin 768 ⫾ 32 mol/l]; metabolic studies B included six healthy subjects (male/female ⫽ 5/1; age 27.0⫾7.4 years; body mass index 21.23⫾3.68 kg/m2, serum albumin 784⫾36 mol/l). The female subject was studied in the follicular phase of the menstrual cycle. None of the subjects had a family history of diabetes mellitus or were taking any medication. All stopped moderate regular exercising at least 3 days prior to the experiments and were on an isocaloric diet [30 kcal/(kg 䡠 day); carbohydrate/protein/fat: 60/20/20%] for 3 days, then fasted overnight for at least 8 h prior to the start of the experiments. Written informed consent was obtained from all subjects after explanation of the nature of the studies, which were approved by the Human Ethics Committee of the University of Vienna. 940
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Mol %a
0.10 12.23 0.14 5.55 2.59 21.13 50.26 6.37 0.35 0.16 0.14 0.34 0.05 0.06 0.06 0.16 0.11 18.67 81.33 24.02 57.31
a Because of rounding and disregard of fatty acid species occurring in trace amounts, mol % of individual fatty acids do not total b exactly 100. The number of double bonds is indicated by the digit behind the colon.
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[0.50 pmol/(kg 䡠 min)] and glucagon [0.19 pmol/(kg 䡠 min)] were infused to maintain their postabsorptive serum concentrations (26). Blood was collected at 0, 180, and 360 min for mononuclear cell preparation, experimental sera, and chemical analyses. To mimic potential effects of elevated serum glycerol generated during lipolysis, control conditions included i.v. administration of glycerol [135–240 min, 2.5 mol/(kg 䡠 min); 240 –360 min, 5.1 mol/(kg 䡠 min)] in lieu of lipid infusion. Cell culture and mononuclear cell preparation The human T cell line Jurkat E6 –1 (American Type Culture Collection, Rockville, Md.) was grown under standard conditions in RPMI 1640 medium (Gibco BRL, Gaithersburg, Md.) supplemented with 10% heat-inactivated bovine calf serum (HyClone, Logan, Utah), penicillin/streptomycin (50 U/ml and 50 g/ml, respectively), and 2 mM glutamine (all Gibco BRL) at 37°C in humidified atmosphere in the presence of 5% CO2 (22). Peripheral blood mononuclear cells were isolated by gradient centrifugation (LymphoPrep, Nyegaard, Oslo, Norway) immediately after blood collection as detailed previously (28). Cell viability was ⬎95% as estimated by trypan blue exclusion. Determination of calcium response Pure FFA experiments The stimulated rise in cytoplasmic calcium concentration was quantified essentially as described (22). Briefly, Jurkat T cells were labeled with the fluorescent Ca2⫹ indicator indo-1-AM (Molecular Probes, Eugene, Oreg.) and subsequently washed three times in Hanks buffered salt solution including 10 mM HEPES (pH 7.4). FFA of highest available quality (Sigma; 5 to 80 M as indicated) were added from stock solutions in ethanol [final concentration ⫽ 0.8% (v/v)] to 106 Jurkat cells in 250 l washing buffer. Ethanol by itself had no effect on calcium signaling (not shown). Jurkat cells were preincubated with FFA for 3 min at 37°C and subsequently stimulated via CD3 by adding 1 g OKT3 mAb under flow cytometric monitoring. For stimulation via CD59, cells were primed with 5 g MEM-43 mAb for 16 min at room temperature before addition of fatty acids for 2 min at 37°C and stimulation by cross-linking with 15 g F(ab⬘)2 fragments of GAM-IgG (22). Flow cytometric analysis was performed on a FACStarplus (Becton Dickinson) under a constant temperature of 37°C using multiline UV excitation by argon laser. The ratio of the emission at 530 nm (calcium-free indo-1) and 395 nm (calcium bound