1 Centre for Rheumatic Diseases, Akersbakken 27, Rikshospitalet, 0172 Oslo,
2 Department of Pharmacology, University of Oslo and
3 Department of Immunology and Transfusion Medicine, Ullevaal University Hospital, N-0407 Oslo, Norway
Correspondence to:
D. A. Fraser, Department of Immunology and Transfusion Medicine, Ullevaal University Hospital, N-0407 Oslo, Norway.
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Abstract |
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Methods. The concentration and composition of plasma FFAs were measured in nine RA patients at the conclusion of a 7 day fast. A FFA mixture was made up based on these findings (20% linoleic, 43% oleic, 10% stearic, 27% palmitic acid). Mitogen-induced lymphocyte proliferative responses were measured after co-culture of peripheral blood mononuclear cells (PBMC) from healthy individuals in the presence of increasing concentrations of this FFA mixture (from 0 to 2000 µM) and in the presence of FFA mixtures where the relative proportions of fatty acids varied.
Results. Both the concentration of the FFA mixture and the ratio between the unsaturated and saturated fatty acids significantly influenced in vitro lymphocyte proliferation (P<0.0001). Unexpectedly, the highest concentrations of the FFA mixture increased lymphocyte proliferation. At equimolar concentrations (600 µM), manipulating the amounts of oleic and linoleic fatty acids relative to stearic and palmitic fatty acids had a potent inhibitory effect upon lymphocyte proliferation.
Conclusion. Fasting-associated increases in total plasma FFA concentrations do not inhibit, but rather enhance, in vitro lymphocyte proliferation. An inhibitory effect could only be achieved by manipulating the balance between the unsaturated and saturated fatty acids.
KEY WORDS: Rheumatoid arthritis, Fasting, Fatty acids, Lymphocyte proliferation.
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Introduction |
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Whilst it is known that long-chain n-3 and n-6 fatty acids have immunomodulatory effects, it is not known whether changes in the total concentration of circulating free fatty acids (FFAs) influence immune responses. Substantial increases in the total concentrations of circulating FFAs occur under certain environmental and metabolic conditions, such as prolonged fasting [7] and insulin resistance, and may be of pathological significance [8].
For unknown reasons, fasting has significant beneficial effects upon both clinical and laboratory variables of disease activity in rheumatoid arthritis (RA) [9, 10]. We hypothesized that fasting-induced changes in the concentrations of serum FFAs might inhibit lymphocyte function and could contribute to the observed anti-inflammatory effects.
We have measured the concentration of plasma FFAs in nine RA patients before and after a 7 day fast. Lymphocyte proliferative responses after mitogenic stimulation with anti-CD3 were measured in the presence of increasing concentrations of a FFA mixture, the composition of which was based on our in vivo findings. As it has been suggested that the clinical improvements in RA patients during fasting may be related to changes in the fatty acid composition of cell membranes [10], we also measured the cellular fatty acid composition of peripheral blood mononuclear cells (PBMC) pre- and post-fasting.
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Patients and methods |
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Sample collection and storage
Pre- and post-fasting blood samples, drawn between 10.00 and 11.30 a.m., were obtained. Ethylenediaminetetraacetic acid (EDTA)whole blood was collected and placed on ice. Plasma was separated and frozen at -70°C for subsequent analysis of FFA composition and concentrations. PBMC were isolated from the RA patients pre- and post-fasting, and were frozen at -70°C until subsequent analysis of total cellular fatty acid composition.
For proliferation assays, PBMC were isolated from healthy controls and were resuspended in RPMI 1640 containing 10% fetal calf serum and 0.6 mM bovine serum albumin (BSA; essentially fatty acid free; Sigma Chemical Co., St Louis, MO, USA) at a concentration of 2x106 cells/ml. More than 96% of the cells were viable as assessed by the trypan blue exclusion test.
Fatty acid analysis
FFAs from EDTAplasma and total cellular lipids were extracted and measured by gasliquid chromatography (GLC) as fatty acid methyl esters. After extraction of plasma [11], the FFAs were incubated with 1 ml methanolic HCl (3 M ) for 15 min at 37°C. The mixtures were neutralized by 2 ml NaHCO3 (0.7 M ) and extracted twice with 2 ml n-hexane. After evaporation of the solvent, the lipids were redissolved in n-hexane, and an aliquot was injected into the chromatograph (Shimadzu, GC-14A), equipped with a polar capillary column (Supelco SPE 2560, length 100 m). The oven temperature was programmed to rise from 135 to 193°C at 56°C/min. Identification of individual methyl esters was performed by comparisons with authentic standard mixtures. Heptadecanoic acid (C17:0) was used as an internal standard for quantification of unesterified fatty acids.
After extraction of PBMC, the residual lipids were redissolved in 1 ml benzene and methylated overnight in 2 ml methanolic HCl (3 M) and 200 ml 2,2-dimethoxypropane. The mixtures were neutralized by 4 ml NaHCO3 (0.7 M ) and the procedure for analysis of FFAs was followed as above.
