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1 From the Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Reading, United Kingdom (ST, SK, CMW, and PY), and the Institute of Human Nutrition, University of Southampton, Southampton, United Kingdom (GCB, TB, JJR, RFG, and PCC)
2 Supported by grant EFH/16 from the BBSRC, DEFRA, SEERAD, and the Milk Development Council under the Eating, Food and Health LINK scheme (to PCC, PY, RFG, and CMW). The capsules used in the study were a gift from Natural AS, Hovdebygda, Norway. 3 Address reprint requests to P Yaqoob, Hugh Sinclair Unit of Nutrition, School of Food Biosciences, The University of Reading, Whiteknights, Reading RG6 6AP, United Kingdom. E-mail: p.yaqoob{at}reading.ac.uk.
ABSTRACT
Background: Animal studies have suggested that conjugated linoleic acid (CLA), a natural component of ruminant meat and dairy products, may confer beneficial effects on health. However, little information on the effects of CLA on immune function is available, especially in humans. Furthermore, the effects of individual isomers of CLA have not been adequately investigated.
Objective: This study investigated the effects of supplementing the diet with 3 doses of highly enriched cis-9,trans-11 CLA (0.59, 1.19, and 2.38 g/d) or trans-10,cis-12 CLA (0.63, 1.26, and 2.52 g/d) on immune outcomes in healthy humans.
Design: The study had a randomized, double-blind, crossover design. Healthy men consumed 1, 2, and 4 capsules sequentially that contained 80% of either cis-9,trans-11 CLA or trans-10,cis-12 CLA for consecutive 8-wk periods. This regimen was followed by a 6-wk washout and a crossover to the other isomer.
Results: Both CLA isomers decreased mitogen-induced T lymphocyte activation in a dose-dependent manner. There was a significant negative correlation between mitogen-induced T lymphocyte activation and the proportions of both cis-9,trans-11 CLA and trans-10,cis-12 CLA in peripheral blood mononuclear cell lipids. However, CLA did not affect lymphocyte subpopulations or serum concentrations of C-reactive protein and did not have any consistent effects on ex vivo cytokine production.
Conclusion: CLA supplementation results in a dose-dependent reduction in the mitogen-induced activation of T lymphocytes. The effects of cis-9,trans-11 CLA and trans-10,cis-12 CLA were similar, and there was a negative correlation between mitogen-induced T lymphocyte activation and the cis-9,trans-11 CLA and trans-10,cis-12 CLA contents of mononuclear cells.
Key Words: Conjugated linoleic acid • cytokines • immunity • inflammation • lymphocytes
INTRODUCTION
The term conjugated linoleic acid (CLA) collectively refers to a group of positional and geometrical isomers of the essential fatty acid linoleic acid. These conjugated fatty acids are formed as a result of the biohydrogenation of linoleic acid in ruminants and are deposited in tissues and milk. CLA has received considerable attention as a result of animal experiments, which report anticarcinogenic (1–4), antiatherosclerotic (5, 6), and antidiabetic (7) effects, although the findings of different studies have not always been consistent.
CLA has also been reported to modulate the immune response. The first reported effects of CLA were enhanced phytohemagglutinin-induced foot pad swelling, macrophage phagocytic activity, and spleen lymphocyte blastogenesis in rodents, while protecting against weight loss induced by bacterial LPS injection (8, 9). More recently, CLA has been reported to alter cytokine production (10–14) and to increase lymphocyte proliferation (10, 14) in vitro and in animal studies. A dietary study in pigs suggested that CLA feeding resulted in an alteration in the phenotype of cytotoxic T lymphocytes (15). Most studies conducted to date have involved feeding animals with mixtures of CLA isomers that contained predominantly cis-9,trans-11 CLA and trans-10,cis-12 CLA in roughly equal amounts. However, emerging evidence suggests that different CLA isomers may have different effects on human health (16, 17).