indo-1) was computed as a direct estimate of the cytoplasmic calcium concentration (29). For quantitation of stimulation and to allow comparability between experiments, the stimulation was expressed in percent of the ethanol control (22). Metabolic studies A Jurkat T cells were prepared as in experiments with pure FFA, but instead of commercial FFA, 210 l of autologous sera collected at the indicated time points during the clamps was added to 106 Jurkat T cells suspended in 40 l buffer [84% (v/v) final serum concentration]. The calcium responses at different time points were compared to the control response achieved at the beginning of the experiment and expressed in percent of control (22). ELEVATED SERUM FATTY ACIDS INHIBIT T LYMPHOCYTES
Metabolic studies B For detection of calcium response in T cell subsets of peripheral blood, indo-1-labeled mononuclear cells derived from subjects during clamp studies were immediately stained with phycoerythrin-labeled antibodies against CD33 (expressed on monocytes), CD20 (B lymphocytes), and CD56 (NK cells) resulting in negative labeling of peripheral T lymphocytes. Phycoerythrin-labeled antibodies against either CD8 or CD4 were added for negative labeling of the CD4⫹ or CD8⫹ T cell subpopulation, respectively. The immunostaining enabled exclusion of the expressing cell populations during flow cytometry using dual laser technique (see below), resulting in selective analysis of peripheral T cells or T cell subsets, respectively (30). After three additional washes, 5 䡠 106 cells were preincubated with autologous sera [84% (v/v) final concentration] from clamp studies for 2 min at 37°C before starting flow cytometric measurements. One minute later, OKT3 mAb (CD3) was added; 2 min later, GAM-IgG was given to stimulate cells by antibody cross-linking and measurement was continued for another 4 min. In the flow cytometer chamber, cells first passed the argon laser (488 nm line, 100 mW) for excitation of phycoerythrin, immediately followed by an HeKd laser (325 nm, 50 mW) for excitation of indo-1. Cells were gated on lymphocytes by scatter analysis and all lymphoid cells with any phycoerythrin fluorescence (575⫾13 nm) above background were excluded from the analysis of calcium response (30). Addition of GAM-IgG without priming with CD3 mAb did not result in any rise in cytoplasmic calcium concentration (not shown). Chemical analyses Plasma glucose concentrations were measured by the glucose oxidase method (Glucose analyzer II, Beckman, Fullerton, Calif.), plasma insulin was measured by double antibody RIA (Serono Diagnostics, Freiburg, Germany). Serum FFA were quantified by an enzyme assay employing acyl-CoA synthase and acyl-CoA oxidase with subsequent colorimetric determination of the resulting hydrogen peroxide (Wako Chem. USA, Inc., Richmond, Va.). Plasma triglycerides were hydrolyzed by lipase and the released glycerol was measured by a peroxidase-coupled colorimetric assay (27). Fatty acid composition of the lipid infusion was analyzed by gas chromatography as detailed previously (31). Statistics Data are presented as means ⫾ se unless stated otherwise. In metabolic studies A, results from identical time points obtained with the high FFA group were compared with those from the low FFA group by unpaired Student’s t test. In metabolic studies B, all results were compared with time zero by paired or one-sample Student’s t test, as appropriate. Correlations were estimated by the parametric correlation coefficient (r) or the nonparametric Spearman rank correlation coefficient () as indicated. A two-tail probability of less than 5% was considered statistically significant.
