The effects of conjugated linoleic acid supplementation on immune function in healthy volunteers

Abstract

Objective:

To assess the effects of dietary supplementation using two isomeric blends of conjugated linoleic acid (CLA) on immune function in healthy human volunteers.

Design:

Double-blind, randomised, placebo-controlled intervention trial.

Subjects and intervention:

A total of 55 healthy volunteers (n=20 males, n=35 females) were randomised into one of three study groups who received 3 g/day of a fatty acid blend containing a 50:50 cis-9, trans-11: trans-10, cis-12 CLA isomer blend (2 g CLA), and 80:20 cis-9, trans-11: trans-10, cis-12 (80:20) CLA isomer blend (1.76 g CLA) or linoleic acid (control, 2 g linoleic acid) for 8 weeks.

Results:

Supplementation with the 80:20 CLA isomer blend significantly (P≤0.05) enhanced PHA-induced lymphocyte proliferation. CLA decreased basal interleukin (IL)-2 secretion (P≤0.01) and increased PHA-induced IL-2 and tumor necrosis factor α (TNFα) production (P≤0.01). However, these effects were not solely attributable to CLA as similar results were observed with linoleic acid. CLA supplementation had no significant effect on peripheral blood mononuclear cells IL-4 production, or on serum-soluble intercellular adhesion molecule-1 (sICAM-1) or plasma prostaglandin E2 (PGE2) or leukotreine B4 (LTB4) concentrations.

Conclusions:

This study shows that CLA supplementation had a minimal effect on the markers of human immune function. Furthermore, supplementation with CLA had no immunological benefit compared with linoleic acid.

Sponsorship:

CLA supplements were provided by Loders Croklaan.

Introduction

Conjugated linoleic acid (CLA) refers to a mixture of geometric and positional conjugated dieonic isomers of linoleic acid (C18:2, n-6). CLA is synthesised endogenously in ruminant animals through the action of the enzyme Δ9 desaturase on vaccenic acid, and, to a lesser extent, during microbial biohydrogenation of linoleic and linolenic acids (Kepler et al, 1966; Griinari et al, 1998, 2000). Hence, dairy products and ruminant meats are the natural dietary sources of CLA, where most (80%) of the CLA in meat and dairy products is present as the cis-9, trans-11 isomer (c9,t11-CLA) (Chin et al, 1992; Kramer et al, 1997; Parodi, 1997). Estimated average daily intakes of CLA in humans range from 95 to 200 mg/day (Chin et al, 1992; Ens et al, 2001; Ritzenthaler et al, 2001). Commercially, CLA can be produced by base-catalysed isomerisation of linoleic acid (Banni & Martin, 1998), resulting in a mixture in which the predominant isomer types include the c9,t11-CLA (35%) and a trans-10, cis-12 CLA isomer (t10,c12-CLA) (35%).

Research over the last 10 y has demonstrated that CLA can influence lipoprotein metabolism, body composition, carcinogenesis, and immune function (Roche et al, 2001). In particular, CLA appears to possess intrinsic immuno-modulatory properties. In vitro studies show that a CLA mix enhanced porcine blood lymphocyte proliferation in response to the T-cell mitogen phytohemagglutinin (PHA), inhibited concanavalin A (Con A)-induced interleukin (IL)-2 production and suppressed the phagocytic activity of murine macrophages (Chew et al, 1997). In vivo studies in chickens, mice and rats demonstrate a significant increase in mitogen-stimulated lymphocyte blastogenesis following supplementation with a CLA blend (Cook et al, 1993; Miller et al, 1994; Turek et al, 1998; Hayek et al, 1999). Indeed dietary CLA, particularly c9,t11-CLA, exerts specific effects on the CD8+ T-cell subset, increasing the number and function of these T cells in pigs and mice (Bassaganya-Riera et al, 2001, 2002; Yamasaki et al, 2003). CLA blends are also reported to depress macrophage phagocytosis (Cook et al, 1993; Miller et al, 1994) and to modulate eicosanoid production, particularly prostaglandin E2 (PGE2) (Li & Watkins, 1998; Kavanaugh et al, 1999). In addition, CLA isomeric blends enhanced IL-2 production in mice (Wong et al, 1997; Hayek et al, 1999; Yang & Cook, 2003), but reduced IL-4, IL-6 and tumor necrosis factor α (TNFα) production in BALB/c mice and in Sprague–Dawley rats (Turek et al, 1998; Yang & Cook, 2003). Most of the above studies used CLA isomeric blends.