There is very little information in relation to the effects of CLA supplementation on immune and inflammatory responses in humans. Two human studies have shown that CLA supplementation had no effect on many indexes of immune function, including the number of circulating leukocytes, lymphocyte subsets, lymphocyte proliferation, eicosanoid and cytokine secretion, delayed-type hypersensitivity (DTH), or serum antibody titers in healthy women (18–20). These studies used between 3 and 6 g/d of CLA supplemented as Tonalin capsules, which contained a range of CLA isomers, in which cis-9,trans-11 CLA and trans-10,cis-12 CLA together represented 20% of total fatty acids. A more recent study compared the effects of 1.7 g/d of a 50:50 mix and 1.6 g/d of an 80:20 mix of cis-9,trans-11 CLA and trans-10,cis-12 CLA for 84 d in 71 healthy men but reported no effect of CLA on the DTH response, natural killer cell activity, lymphocyte proliferation, production of prostaglandin E2, or production of cytokines (21). However, the study showed that almost twice as many subjects reached protective antibody concentrations to hepatitis B after supplementation with 1.7 g/d of a 50:50 mix of cis-9,trans-11 CLA and trans-10,cis-12 CLA compared with control subjects (21). The effect was not observed in the 80:20 group, which led the authors to suggest that trans-10, cis-12 CLA was the active isomer (21). However, there has been no direct comparison of highly enriched cis-9,trans-11 CLA and trans-10,cis-12 CLA to date.
We previously showed that both cis-9,trans-11 CLA and trans-10,cis-12 CLA are incorporated into peripheral blood mononuclear cell (PBMC) lipids in a dose-dependent manner (22). The aim of the current study was to determine 1) whether supplementation with CLA isomers affects immune function in healthy humans in a dose-dependent manner and 2) whether cis-9,trans-11 CLA and trans-10,cis-12 CLA differ in their effects on immune function.
SUBJECTS AND METHODS
Materials
HEPES-buffered RPMI medium was obtained from Autogen Bioclear (Calne, United Kingdom). Histopaque, glutamine, antibiotics (penicillin and streptomycin), concanavalin A (Con A), and Escherichia coli 0111:B4 LPS were purchased from Sigma Chemical Co Ltd (Poole, United Kingdom). Fluorescein isothiocyanate-labeled mouse anti-human CD3, CD14, and CD19 and R-phycoerythrin-labeled mouse anti-human CD4, CD8, CD16, CD19/anti-, CD54, and CD69 were purchased from Serotec Ltd (Kidlington, United Kingdom). Kits for measurement of cytokines in culture supernatant fractions [human T helper subsets 1 and 2 and inflammation cytokine Cytometric Bead Array (CBA) kits] were purchased from Becton Dickinson (Oxford, United Kingdom). The solvents were purchased from Fisher Ltd (Loughborough, United Kingdom), and all other reagents were from Sigma Chemical Co, Ltd.
Subjects and study design
The study was conducted at the University of Southampton and The University of Reading, with approval from the Research and Ethics Committee of The University of Reading and the South and West Hampshire Local Research Ethics Committee. Healthy men aged 20–47 y were recruited by advertisements, and potential volunteers were selected after confirming that they were healthy; had a body mass index (BMI; in kg/m2) >18 and <32; had no diagnosis of cardiovascular disease, diabetes, liver or endocrine dysfunction, or chronic inflammatory disease; were not taking any medication; were not vegetarians or vegans; were not heavy smokers (>10 cigarettes/d); were not heavy consumers of alcohol (<10 units/wk); and were not consuming any supplements (eg, vitamins, fish oils, or evening primrose oil). Subjects fitting these criteria were then screened for fasting plasma concentrations of cholesterol (3.0–5.2 mmol/L), triacylglycerol (0.45–1.81 mmol/L), and glucose (3.9–5.8 mmol/L) and for markers of liver dysfunction (total bilirubin, alanine aminotransferase, and aspartate aminotransferase). All volunteers gave informed consent and completed a health and lifestyle questionnaire before entering the study.