RESULTS Unbound FFA inhibit T cell calcium signaling Stimulation of Jurkat T cells via the T cell receptor (TCR)/CD3 complex or the costimulatory glycosyl 941
phosphatidylinositol (GPI)-anchored molecule CD59 results in a rise in cytoplasmic calcium concentration, which is needed for cell activation (21–23). In the absence of albumin, addition of cis-unsaturated longchain FFA almost instantaneously inhibited the response elicited via both cell surface proteins, whereas saturated and trans-unsaturated long-chain FFA were without effect (Fig. 1A, B). Figure 1 shows results obtained with those saturated and cis-unsaturated fatty acids that are most abundant in the lipid infusion used in metabolic studies altogether making up ⬎95 mol% of fatty acids (cf. Table 1). Inclusion of fatty acid-free albumin, which effectively binds FFA, totally abolished this effect (not shown), indicating that only the unbound fraction of FFA is responsible for immediate inhibition of T cell signaling (3, 6). Unsaturated FFA affected stimulation via both surface proteins, but the extent of inhibition was often greater with the GPIanchored protein CD59 (Fig. 1). Thus, unbound cisunsaturated FFA inhibit calcium signaling in T cells stimulated via various cell surface molecules. Elevated serum FFA inhibit T cell signaling Human blood contains a considerable amount of albumin (⬃770 M), which binds by far most of the FFA present in serum to avoid toxic effects. Therefore, we addressed the question of whether a metabolically induced rise in serum FFA concentration could be sufficient for functional alterations in cultured T cells. Young healthy subjects were given an i.v. lipid infusion with (high FFA) or without i.v. heparin (low FFA) to activate lipoprotein lipase thereby generating considerable amounts of FFA (27). Despite comparable concentrations of serum triglycerides and insulin, the concentration of FFA was selectively elevated in the high FFA group (Fig. 2). According to the fatty acid composition of the lipid infusion, which increased serum triglycerides by a factor of about three and therefore was the predominant substrate for lipoprotein lipase in this setting, ⬃80% of the FFA generated should be cisunsaturated (Table 1) and therefore capable of altering T cell function. Calcium response of Jurkat T cells was significantly diminished in presence of sera from the high FFA group whereas sera from the low FFA group or heparin by itself did not affect T cell signaling (Fig. 3 and data not shown). The response to both stimuli (i.e., CD3 and CD59) was affected in a similar time course, though the extent of inhibition was more pronounced in case of GPIanchored CD59 (Fig. 3; mean difference between high and low FFA at 3 h: CD3: ⫺5.8 percentage points; CD59: ⫺40.5 percentage points). There was a clear-cut negative correlation between the serum FFA concentration and calcium response elicited via both T cell surface molecules (Fig. 4; CD3: r ⫽ ⫺0.6201, ⫽ ⫺0.5820; CD59: r ⫽ ⫺0.6972, ⫽ 942
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Figure 1. Inhibition of calcium response in cultured T cells by cis-unsaturated free fatty acids (FFA). Jurkat T cells exposed to indicated concentrations of various FFA (n⫽4 –7) in the absence of albumin were stimulated via A) transmembrane CD3 or B) the glycosyl phosphatidylinositol (GPI)-anchored protein CD59, and the rise in cytoplasmic calcium concentration was monitored. The maximal response achieved in the after 4 min was detected and related to the ethanol control lacking FFA, which was set to 100%. In FFA abbreviations, the digit behind the colon refers to the number of cis-double bonds unless indicated as trans (t).
⫺0.4735; all Ps ⬍ 0.0005), demonstrating that metabolic serum FFA elevations interfere with T lymphocyte function despite the presence of large amounts of albumin.
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Elevated serum FFA inhibit signaling of autologous T cells Peripheral blood T cells have somewhat altered requirements for stimulation compared to cultured Jurkat T cells and potentially differ in the sensitivity for the inhibitory effect of unbound FFA. Therefore, we evaluated whether signaling of autologous peripheral T cells may be abolished in states of high serum FFA. To this end, blood for mononuclear cell isolation and serum preparation was collected at the
Figure 3. Metabolic studies A: alterations of calcium signaling in cultured T cells. Jurkat T cells were exposed to sera obtained from low and high FFA groups at indicated time points (n⫽7 for each group). Cells were stimulated via A) CD3 or B) GPI-anchored CD59, and the rise in cytoplasmic calcium concentration was monitored. The maximal response achieved in the ensuing 4 min was detected and related to the control at time zero, which was set to 100%. Significant differences between the high and low FFA groups are indicated (*P⬍0.05; **P⬍0.01; ***P⬍0.001).