More recently, animal studies have focused on the potential isomer-specific effects of CLA. C57BL/6N mice exhibited no significant difference in ex vivo PGE2 secretion, Con A-stimulated IL-2 production, or Con A- and lipopolysaccharide (LPS)-stimulated splenocyte proliferation, following an 8-week diet enriched with purified c9,t11-CLA or t10,c12-CLA (Kelley et al, 2002). However, both of the CLA isomers increased ex vivo LPS-stimulated TNFα and IL-6 production and decreased Con A-induced IL-4 secretion. The authors concluded that the two CLA isomers had similar effects on the immune response in these mice (Kelley et al, 2002). Yamasaki et al (2003) supplemented C57BL/6J mice purified c9,t11-CLA, t10,c12-CLA, or a 50:50 blend of these two isomers for 3 weeks. Supplementation with the c9,t11-CLA isomer alone increased Con A-stimulated TNFα production: no differences were observed between the groups with respect to IL-2, IL-4, IL-5 and interferon-γ (IFN-γ) production. However, an increase in B-cell number and function was reported following supplementation with the t10,c12-CLA isomer (Yamasaki et al, 2003). Nevertheless, it remains unclear whether the immuno-modulatory effects observed in the aforementioned studies can be ascribed to a particular CLA isomer.

There have been relatively few studies that have investigated the effect of CLA on humans. At the outset of this study, none were published. More recently, Kelley et al (2000, 2001) demonstrated no effect of CLA supplementation on lymphocyte proliferation, serum antibody titers, delayed-type hypersensitivity (DTH), ex vivo LPS-stimulated secretion of PGE2, leukotriene B4 (LTB4), IL-1β, TNFα or PHA-induced IL-2 production. CLA supplementation also had no effect on the proportion of T cells producing IL-2 and IFN-γ, or on the percentage monocytes secreting TNFα (Kelley et al, 2000, 2001). While this study was conducted in a highly controlled metabolic suite, their findings were based on a very small sample size (n=10) and the CLA blend used consisted of a heterogenous mix of four CLA isomers. In addition, Albers et al (2003) demonstrated that a 50:50 blend of the c9,t11-CLA: t10,c12-CLA isomers for 12 weeks augmented seroprotective antibody levels in response to hepatitis B, suggesting that CLA may enhance B-cell function in humans. At the time of commencing this study, there was no information as to the influence of the individual CLA isomers on human immune function. The objective of the present study was to determine whether CLA supplementation modulates the immune response in healthy free-living humans. This double-blind placebo-controlled trial investigated the effects of dietary supplementation with two isomeric blends of CLA on lymphocyte function and cytokine production in healthy free-living volunteers. The CLA blends provided different levels of the c9,t11-CLA and t10,c12-CLA isomers. One group received a 50:50 isomeric blend of the c9,t11-CLA: t10,c12-CLA isomers (50:50 CLA group), while another group received an 80:20 isomeric blend of c9,t11-CLA: t10,c12-CLA isomers (80:20 CLA group).

Subjects and methods

Subjects and experimental design

The study was approved by the Ethics Committee of the Federated Dublin Voluntary Hospital, Ireland. Written informed consent was obtained from all volunteers before commencing the trial. A total of 55 healthy volunteers participated in this study; these subjects were recruited from the personnel of Trinity College Dublin and St James's Hospital, Dublin. A screening blood sample ensured that all subjects conformed with the following biochemical exclusion criteria: fasting plasma cholesterol <7.0 mmol/l, plasma triglyceride <2.0 mmol/l, glucose <5 mmol/l, gammaglutamyltransferase <60 IU/l, haemaglobin >12.0 g/dl and BMI <25 kg/m2. This study was double blinded and placebo controlled. Subjects were randomly assigned to one of three intervention groups who received 3 g/day of capsules containing either a 80:20 CLA isomer blend, a 50:50 CLA isomer blend or linoleic acid (control). Each capsule weighed 500 mg and was composed of 67.2% total CLA (50:50 blend), 58.8% total CLA (80:20 blend) or 66.9% linoleic acid (no CLA). Each volunteer was allotted six capsules per day. Thus daily intake of CLA was approximately 2.016 g in the 50:50 blend group and 1.764 g in the 80:20 blend group. Linoleic acid intake in the control group was 2 g. Percentage intakes of the c9,t11-CLA and t10,c12-CLA isomers were 50:50 and 80:20, respectively in the CLA treatment groups. All capsules were stabilised with 500 p.p.m. Tocomix L 50, which contained a mixture of α, β, γ and δ tocopherols in sunflower oil, 1:1. The fatty acid compositions of the supplements of the three study groups are shown in Table 1. Compliance was verified by measurement of plasma total fatty acid composition by gas chromatography and by conducting a capsule count of which 96% were consumed. There was no difference in capsule intake between the study groups. All participants were nonsmokers, had no history of inflammatory disorders and avoided taking prescribed medications or dietary supplements. Volunteers also completed a basic lifestyle and dietary questionnaire, which screened for vegetarians, individuals with excessive dairy and meat consumption, or vigorous physical activity patterns. Participants were asked to maintain their typical dietary habits and lifestyle patterns throughout the study period.