The study had a randomized, double-blind, crossover design. Randomization was stratified for age, BMI, and fasting triacylglycerol concentration. Forty-nine subjects participated in the study. Because of the long study duration (13 mo), some subjects were unable to make every visit. Therefore, the sample number for each time point ranges between 39 and 49 subjects. Subject characteristics at baseline were as follows: mean age (31.0 ± 1.1 y), BMI (24.6 ± 0.4), fasting plasma triacylglycerol concentration (0.9 ± 0.03 mmol/L), and total cholesterol concentration (4.4 ± 0.1 mmol/L). Subjects were asked to consume either encapsulated trans-10,cis-12 CLA or cis-9,trans-11 CLA for 3 consecutive 8-wk periods, with increasing doses before crossing over to the other isomer.
Subjects therefore consumed each isomer for 6 mo, separated by a 6-wk washout. The CLA isomers were provided in 750-mg gelatin-coated capsules that contained 80–85% of either cis-9,trans-11 or trans-10,cis-12 CLA in triacylglycerol form. The cis-9,trans-11 CLA-enriched capsules contained 79.3% cis-9,trans-11 CLA, 7.8% trans-10,cis-12 CLA, 5.8% 18:1n–9, and 7.1% other fatty acids. The trans-10,cis-12 CLA-enriched capsules contained 84.1% trans-10,cis-12 CLA, 10.6% cis-9,trans-11 CLA, 1.7% 18:1n–9, and 5.3% other fatty acids. The fatty acid composition of the capsules was analyzed by gas chromatography, as described previously (22). The capsules used were identical in appearance and were packaged in the same manner.
Subjects consumed 1, 2, or 4 capsules/d, which provided 0.59, 1.19, or 2.38 g/d cis-9,trans-11 CLA and 0.63, 1.26, or 2.52 g/d trans-10,cis-12 CLA, respectively. Mean compliance assessed by capsule counting (92%) was not significantly different between doses or isomer treatment. Furthermore, detailed analysis of the fatty acid composition of plasma phospholipids and cholesteryl esters has shown an excellent dose-response relation for the incorporation of both isomers of CLA (22). The capsules were well tolerated.
C-reactive protein analysis
Blood samples were collected into SST evacuated tubes between 0800 and 1000 after fasting for 10 h. Serum was prepared and stored at –20°C before analysis. Serum CRP was measured in an ILab 600 clinical chemistry analyzer (Instrumentation Laboratories Ltd, Warrington, United Kingdom) with the use of high-sensitivity CRP kits (Biokit SA, Barcelona, Spain).
Analysis of PBMC subsets
For the determination of PBMC subsets, whole blood (100 µL) was incubated with various combinations of fluorescently labeled monoclonal antibodies (10 µL of each antibody) for 30 min at 4°C. The monoclonal antibody combinations used were anti-CD3/anti-CD4 (to distinguish T lymphocytes as CD3+ and T helper lymphocytes as CD3+CD4+), anti-CD3/anti-CD8 (to distinguish cytotoxic T lymphocytes as CD3+CD8+), anti-CD3/anti-CD16 (to distinguish natural killer cells as CD3–CD16+), anti-CD14/anti-CD54 [to distinguish monocytes as CD14+ and to determine the level of expression of the intercellular adhesion molecule-1 (ICAM-1; CD54) on monocytes], and anti-CD19/anti- (to distinguish B lymphocytes expressing the immunoglobulin light chain as CD19+/+). Erythrocytes were then lysed with the use of 2 mL lysing solution (3.75 mL formaldehyde, 4.5 mL diethylene glycol, and 1.75 mL of 0.2 mol/L Tris made up to 1 L with distilled water) and the leukocytes were washed and then fixed with 0.2 mL fixing solution (phosphate-buffered solution containing 2 mL/100 mL formaldehyde). Fixed leukocytes were analyzed with a FACSCalibur flow cytometer (Becton Dickinson). Fluorescence data were collected on 1 x 104 cells and analyzed with Cellquest software. All flow cytometry methods were cross-validated across the 2 sites.