Figure 2. Metabolic studies A: alterations in metabolic data. Lipid infusion was administered to healthy young men with (high FFA) or without (low FFA) simultaneous infusion of heparin for induction of lipolysis (n⫽7 for each group). The resulting serum concentrations of A) FFA, B) triglycerides, and C) insulin are shown. Significant differences between the high and low FFA groups are indicated (*P⬍0.05; ***P⬍0.001). ELEVATED SERUM FATTY ACIDS INHIBIT T LYMPHOCYTES
same time points during the metabolic intervention; after sample preparation, corresponding cells and sera were again combined prior to the stimulation assay. Using double laser flow cytometry, peripheral T cells (CD20⫺CD33⫺CD56⫺) and their CD4⫹ and CD8⫹ subsets were evaluated for calcium response after CD3 stimulation. Analysis of all T cells together as well as separate evaluation of CD4⫹ and CD8⫹ subpopulations revealed a striking inhibition of calcium response during conditions with high FFA as occurring 3 and 6 h after starting the lipid/heparin infusion (Fig. 5; significance of differences in calcium response compared to time zero at 3 h: all T 943
autologous T lymphocytes by increasing serum FFA concentrations. DISCUSSION Previous data revealed an inhibitory effect of unbound cis-unsaturated FFA on T lymphocyte function in vitro (2– 6), but until now it was questionable
Figure 4. Metabolic studies A: negative correlation of serum FFA concentration and calcium response in cultured T cells. Data on calcium response via A) CD3 or B) GPI-anchored CD59 obtained during metabolic studies A (see Fig. 3) were significantly correlated to the corresponding serum FFA concentrations (see Fig. 2; CD3 stimulation: r ⫽ ⫺0.6201, ⫽ ⫺0.5820; CD59 stimulation: r ⫽ ⫺0.6972, ⫽ ⫺0.4735; all Ps ⬍ 0.0005).
cells, P⫽0.114; CD4⫹, P⫽0.066; CD8⫹, P⫽0.056; at 6 h: all T cells, P⫽0.016; CD4⫹, P⫽0.012; CD8⫹, P⫽0.009). Control samples obtained by application of glycerol instead of lipid infusion maintained normal fasting FFA levels and did not inhibit calcium response of autologous T cells or their subsets (not shown). Furthermore, clear-cut negative correlations between the serum concentration of FFA and the calcium response in T lymphocytes and T cell subpopulations emphasized the inhibitory effect of FFA on T cell calcium response (Fig. 6; all T cells: ⫽ ⫺0.6110, P⫽0.016; CD4⫹ T cells: ⫽ ⫺0.5710, P⫽0.005; CD8⫹ T cells: ⫽ ⫺0.5965, P⫽0.019). Thus, signal transduction measured by the rise in cytoplasmic calcium concentration is inhibited in 944
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Figure 5. Metabolic studies B: elevation of serum FFA concentration and inhibition of calcium response in autologous T lymphocytes and their subsets. Lipid infusion was administered in stepwise increasing rates to healthy young men with simultaneous infusion of heparin for induction of lipolysis (n⫽6). The FFA concentration of sera from indicated time points is given in panel A and compared to time zero (*P⬍0.05; **P⬍0.01). B) Autologous mononuclear cells were exposed to sera from corresponding time points and stimulated via CD3 while monitoring the rise in cytoplasmic calcium concentration in all T cells or their CD4⫹ or CD8⫹ subpopulations as indicated. The significance of differences in maximal calcium response compared to time zero (set to 100%): at 3 h, all T cells, P⫽0.114; CD4⫹, P⫽0.066; CD8⫹, P⫽0.056; at 6 h, all T cells, P⫽0.016; CD4⫹, P⫽0.012; CD8⫹, P⫽0.009).