Table 1 Capsule composition of the 50:50 and 80:20 cis-9, trans-11: trans-10, cis-12 isomer blends and the control treatment, linoleic acida,b

At week 0 and week 8, each volunteer fasted for 12 h and donated a fasting blood sample. Subjects abstained from alcohol and refrained from strenuous exercise for 24 h before the investigation. Body weight and height were recorded on both occasions.

Preparation of peripheral blood mononuclear cells

Fasting blood samples (50 ml) were collected in lithium heparin-coated tubes (Beckton Dickinson UK Ltd, Cowley, Oxford, UK) and diluted 1:1 with HEPES-buffered Hanks balanced salt solution (HBSS, Gibco, Grand Island, New York, USA). The diluted blood was gently layered onto lymphoprep (density 1.077 g/l, ratio of diluted blood:lymphoprep 1:5, Nycomed Pharma, Oslo) and centrifuged at 800 × g for 30 min. Peripheral blood mononuclear cells (PBMCs) were removed from the interface and washed twice in RPMI complete medium supplemented with 2 mmol L-glutamine/l, 100 μg streptomycin/ml and 100 μg penicillin/ml (Gibco, Grand Island, New York, USA). Cell viability was assessed by fluorescence microscopy using ethidium bromide/acridine orange (Lee et al, 1975), cells were counted and resuspended in complete medium at 2 × 106 cells/ml for both lymphocyte proliferation and cytokine assays.

Lymphocyte proliferation assays

Triplicate aliquots of cells (100 μl, 2 × 106 cells/ml) were seeded in 96-well round bottom plates with 5 μl autologous serum (2.5% by vol) and subsequently cultured in the presence and absence of the T-cell mitogens; Phytohemagglutinin (PHA-P, 10 μg/ml, Sigma-Aldrich, Poole, Dorset, UK), Con A (10 μg/ml, Sigma- Aldrich), the murine monoclonal anti-human CD3 antibody OKT3 (10 μg/ml, CRL 8001, ATCC, Rockville, MD, USA), the murine monoclonal anti-mouse MHC antibody (antibody control), anti-IE (30 μg/ml, HB179, ATCC, Rockville) or negative control (no mitogen, extra RPMI added). Additional RPMI was added to each well to yield a total volume of 200 μl/well and cell concentration of 1 × 106 cells/ml. Plates were incubated at 37°C, 5% CO2, 95% humidity for 72 h. Lymphocyte proliferation was assessed as the incorporation of 3[H] thymidine (0.3 μCi/well, specific activity 6.7 Ci/mmol, New England Nuclear, Boston, MA, USA) during the final 18 h of the culture period. At the end of the incubation period, plates were immediately frozen at −70°C, defrosted within 3 months, and cells harvested onto glass fibre filters using a cell harvester (Inotech, Dottikon, Switzerland). Radiolabel uptake was assessed using a liquid scintillation counting system (Wallac, Turku, Finland). Previous experiments in our laboratory confirmed no significant differences in radiolabel uptake between freshly harvested and frozen PBMCs. Results are expressed as actual counts per minute of radioactive decay (cpm).