Measurement of mitogen-induced T lymphocyte activation in whole blood
Mitogen-induced lymphocyte activation was determined by measuring the expression of CD69, a cell surface marker whose expression is up-regulated in response to stimulation (23). The method used was essentially as described by Lim et al (24). Briefly, blood was diluted 1:1 with culture medium and then cultured for 24 h with Con A at final concentrations of 0, 6.25, 12.5, or 25 mg/L; the final volume of the culture was 250 µL. For the determination of CD69 expression on lymphocytes, the stimulated, diluted whole blood (200 µL) was incubated with fluorescently labeled monoclonal antibodies for 30 min at 4°C. The monoclonal antibodies used were anti-CD69 in combination with either anti-CD3, anti-CD4, or anti-CD8. At the completion of incubation, the erythrocytes were lysed and the leukocytes were fixed. The cell preparations were then analyzed by flow cytometry in a Becton Dickinson FACSCalibur flow cytometer (Becton Dickinson). Fluorescence data were collected on 2 x 104 cells and analyzed with Cellquest software. The proportions of all activated T lymphocytes (CD3+), T helper cells (CD4+), and cytotoxic T cells (CD8+) expressing CD69 on their surface were determined.
Preparation of PBMCs
Blood samples were collected into heparin-treated evacuated tubes between 0800 and 1000 after the subjects fasted for 10 h. The blood was layered onto Histopaque (density: 1.077 g/L; ratio of blood to Histopaque: 1:1) and centrifuged for 15 min at 800 x g at 20°C. PBMCs were collected from the interface and washed once with RPMI medium containing 0.75 mmol/L glutamine and antibiotics (penicillin and streptomycin) (culture medium). After resuspension in 4-mL culture medium, the cells were layered onto 4 mL Histopaque and then centrifuged once more (15 min, 800 x g, 20°C) to achieve a lower degree of erythrocyte contamination, washed with culture medium, and finally resuspended and counted with a Coulter Z1 Cell Counter (Beckman Coulter Ltd, Luton, United Kingdom).
Measurement of the production of cytokines by PBMC cultures
PBMCs (2 x 106) were cultured for 48 h in culture medium, supplemented with 50 mL autologous plasma/L, and either unstimulated or stimulated with 12.5 mg Con A/L or 5 mg LPS/L; the final culture volume was 2 mL. At the end of the incubation, the plates were centrifuged (800 x g, 10 min, room temperature) and the culture medium was collected and frozen in aliquots. The concentrations of cytokines were measured by flow cytometry with the use of cytometric bead arrays (Becton Dickinson). Tumor necrosis factor (TNF-), interleukin (IL) 1ß, IL-6, IL-8, and IL-10 were measured in the supernatant fractions of cells stimulated with LPS; IL-2, IFN-, IL-10, IL-5, TNF-, and IL-4 were measured in the supernatant fractions of cells stimulated with Con A. The limits of detection for the LPS assays were 3.7 pg/mL (TNF-), 3.3 pg/mL (IL-10), 2.5 pg/mL (IL-6), 7.2 pg/mL (IL-1ß), 3.6 pg/mL (IL-8), and 7.1 pg/mL (IFN-); those for the Con A assays were 2.8 pg/mL (TNF-), 2.8 pg/mL (IL-10), 2.4 pg/mL (IL-5), 2.6 pg/mL (IL-4), and 2.6 pg/mL (IL-2) (data supplied by the manufacturer of the kits). Fluorescence data were collected for 180 of each cytokine per capture bead of R1-gated events and analyzed with the use of Becton Dickinson CBA software (Becton Dickinson). The interassay and intraassay CVs were <10% for all cytokine bead arrays.