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Figure 6. Metabolic studies B: negative correlation of serum FFA concentration and calcium response in autologous T lymphocytes and their subsets. Autologous T cells were stimulated for calcium response via CD3, as detailed in legend to Fig. 5, and resulting data were correlated to the corresponding serum FFA concentrations (n⫽6; all T cells: ⫽ ⫺0.6110, P⫽0.016; CD4⫹ T cells: ⫽ ⫺0.5710, P⫽0.005; CD8⫹ T cells: ⫽ ⫺0.5965, P⫽0.019).
whether metabolic increases in serum FFA concentrations could inhibit T lymphocytes despite the high albumin concentration in human serum. Our data clearly show that those in vitro effects are most probably relevant in vivo as well. The metabolic studies performed here aimed at specifically elevating unsaturated serum FFA concentrations while avoiding other metabolic changes. The only other difference between high and low FFA groups in metabolic studies A was the infusion of heparin, which by itself did not affect the lymphocyte calcium response. Since heparin was given at a constant dose throughout the high FFA experiment and other metabolic parameters changed comparably in both low and high FFA groups, the inhibition of T cell signaling with time in the high FFA group was most probably due to the rise in unsaturated serum FFA. Moreover, a direct influence of serum FFA on lymphocyte signaling was further emphasized by the striking correlation between calcium response and serum FFA concentration. In addition, metabolic studies B revealed that elevated FFA not only affect signaling in cultured lymphoid cells, but also strikingly inhibit stimulation of autologous T cells and their major subsets. Thus, activation of lymphocytes could be altered in vivo by unsaturated serum FFA elevation. Unbound cis-unsaturated FFA probably inhibit lymphocyte function by membrane partition of these highly hydrophobic molecules and subsequent perturbation of the lipid environment (20). The steric conformation of FFA seems to be of primary imporELEVATED SERUM FATTY ACIDS INHIBIT T LYMPHOCYTES
tance for immediate functional effects, since transunsaturated fatty acids similar to saturated fatty acids did not alter T cell signaling (6) and almost no fatty acid esterification could occur during the short time of exposure. Moreover, the inhibitory effect of unsaturated FFA was attained within seconds after addition of the FFA, and normal function was promptly restored when removing unbound FFA by binding to albumin (20, and data not shown), indicating that the effect of FFA was due to physicochemical alterations. Supposing that FFA membrane partition underlies lymphocyte inhibition, serum FFA elevation apparently raises the membrane concentration of FFA to sufficiently high levels to evoke functional alterations in lymphocytes despite the very small detectable concentration of truly unbound serum FFA (1). In addition to lymphocytes, metabolic elevation of serum FFA has recently been shown to impair endothelial function (32), suggesting a more general effect of high circulating FFA levels on cells. Activation of lymphocytes is prerequisite for specific immune responses. The elevation of cytoplasmic calcium concentration is one of the obligatory events in this process required for proliferation, T cell-mediated cytolysis, and production of cytokines (21, 23, 33). Thus, detection of calcium response has become a widespread parameter to determine signaling function in lymphoid cells. Unbound FFA may abolish the stimulated rise in cytoplasmic calcium concentration by inhibiting capacitative calcium influx and/or enhancing calcium extrusion from the cytosol (6, 34). This proposed mechanism is consistent with our observation that calcium response triggered via different cell surface molecules, the TCR/CD3 complex and the GPI-anchored protein CD59, were affected by unbound unsaturated FFA, indicating that FFA interact with various if not all signaling molecules on T lymphocytes. T lymphocytes require a second signal