Cytokine assays (IL-2, TNF α and IL-4)

To ascertain IL-2 and TNF α activity, 2 × 106 cells/ml in RPMI (500 μl) with 2.5% v/v autologous serum (25 μl) were cultured in 24-well flat bottomed plates in the presence and absence of the mitogens PHA-P (10 μg/ml), LPS (10 μg/ml, Sigma Aldrich), OKT3 (10 μg/ml) or anti-IE (30 μg/ml). IL-4 production was investigated in PHA-P-stimulated cells only. A large batch of all mitogens and antibodies was prepared prior to commencing the study; this stock of stimulants was used for both the lymphocyte proliferation and cytokine assays pre- and post-supplementation. In-house experiments identified these mitogen/antibody concentrations to cause maximal lymphocyte proliferation and cytokine production using our culture system. Again additional RPMI was added to each well to yield a final volume and cell concentration of 1 ml and 1 × 106 cells/ml, respectively. At 24 h supernatants were collected and frozen at −70°C for single-batch analysis by ELISA at the end of the intervention study. Samples were processed in a random fashion so that the groups were analysed simultaneously and each individuals pre- and post-samples were analysed on the same plate. IL-2, TNF α and IL-4 concentrations (pg/ml) quantified using commercial ELISA kits (R&D Systems, Abingdon, Oxon, UK). Lowest detectable levels of IL-2, TNFα and IL-4 were 15.6, 15.6 and 7.8 pg/ml, respectively.

Soluble cell adhesion molecule expression

In all, 5 ml of blood was collected at weeks 0 and 8 in additive-free vacutainers (Beckton Dickinson UK Ltd, Cowley, Oxford, UK). Serum was isolated and frozen at −70°C for subsequent analysis by commercial ELISA (R&D Systems). Lowest detectable limits of these assays were 0.35 ng/ml.

Circulating prostaglandin and leukotriene concentrations

In all, 10 ml blood was collected in EDTA tubes (Beckton Dickinson UK Ltd), plasma separated and frozen at −70°C for future determination of circulating PGE2 and LTB4 concentrations by commercial ELISA (R&D Systems). The sensitivity of these assays was 8.25 and 19.5 pg/ml for the PGE2 and LTB4 assays, respectively.

Fatty acid analysis

Total lipids were extracted according to the method derived by Folch et al (1957). Lipid present in the organic phase was dried using a vortex evaporator (AGB Scientific, Dublin, Ireland). The dried samples were flushed with nitrogen, sealed to prevent oxidation and stored at −20°C. Fatty acid methyl esters were prepared according to Noone et al (2002) and Black et al (2002). Briefly, total plasma lipid was extracted with 0.5 ml 0.01 M NaOH in dry methanol. To transmethylate the fatty acids, 0.75 ml of 14% BF3 was added to the samples, vortexed and incubated at 60°C for 15 min. The resultant fatty acid methyl esters were extracted three times using 0.5 ml hexane, and the samples dried in a vortex evaporator and stored under nitrogen at −20°C until analysis.

The fatty acid methyl ester composition of total plasma lipids were analysed for incorporation of CLA isomers using a Shimadzu GC-14A gas liquid chromatograph (Mason Technologies, Dublin), which was fitted with a Shimadzu C-16A integrator. A CP Sil 88 fused Silica Column (50 m × 0.22 μm file thickness; Chrompack Ltd, Middleburg, The Netherlands) was installed. Nitrogen was used as the carrier gas. Oven temperature conditions for each run were an initial column temperature of 120°C, increasing to 180°C at a rate of 8°C per minute. Column temperature was held at 180°C for 40 min, subsequently increased to 220°C at a rate of 4°C per min and held at 220°C for 15 min. A FAME standard spiked with known concentrations of the c9,t11-CLA and t10,c12- CLA isomers was used for peak identification. Fatty acids were identified with retention times compared to standards. Fatty acid compositions were calculated as a percentage of the total fatty acids.

Statistical analysis

Results are expressed as means±s.e.m. for the number of observations indicated. All statistical analysis was performed with the Apple Macintosh compatible statistical package DataDesk 4.1 (Data Description Inc., NY, USA). The distribution of the data for each variable was assessed and some of the variables transformed to normalise the distribution of some of the data sets. One-way ANOVA confirmed no difference in baseline variables between the study groups. Repeated measures ANOVA was used to identify significant treatment effects (CLA vs placebo effects) or treatment × time interactions (CLA or placebo effects pre- vs post-supplementation) for changes in biochemical parameters following supplementation. Post hoc statistical analysis was completed using a protected least significant difference (LSD) test. A P-value of ≤0.05 was considered statistically significant.