Statistical analysis
All statistical tests were performed with the use of SPSS (version 11.0; SPSS Inc, Chicago), and a P value <0.05 indicated statistical significance. Preliminary statistical analyses were used to determine whether there were any period effects or treatment-period interactions as a result of the crossover design. This analysis was simplified by comparing the change for each parameter tested, during each period, by using independent-sample t tests. There was no period effect and no treatment-period interaction (data not shown); thus, all data were treated as paired samples from a crossover study. Data that were not normally distributed were logarithmically transformed. Data were analyzed by using a two-factor repeated-measures analysis of variance (ANOVA) followed by a post hoc analysis where relevant (one-factor repeated-measures ANOVA, followed by Tukey's tests for a significant effect of dose and paired t tests for a significant effect of isomer). For the lymphocyte activation experiment, data were first analyzed by using a three-factor repeated-measures ANOVA to test for the effects of CLA dose, CLA isomer, and Con A concentration. Correlations between the incorporation of cis-9,trans-11 and trans-10,cis-12 CLA (after treatment) into PBMC lipids and measures of immune function were determined as Spearman's rank-order correlation coefficients ().
RESULTS
Effects of cis-9,trans-11 CLA and trans-10,cis-12 CLA on PBMC subsets
There were no consistent effects of CLA on the proportions of T lymphocytes, helper T cells, cytotoxic T cells, B lymphocytes, natural killer cells, or monocytes, although in some cases there were significant effects of isomer only or dose only (Table 1). However, there was a significant effect of dose (P < 0.001) and an isomer x dose interaction (P < 0.05) on the proportion of monocytes expressing ICAM-1. The expression of ICAM-1 was lower after supplementation with the highest dose of both isomers than at baseline or after the 2 lower doses of CLA (comparison of marginal means for the 2 isomers combined across doses; Table 1).
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TABLE 1. Effect of cis-9,trans-11 conjugated linoleic acid (CLA) and trans-10,cis-12 CLA on peripheral blood mononuclear cell (PBMC) subsets1
Effects of cis-9,trans-11 CLA and trans-10,cis-12 CLA on mitogen-induced T lymphocyte activation
Mitogen-induced activation of lymphocytes was assessed by the expression of the early T cell activation marker, CD69, on T lymphocytes (CD3+/CD69+ cells), helper T lymphocytes (CD4+/CD69+ cells), and cytotoxic T lymphocytes (CD8+/CD69+) (Table 2). Expression of CD69 correlates strongly with lymphocyte proliferation, as assessed by incorporation of [3H]thymidine (data not shown), and it has the advantage that it does not require long cell culture periods, during which the effects of fatty acid manipulation could be lost. There were significant effects of Con A concentration and dose of CLA on CD69 expression, and a significant dose x Con A concentration interaction (three-factor repeated-measures ANOVA, P 0.001). However, there was no effect of isomer. Comparison of marginal means for the 2 isomers combined across doses indicated that increasing doses of CLA resulted in decreased lymphocyte activation at 12.5 and 25 mg Con A/L for T lymphocytes and T helper cells and 12.5 mg Con A/L only for cytotoxic T lymphocytes. Thus, both isomers of CLA resulted in a dose-dependent decrease in lymphocyte activation over a range of Con A concentrations.
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TABLE 2. Effect of cis-9,trans-11 conjugated linoleic acid (CLA) and trans-10,cis-12 CLA on activation of T lymphocytes, T helper lymphocytes, and cytotoxic T lymphocytes in response to concanavalin (Con A) stimulation1
The percentage change from baseline CD69 expression by T lymphocytes in response to CLA isomers, after stimulation of lymphocytes with 12.5 mg/L Con A, is shown in Figure 1. There was a significant effect of dose, but not of isomer, on the change from baseline CD69 expression (P < 0.001). Comparison of marginal means for the 2 isomers combined across doses confirmed that both isomers decreased CD69 expression in a dose-dependent manner.