in addition to that via the TCR/CD3 complex to result in full activation (35). GPI-anchored proteins such as CD59 are capable of providing costimulatory signals, but due to lipid anchorage and their enrichment in membrane domains of particular lipid composition (36), signaling via these proteins seems predisposed to be affected by lipid alterations (22, 37). Accordingly, calcium response in Jurkat T cells stimulated via GPI-anchored CD59 was often blocked to a considerably greater extent than the response stimulated via CD3 (Fig. 3A, B). Different from Jurkat T cells, CD3 stimulation of freshly isolated peripheral blood T lymphocytes was suppressed by more than 40% under conditions with high-serum FFA compared to stimulation under fasting FFA levels (Fig. 5) indicating that serum FFA elevation could substantially abolish T lymphocyte 945
activation. Activating stimuli are in most cases very weak in vivo compared to in vitro experiments, indicating that the inhibitory effect of unbound FFA on lymphocyte signaling may be even more marked in the in vivo situation. Moreover, as discussed above, serum FFA elevation simultaneously inhibits several T cell signaling pathways and hence may potentiate its suppressive effect on lymphocyte activation in vivo. Serum FFA elevation occurs in insulin resistance and diabetes mellitus type 2, to which it seems causally linked (38), but is also present in neoplastic disease (8, 9) and congestive heart failure (11). Such diseases are often accompanied by clinically important disturbances in immune function (12, 13), predisposing these patients to infectious complications. A direct effect of FFA on lymphocyte activation was once presumed in leukemia patients (8). Serum FFA concentrations in diabetes mellitus and cancer of ⬃800 mol/l, and even exceeding 1500 mol/l, respectively (7, 9), are in the range of those achieved in our metabolic studies and could influence lymphocyte function considerably according to the data obtained. Even when taking into account that the lipid infusion used in this study contains a somewhat higher proportion of unsaturated fatty acids (81 mol%; Table 1) compared to fasting sera from healthy and diabetic individuals (⬃65 mol%) (1, 39), the absolute serum concentrations of cis-unsaturated FFA in the aforementioned patients correspond well to those required for inhibition of lymphocyte activation. Thus, the disturbed immune function in situations with high-serum FFA may be at least partly explained by direct effects of FFA on lymphocyte function. Diets rich in unsaturated fatty acids are generally recommended because of their beneficial effect on the development of cardiovascular disease (40). Unsaturated fatty acid-enriched diets primarily induce changes in the ratio of various unsaturated fatty acid species with only minor alterations in the proportion of total unsaturated vs. saturated fatty acids (41, 42). Thus, serum FFA elevations under pathological conditions usually result in considerably higher absolute serum concentrations of unsaturated FFA than those achieved by dietary alterations of fatty acid composition. Since direct effects of elevated serum FFA on lymphocytes occur to a similar extent with different mono- and polyunsaturated fatty acid species (Fig. 1), dietary unsaturated fatty acids may not add greatly to the direct inhibitory effect of FFA on lymphocyte activation. In conclusion, our data demonstrate that serum FFA elevations can acutely alter lymphocyte function and thus underscore a pathophysiological relevance of high serum FFA concentrations for an altered immune response. More general, the data emphasize 946
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the interrelation of lipids and the immune system beyond the function of particular lipid molecules as intercellular and intracellular messengers (43, 44). This work was supported by the Austrian National Bank (project no. 7196, T.M.S.), the Austrian Science Foundation, project no. P13507-MED (T.M.S.) and no. P13213-MOB (M.R.), and the ICP Program for Molecular Medicine of the Austrian Ministry for Science and Transport (W.W. and T.M.S.).