Results

Subject characteristics

All 55 volunteers who entered the study (20 male, 35 female) completed it. The total study group had a mean age 31.5 (s.e.m. 1.3) y, mean weight 69.3 (s.e.m. 7.4) kg and mean BMI 23.9 (s.e.m. 0.05) kg/m2. Subject characteristics are shown in Table 2. There were no significant differences in body weight and BMI between any of the treatment groups at baseline, or as a result of the dietary supplementation. No untoward side effects of ingesting the CLA or control supplements were reported.

Table 2 Subject characteristics pre- and post-supplementationa

CLA enhanced PHA-induced lymphocyte proliferation

Figure 1 presents the effect of CLA supplementation on PHA-, Con A- and OKT3-induced PBMC proliferation pre- and post-intervention. Supplementation with the 80:20 CLA isomer blend significantly (P≤0.05) increased PHA-induced proliferation. Proliferation was not significantly altered in either of the CLA groups following stimulation with Con A, the anti-CD3 monoclonal antibody OKT3 or with the antibody control, anti-IE. Dietary supplementation with the control fatty acid (linoleic acid) had no significant effect on lymphocyte proliferation in response to any of the stimulants tested.

Figure 1
figure1

Effects of supplementation with 50:50 CLA isomer blend, 80:20 CLA isomer blend and linoleic acid on mitogen-stimulated lymphocyte proliferation. Results are expressed as counts per minute of radioactive decay (cpm). aIndicates a statistical difference between group mean values pre- and post-supplementation.

CLA has no effect on cytokines and eicosanoids and soluble cell adhesion molecule expression

Table 3 shows the effects of supplementation with 50:50 CLA isomer blend, 80:20 CLA isomer blend and linoleic acid on ex vivo PBMC IL-2 production in unstimulated (basal) and PHA- and OKT3-stimulated cells. Repeated measures ANOVA demonstrated a significant treatment effect (P≤0.01) whereby supplementation with the 50:50 blend, 80:20 CLA blend and the control fatty acid (linoleic acid) significantly increased IL-2 secretion in response to PHA. In contrast, IL-2 production was significantly reduced in resting cells (P≤0.01) in all three supplement groups and in response to the negative antibody control, anti-IE (P≤0.01), in the 50:50 CLA and control groups.

Table 3 Effects of supplementation with a 50:50 and 80:20 cis-9, trans-11: trans-10, cis-12 CLA isomer blend or linoleic acid on IL-2 production (pg/ml)

Table 4 presents the effect of supplementation with 50:50 CLA blend, 80:20 CLA blend or linoleic acid on basal, PHA, LPS, and OKT3-stimulated ex vivo PBMC TNFα production. Repeated measures ANOVA showed that PHA-induced TNFα production was significantly increased following supplementation with the 50:50 CLA mix (P≤0.01), the 80:20 CLA mix (P≤0.001) and linoleic acid (control fatty acid) (P≤0.001). However, there was no significant difference in the effect of fatty acid supplementation on TNFα production between the treatment groups. Ex vivo basal, LPS and OKT3-stimulated PBMC TNFα secretion were not altered by CLA supplementation.

Table 4 Effects of supplementation with a 50:50 and 80:20 cis-9, trans-11, trans-10, cis-12 CLA isomer blend or linoleic acid on TNFα production (pg/ml)

PHA-induced PBMC IL-4 production, serum sICAM-1 concentration or plasma PGE2 and LTB4 concentrations were determined. CLA supplementation had no significant effect on these markers of these inflammatory markers (Tables 5 and 6).

Table 5 Effects of supplementation with either a 50:50 or 80:20 cis-9, trans-11: trans-10, cis-12 CLA blend, or linoleic acid (3 g/day for 8 weeks) on PHA-stimulated IL-4 production (pg/ml) and on serum sICAM-1 concentrations (pg/ml)
Table 6 Effects of supplementation with a 50:50 cis-9, trans-11: trans-10, cis-12 CLA isomer blend, an 80:20 cis-9, trans-11, trans-10, cis-12 CLA isomer blend or linoleic acid (3 g/dday for 8 weeks) on plasma PGE2 and LTB4 concentrations (pg/ml)