FIGURE 1.. Mean (±SEM) change from baseline in T (CD3+) lymphocytes expressing CD69+ after supplementation with 1, 2, or 4 capsules/d of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acids (CLAs) in 39–49 subjects. CD69+ expression was assessed as described in Subjects and Methods, with cells stimulated by 12.5 mg concanavalin A/L. There was a significant effect of dose (two-factor repeated-measures ANOVA) but no significant effect of isomer and no isomer x dose interaction for the change in CD69+expression (P < 0.001). The marginal means for the percentage change in CD69+ expression by T lymphocytes for the 2 isomers combined across doses were significantly different (one-way repeated-measures ANOVA, P < 0.001), with a dose of 4 capsules/d producing a significantly greater change than either 1 or 2 capsules/d (post hoc Tukey's test, P < 0.02). One capsule per day provided 0.59 g cis-9,trans-11 CLA/d or 0.63 trans-10,cis-12 CLA/d; 2 capsules/d provided 1.19 cis-9,trans-11 CLA/d or 1.26 trans-10,cis-12 CLA/d; and 4 capsules/d provided 2.38 cis-9,trans-11 CLA/d or 2.52 trans-10,cis-12 CLA/d.
Relation between incorporation of cis-9,trans-11 CLA and trans-10,cis-12 CLA into PBMC lipids and mitogen-induced T lymphocyte activation
We previously reported a dose-dependent incorporation of both cis-9,trans-11 CLA and trans-10,cis-12 CLA into PBMC lipids in the subjects under investigation in the current study (22). The relations between the cis-9,trans-11 CLA and trans-10,cis-12 CLA contents of PBMC lipids and CD69 expression by T lymphocytes after stimulation with 25 mg Con A/L are shown in Figure 2. For both isomers, there was a significant negative relation between the CLA content of PBMCs and mitogen-induced T lymphocyte activation (Figure 2). The same relations were observed at 6.25 and 12.5 mg Con A/L, although they were not statistically significant (data not shown).
FIGURE 2.. Relation between the cis-9,trans-11 conjugated linoleic acid (CLA) (A) and trans-10,cis-12 CLA (B) contents of peripheral blood mononuclear cell (PBMC) lipids and T lymphocyte activation. Fatty acid composition data before and after supplementation with cis-9,trans-11 CLA or trans-10,cis-12 CLA were published previously (22). *P < 0.05 (Spearman's correlation).
Effects of cis-9,trans-11 CLA and trans-10,cis-12 CLA on cytokine production by PBMCs
The effects of each of the CLA isomers on the production of inflammatory cytokines (TNF-, IL-10, IL-6, IL-1ß and IL-8) are shown in Table 3. There were no significant effects of isomer on the production of these cytokines (two-factor repeated-measures ANOVA), but there was a significant effect of dose on the production of TNF- and IL-1ß (P < 0.001 and P < 0.05, respectively). However, there was no significant isomer x dose interaction on the production of any of these cytokines. The effects of CLA on the production of lymphocyte-derived T helper 1 and T helper 2 cytokines (IFN-, TNF-, IL-10, IL-5, IL-4, and IL-2) are shown in Table 4. There were no significant effects of isomer or dose on the production of IL-4 or IL-2. However, there was a significant effect of dose on the production of IFN-, TNF-, IL-10, and IL-5 (two-factor repeated-measures ANOVA, P < 0.01), but no significant dose x isomer interaction.
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TABLE 3. Effect of cis-9,trans-11 conjugated linoleic acid (CLA) and trans-10,cis-12 CLA on production of inflammatory cytokines by peripheral blood mononuclear cells in response to lipopolysaccharide1
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TABLE 4. Effect of cis-9,trans-11 conjugated linoleic acid (CLA) and trans-10,cis-12 CLA on production of T helper 1 and 2 cytokines by peripheral blood mononuclear cells in response to concanavalin A1
Effects of cis-9,trans-11 CLA and trans-10,cis-12 CLA on serum CRP concentrations
There were no effects of either isomer of CLA on serum CRP concentrations (data not shown).