REFERENCES 1. 2. 3. 4. 5.
6.
7.
8.
9. 10. 11. 12. 13. 14.
15.
16. 17. 18. 19.
Richieri, G. V., and Kleinfeld, A. M. (1995) Unbound free fatty acid levels in human serum. J. Lipid Res. 36, 229 –240 Richieri, G. V., and Kleinfeld, A. M. (1989) Free fatty acid perturbation of transmembrane signaling in cytotoxic T lymphocytes. J. Immunol. 143, 2302–2310 Chow, S. C., Ansotegui, I. J., and Jondal, M. (1990) Inhibition of receptor-mediated calcium influx in T cells by unsaturated non-esterified fatty acids. Biochem. J. 267, 727–732 Richieri, G. V., and Kleinfeld, A. M. (1990) Free fatty acids inhibit cytotoxic T lymphocyte-mediated lysis of allogeneic target cells. J. Immunol. 145, 1074 –1077 Richieri, G. V., Mescher, M. F., and Kleinfeld, A. M. (1990) Short term exposure to cis unsaturated free fatty acids inhibits degranulation of cytotoxic T lymphocytes. J. Immunol. 144, 671– 677 Breittmayer, J. P., Pelassy, C., Cousin, J. L., Bernard, A., and Aussel, C. (1993) The inhibition by fatty acids of receptormediated calcium movements in Jurkat T-cells is due to increased calcium extrusion. J. Biol. Chem. 268, 20812–20817 Reaven, G. M., Hollenbeck, C., Jeng, C. Y., Wu, M. S., and Chen, Y. D. (1988) Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37, 1020 –1024 Brown, R. E., Steele, R. W., Marmer, D. J., Hudson, J. L., and Brewster, M. A. (1983) Fatty acids and the inhibition of mitogeninduced lymphocyte transformation by leukemic serum. J. Immunol. 131, 1011–1016 Legaspi, A., Jeevanandam, M., Starnes, H. F., Jr., and Brennan, M. F. (1987) Whole body lipid and energy metabolism in the cancer patient. Metabolism 36, 958 –963 Hendrickson, S. C., St. Louis, J. D., Lowe, J. E., and AbdelAleem, S. (1997) Free fatty acid metabolism during myocardial ischemia and reperfusion. Mol. Cell. Biochem. 166, 85–94 Lommi, J., Kupari, M., and Yki-Jarvinen, H. (1998) Free fatty acid kinetics and oxidation in congestive heart failure. Am. J. Cardiol. 81, 45–50 Moutschen, M. P., Scheen, A. J., and Lefebvre, P. J. (1992) Impaired immune responses in diabetes mellitus: analysis of the factors and mechanisms involved. Diabetes Metab. 18, 187–201 Levey, D. L., and Srivastava, P. K. (1996) Alterations in T cells of cancer-bearers: whence specificity? Immunol. Today 17, 365–368 Richieri, G. V., Anel, A., and Kleinfeld, A. M. (1993) Interactions of long-chain fatty acids and albumin: determination of free fatty acid levels using the fluorescent probe ADIFAB. Biochemistry 32, 7574 –7580 Rose, H., Conventz, M., Fischer, Y., Ju¨ngling, E., Hennecke, T., and Kammermeier, H. (1994) Long-chain fatty acid-binding to albumin: re-evaluation with directly measured concentrations. Biochim. Biophys. Acta 1215, 321–326 Spector, A. A. (1975) Fatty acid binding to plasma albumin. J. Lipid Res. 16, 165–179 Abumrad, N. A., Perkins, R. C., Park, J. H., and Park, C. R. (1981) Mechanism of long chain fatty acid permeation in the isolated adipocyte. J. Biol. Chem. 256, 9183–9191 Potter, B. J., Sorrentino, D., and Berk, P. D. (1989) Mechanisms of cellular uptake of free fatty acids. Annu. Rev. Nutr. 9, 253–270 Bojesen, I. N., and Bojesen, E. (1994) Binding of archidonate and oleate to bovine serum albumin. J. Lipid Res. 35, 770 –778
The FASEB Journal
STULNIG ET AL.
20. 21. 22.
23. 24.
25.
26.
27.
28.
29. 30.
31.