Lymphocyte proliferation assay
Fifty microlitres (1x105 cells) of the cell suspension were added to each well of a round-bottom 96 well microtitre plate (Falcon Plastics). A FFA mixture (final concentration 2666 µM ) based on the average percentage composition of FFAs in vivo was made up. The mixture consisted of 20% linoleic acid, 43% oleic acid, 10% stearic acid and 27% palmitic acid (all Sigma Chemical Co., St Louis, MO, USA) and contained 0.6 mM BSA (equivalent to in vivo albumin concentration). Various amounts of the fatty acid mixture (from 0 to 150 µl) were added to the cell suspension as required and the volume in each well was brought up to 200 µl by adding RPMI 1640 containing 0.6 mM BSA.
For additional experiments, two fatty acid mixtures were made: an unsaturated mixture containing equimolar concentrations of oleic acid and linoleic acid, and a saturated mixture containing equimolar concentrations of palmitic and stearic acid. BSA was added at a final concentration of 0.6 mM. Cell cultures were incubated in 20% O2 /5% CO2 at 37°C. After 24 h, an anti-CD3 (OKT3D) monoclonal antibody (MAb) was added, and the mixtures were incubated for a further 72 h under identical conditions.
Viability was assessed by the trypan blue exclusion test; 1 mCi methyl [3H]thymidine was added for the final 18 h before harvesting and [3H]thymidine incorporation was measured by liquid scintillation. All experiments were carried out in triplicate and results were expressed as counts per minute (c.p.m.).
Statistical analyses
Within-group differences at fixed time points were tested by Wilcoxon's signed rank test. To test whether different concentrations or compositions of fatty acids influenced lymphocyte proliferation in vitro, we used analysis of variance. A randomized complete block model using triplicate values as a blocking factor was used. Fisher's protected least square difference was used to identify which combinations of the unsaturated fatty acid mixture and the saturated fatty acid mixture differed significantly from the representative in vivo combination. Two-sided P values of <0.05 were considered as significant. The analyses were carried out using STATVIEW 4.1 and Super ANOVA 1.11 (both Abacus Concepts, Berkeley, CA, USA).
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Results |
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Altering the ratio between the unsaturated fatty acid mixture and the saturated fatty acid mixture significantly influenced lymphocyte proliferation (P<0.001) (Fig. 1b). The post hoc analysis revealed that as compared to 400 µM unsaturates+200 µM saturates together (representative of the in vivo ratio), 600 µM of the unsaturates alone had a significant inhibitory effect (68% reduction; P<0.001). Substitution of only 50 µM of the unsaturates with 50 µM of the saturates reversed this inhibitory effect. Incubation of the cells in the presence of saturates only also had a significant inhibitory effect as compared to the in vivo ratio (65% reduction; P<0.001). Substitution of 50 µM of the saturated mixture with 50 µM of the unsaturated mixture (50 µM unsaturated+550 µM saturated) had a less potent but still significant inhibitory effect as compared to the in vivo ratio (44% reduction; P<0.001).
Cell viability
There were no significant differences in cell viability related either to the type or concentration of fatty acids used in culture. The mean cell viability in the cell cultures activated with anti-CD3 was 84% (range 7988%). Mean cell viability in PBMC isolated from wells containing resting cells was 94%.
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Discussion |
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That in vitro lymphocyte proliferation increased with increasing concentrations of the FFA mixture does not support the idea that fasting-induced increases in circulating FFAs inhibit lymphocyte proliferation. The concentrations of FFAs we selected for our in vitro experiments approximated the observed changes in vivo, where the lowest concentrations of FFAs pre-fasting and the highest concentration post-fasting were 230 and 2400 µM, respectively. Thus, in contrast to the dose-dependent inhibitory effect upon lymphocyte proliferation which occurs with individual fatty acids [15], the opposite occurs when the FFAs are presented as a mixture of saturated and unsaturated fatty acids. Possible explanations for this effect may be that increased concentrations of FFAs provide a greater energy source for proliferating lymphocytes [16]. The increasing FFA/albumin ratio (from 0.37 at the 250 µM concentration to 3.33 at the 2000 µM concentration) may also be of significance.
To achieve an inhibitory effect upon lymphocyte proliferation in vitro using the predominant circulating FFAs, it was necessary to alter the balance of fatty acids towards either an excess of unsaturated or saturated fatty acids. Similar findings have been reported in concanavalin A-stimulated rat lymph node lymphocytes [12]. The inhibitory effects we observed may occur secondary to changes in membrane fluidity induced by an excess of either unsaturated fatty acids or saturated fatty acids. The fluidity and flexibility of the cell membrane play a central role in both receptor expression and protein movement in the membrane [17], both of which are intimately involved in T-cell activation.
The practical implications of these observations to the in vivo situation are unclear. Although circulating FFAs are ultimately determined by diet, it is unlikely that the ratio of unsaturates to saturates required for T-cell inhibition to occur in vitro could be achieved in vivo. However, it has been reported that a very low intake of saturated fats is beneficial in multiple sclerosis, where, like in RA, CD4+ lymphocytes are thought to play a pathogenic role [18]. Whether such a diet could have immunosuppressive effects in vivo and could thus be of benefit in the treatment of RA should be investigated.
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Acknowledgments |
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References |
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