DISCUSSION
Animal studies that have examined the influence of CLA at doses of 0.1–1.5% (by weight) of the diet on immune function have reported effects on DTH, lymphocyte proliferation, antibody responses, and production of cytokines (8–15). However, a recent review concluded that, given the inconsistencies in the data, it was not possible to determine whether CLA affected lymphocyte proliferation in response to mitogens, that the data did not support the claim that CLA feeding improves antibody responses, and that there were discrepancies in the reported effects of CLA on ex vivo cytokine production (25). Kelley and Erickson (25) questioned the accuracy of the reported compositions for the CLA mixtures used in these animal studies and highlighted the fact that most of them tested the effects of CLA in growing animals whose food intake was not controlled. Only one animal study to date has examined the separate effects of cis-9,trans-11 CLA and trans-10,cis-12 CLA on immune function (26). This study, conducted in mice, reported that both CLA isomers increased the ex vivo production of TNF- and IL-6 and decreased that of IL-4 compared with a control group, whereas neither isomer affected the proliferation of splenic lymphocytes.
There is very little information in relation to the effects of CLA supplementation on immune and inflammatory response in humans. Two recent human showed that CLA supplementation had no effect on many indexes of immune function (18–20), although both studies used mixtures containing many CLA isomers, with the cis-9,trans-11 CLA and trans-10,cis-12 isomers each representing 20% of the total CLA. A third study compared the effects of 1.7 g/d of a 50:50 mix and 1.6 g/d of an 80:20 mix of cis-9,trans-11 CLA and trans-10,cis-12 CLA for 84 d in 71 healthy men, but reported no effect of CLA on the DTH response, natural killer cell activity, lymphocyte proliferation, production of prostaglandin E2, or production of cytokines (21). However, the study showed that almost twice as many subjects reached protective antibody concentrations to hepatitis B (>10 IU/L) after supplementation with 1.7 g/d of a 50:50 mix of cis-9,trans-11 CLA and trans-10,cis-12 CLA compared with control subjects, although it is notable that mean antibody titers did not differ significantly between the 3 groups (21). Because the effect was not observed in the 80:20 group, the authors suggested that trans-10, cis-12 CLA was the active isomer and that there may be isomer-specific effects of CLA on immune function (21). However, Kelley and Erickson (25) question the interpretation resulting from the use of arbitrary thresholds for seroprotective titers. Furthermore, in an earlier study, a CLA mixture had no effect on influenza antibody titers (18). Thus, the effects of CLA on human immune function are still unclear and require direct comparison of cis-9,trans-11 CLA and trans-10,cis-12 CLA. The aim of the current study, therefore, was to determine whether supplementation with CLA isomers affects immune function in healthy humans in a dose-dependent manner and whether highly purified preparations of cis-9,trans-11 CLA and trans-10,cis-12 CLA differ in their effects on immune function.
In the current study, there were no effects of either isomer of CLA on PBMC subsets, which is consistent with the finding of Kelley et al (18) and Krieder et al (19). There were no consistent effects of either isomer of CLA on ex vivo cytokine production, which is consistent with the studies of Kelley et al (18) and Albers et al (21). There was also no effect of either isomer of CLA on serum CRP concentrations. Only one other study has examined the effect of CLA on circulating CRP; this study compared the effects of 3.4 g/d of a 50:50 mix of cis-9,trans-11 CLA and trans-10,cis-12 CLA, a highly purified preparation of trans-10,cis-12 CLA, and a placebo (olive oil) in men with metabolic syndrome (27). This study reported an increase in plasma CRP concentrations after supplementation with both CLA preparations, although only the effect of the highly purified trans-10,cis-12 CLA was significantly different from the placebo (27). Because CRP is an independent risk factor for coronary risk (28), the authors highlighted this detrimental effect of trans-10,cis-12 CLA (albeit at a high dose) as a potential cause for concern (27). The highest dose of trans-10,cis-12 CLA used in the current study (2.52 g/d for 8 wk) was lower than that provided in the study by Riserus et al (3.4 g/d for 12 wk; 27), which may account for the lack of effect of CLA on CRP in the current study. However, the lack of effect was more likely due to the fact that the subjects in the current study were healthy volunteers with an average BMI of 24.6 ± 0.4 whereas the subjects in the study by Riserus et al (27) had metabolic syndrome and their BMIs ranged from 27 to 39. It is well known that one of the major determinants of circulating CRP concentrations is BMI (29) and, not surprisingly, the CRP concentrations of subjects in the current study were low. It would appear, therefore, that whereas CRP concentrations are unaffected by trans-10,cis-12 CLA in healthy subjects of normal weight, they are elevated in overweight subjects, who may already have elevated CRP concentrations.