Anel, A., Richieri, G. V., and Kleinfeld, A. M. (1993) Membrane partition of fatty acids and inhibition of T-cell function. Biochemistry 32, 530 –536 Weiss, A., and Littman, D. R. (1994) Signal transduction by lymphocyte antigen receptors. Cell 76, 263–274 Stulnig, T. M., Berger, M., Sigmund, T., Stockinger, H., Horˇejsˇi, V., and Waldha¨usl, W. (1997) Signal transduction via glycosyl phosphatidylinositol-anchored proteins in T cells is inhibited by lowering cellular cholesterol. J. Biol. Chem. 272, 19242–19247 Guse, A. H. (1998) Ca2⫹ signaling in T-lymphocytes. Crit. Rev. Immunol. 18, 419 – 448 Roden, M., Krssak, M., Stingl, H., Gruber, S., Hofer, A., Fu¨rnsinn, C., Moser, E., and Waldha¨usl, W. (1999) Rapid impairment of skeletal muscle glucose transport/phosphorylation by free fatty acids in humans. Diabetes 48, 358 –364 Boden, G., Jadali, F., White, J., Liang, Y., Mozzoli, M., Chen, X., Coleman, E., and Smith, C. (1991) Effects of fat on insulinstimulated carbohydrate metabolism in normal men. J. Clin. Invest. 88, 960 –966 Roden, M., Price, T. B., Perseghin, G., Petersen, K. F., Rothman, D. L., Cline, G. W., and Shulman, G. I. (1996) Mechanism of free fatty acid-induced insulin resistance in humans. J. Clin. Invest. 97, 2859 –2865 Hultin, M., Bengtsson Olivecrona, G., and Olivecrona, T. (1992) Release of lipoprotein lipase to plasma by triacylglycerol emulsions. Comparison to the effect of heparin. Biochim. Biophys. Acta 1125, 97–103 Stulnig, T. M., Klocker, H., Harwood, H. J., Jr., Ju¨rgens, G., Scho¨nitzer, D., Jarosch, E., Huber, L. A., Amberger, A., and Wick, G. (1995) In vivo low-density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase regulation in human lymphocytes and its alterations during aging. Arterioscler. Thromb. Vasc. Biol. 15, 872– 878 Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of Ca2⫹ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440 –3450 Rabinovitch, P. S., June, C. H., Grossmann, A., and Ledbetter, J. A. (1986) Heterogeneity among T cells in intracellular free calcium after mitogen stimulation with PHA or anti-CD3. Simultaneous use of indo-1 and immunofluorescence with flow cytometry. J. Immunol. 137, 952–961 Raederstorff, D., Meier, C. A., Moser, U., and Walter, P. (1991) Hypothyroidism and thyroxin substitution affect the n-3 fatty acid composition of rat liver mitochondria. Lipids 26, 781–787
ELEVATED SERUM FATTY ACIDS INHIBIT T LYMPHOCYTES
32.
33. 34. 35. 36. 37.
38. 39.
40.
41.
42.
43. 44.
Steinberg, H. O., Tarshoby, M., Monestel, R., Hook, G., Cronin, J., Johnson, A., Bayazeed, B., and Baron, A. D. (1997) Elevated circulating free fatty acid levels impair endothelium-dependent vasodilation. J. Clin. Invest. 100, 1230 –1239 Berridge, M. J. (1993) Inositol trisphosphate and calcium signalling. Nature (London) 361, 315–325 Gamberucci, A., Fulceri, R., and Benedetti, A. (1997) Inhibition of store-dependent capacitative Ca2⫹ influx by unsaturated fatty acids. Cell Calcium 21, 375–385 Schwartz, R. H. (1990) A cell culture model for T lymphocyte clonal anergy. Science 248, 1349 –1356 Simons, K., and Ikonen, E. (1997) Functional rafts in cell membranes. Nature (London) 387, 569 –572 Stulnig, T. M., Berger, M., Sigmund, T., Raederstorff, D., Stockinger, H., and Waldha¨usl, W. (1998) Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains. J. Cell Biol. 143, 637– 644 Reaven, G. M. (1988) Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37, 1595–1607 Miwa, H., Yamamoto, M., Nishida, T., Nunoi, K., and Kikuchi, M. (1987) High-performance liquid chromatographic analysis of serum long-chain fatty acids by direct derivatization method. J. Chromatogr. 416, 237–245 (Anonymous). (1994) National Cholesterol Education Program. Second Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel II). Circulation 89, 1329 –1445 Hamazaki, T., Nakazawa, R., Tateno, S., Shishido, H., Isoda, K., Hattori, Y., Yoshida, T., Fujita, T., Yano, S., and Kumagai, A. (1984) Effects of fish oil rich in eicosapentaenoic acid on serum lipid in hyperlipidemic hemodialysis patients. Kidney Int. 26, 81– 84 Herold, P. M., and Kinsella, J. E. (1986) Fish oil consumption and decreased risk of cardiovascular disease: a comparison of findings from animal and human feeding trials. Am. J. Clin. Nutr. 43, 566 –598 Traill, K. N., and Wick, G. (1984) Lipids and lymphocyte function. Immunol. Today 5, 70 –76 Chandra, R. K., and Amorin, S. A. D. (1992) Lipids and immunoregulation. Nutr. Res. 12, S137–S145 Received for publication August 6, 1999. Revised for publication November 24, 1999.
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