Although the current study did not include a placebo treatment, it is important to note that the statistical analysis confirmed that there were no period or treatment effects and no period x treatment interactions. Essentially, this demonstrates that regardless of the order of treatment, the effects of the treatments were the same in both arms of the study. Because each treatment lasted for 6 mo and the entire study lasted 13 mo, any effects of CLA were deemed unlikely to represent a transient effect of time. Furthermore, there was no carryover effect of either treatment. Also, although compliance was reported in the form of capsule counts in this study, we showed an excellent dose-response relation for the incorporation of both isomers of CLA into plasma phospholipids and cholesteryl esters and into PBMC lipids (22).
This study showed for the first time a dose-dependent reduction in the mitogen-induced activation of T lymphocytes by both cis-9,trans-11 CLA and trans-10,cis-12 CLA. Furthermore, there was a significant negative correlation between CD69 expression and the proportions of both cis-9,trans-11 CLA and trans-10,cis-12 CLA in PBMC lipids, which suggests that the lower CD69 expression may be associated with incorporation of both CLA isomers into cellular lipid pools. Because no other fatty acids were altered, we suggest that the effect was specifically due to CLA. However, the mechanisms by which dietary fatty acids modulate immune function at the cellular and molecular level are still poorly understood (30, 31), and this is especially true for CLA. Furthermore, because the function of CD69 is not known, the implications and relevance of the effects of CLA on mitogen-induced lymphocyte activation are not clear. Thus, whereas a reduction in lymphocyte activation may be undesirable in terms of host defense, it may be useful in situations such as allergy and inflammatory disease. It is perhaps important to note that there was little effect of CLA isomers at the lowest dose, which represents an approximate doubling of the estimated daily CLA intakes from dairy products in the United Kingdom (chiefly providing cis-9,trans-11 CLA; 32). Provision of CLA in dairy products at the doses reported to inhibit lymphocyte activation in the current study would probably only be possible in experimental settings. Nevertheless, CLA is available commercially in supplement form, so there is the potential for individuals to be exposed to high doses.
Finally, it is notable that in the current study, the effect of CLA with respect to mitogen-induced lymphocyte activation was not specific to the cis-9,trans-11 or the trans-10,cis-12 isomer. This finding is in contrast with the effects of CLA on blood lipids in the same subject group, in which there was a striking and consistent pattern suggesting opposing effects of the 2 isomers on the ratios of LDL cholesterol to HDL cholesterol and of total cholesterol to HDL cholesterol (17). Overall, there appeared to be a detrimental effect of trans-10,cis-12 CLA, relative to cis-9,trans-11 CLA, on the blood lipid profile (17). This suggests that some of the effects of CLA on human health may be isomer-specific, whereas others are not.
In conclusion, CLA did not affect lymphocyte subpopulations, ex vivo cytokine production, or serum CRP concentrations, but both cis-9,trans-11 CLA and trans-10,cis-12 CLA decreased mitogen-induced lymphocyte activation in a dose-dependent manner.
ACKNOWLEDGMENTS
PCC, PY, CMW, and RFG designed the study and supervised the experimental work. ST, GCB, SK, TB, and JJR screened, recruited, and sampled the volunteers and conducted the experimental work. ST and PY analyzed the data. ST and PY wrote the manuscript, with input from all authors. None of the authors had any financial or personal interest in any company or organization sponsoring the research, including advisory board affiliations.
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