Fatty acid composition of total plasma lipids

The effect of CLA supplementation on the proportion of c9,t11-CLA in total plasma fatty acid is presented in Figure 2. There was a significant increase in plasma c9,t11-CLA levels in the groups who received the 50:50 CLA (P≤0.001) and the 80:20 CLA isomers (P≤0.0001), respectively. The t10,c12-CLA isomer was undetectable in most samples. The increase in the proportion of c9,t11-CLA in these groups was associated with a significant decrease in C18:3, n-3 in the 50:50 CLA group (P≤0.01), and C20:5, n-3 in the 80:20 CLA treatment group (P≤0.05) postdietary intervention. No other significant changes in fatty acid composition were observed following CLA supplementation. The control supplements (linoleic acid) had no significant effect on total plasma fatty acid composition.

Figure 2
figure2

The effect of dietary supplementation using isomeric blends of CLA and linoleic acid at 3 g/day for 8 weeks on total plasma fatty acid composition (g/100 g total plasma lipids). Repeated measures ANOVA shows significant treatment × time interaction aP≤0.001, bP≤0.0001.

Discussion

This study shows that CLA supplementation modulated lymphocyte proliferation; however, other components of the human immune response (IL-2, TNFα, IL-4, sICAM-1, PGE2, LTB4) were not altered. Our study showed that PHA-induced lymphocyte proliferation was significantly enhanced by the 80:20 CLA isomer. The 50:50 CLA blend and linoleic acid did not affect lymphocyte proliferation. While cytokine production was affected by CLA supplementation, this change also occurred in the control group. Therefore, any change in cytokine expression can be attributed to PUFA supplementation and not uniquely to CLA. Careful consideration was given to the choice of control oil used in the present study since every fatty acid has the potential to modulate the immune system. Linoleic acid was chosen since it represented the nonconjugated control for CLA.

To date, the majority of studies investigating the effect of CLA on the immune system have been in vitro or animal studies. The majority of these have shown that CLA enhances lymphocyte proliferation (Cook et al, 1993; Miller et al, 1994; Chew et al, 1997; Turek et al, 1998; Hayek et al, 1999). Our data agree with these data whereby the 80:20 CLA blend enhanced PHA-induced lymphocyte proliferation. In contrast, the 50:50 CLA isomer blend had no effect on Con A-induced lymphocyte proliferation, which concurs with the results of Kelley et al (2002). There are considerable methodological considerations with respect to the lymphocyte proliferation assay, especially when studying the effects of CLA. The aforementioned used foetal or bovine calf sera in their culture systems. These sera are known to mask lymphocyte proliferation (Yaqoob et al, 1994) and are endogenous sources of CLA (Park & Pariza, 1998), factors that may conceal the true effect of exogenous CLA. Hence, we used autologous serum in our study. The fact that the 80:20 but not the 50:50 CLA blend enhanced PHA-induced blastogenesis may be ascribed to an isomer-specific immuno-modulatory effect of CLA. Indeed, Kelley et al (2002) showed that purified t10,c12-CLA tended to reduce LPS-induced splenocyte proliferation at lower endotoxin concentrations. Thus, the greater level of t10,c12-CLA in the 50:50 CLA blend may have negated any proproliferative action of c9,t11-CLA observed in the 80:20 CLA group. However, further studies are required to confirm these findings. Otherwise, the difference in lymphocyte proliferation between the CLA treatments could be due to the different stimulators of proliferation. OKT3 is a monoclonal antibody, which activates T cells by binding specifically to the T-cell receptor (TCR) and activating protein kinase C. PHA and Con A are two polyclonal T-cell mitogens, which ultimately result in protein kinase C activation and T-cell proliferation (Goldsmith & Greene, 1996). Con A has been reported to bind to the α and β chains of the TCR, while PHA-P (the form of PHA used in this study) binds to the TCRαβ and also to CD2 (O'Flynn et al, 1985, 1986). It has been reported that simultaneous activation of CD2 and CD3 may have a synergistic effect on T-cell activtion (Holter et al, 1988; Webb et al, 1990), which would explain the greater proliferative response observed following treatment with PHA, but not Con-A or OKT3.

Since this trial was initiated, the results of two human CLA intervention trials have been published. The first reported no significant effect of a heterogenous mix of four CLA isomers on PHA-induced lymphocyte proliferation (Kelley et al, 2000, 2001). While this study was highly controlled, it was conducted in a very small sample size. The second study demonstrated that 50:50 and 80:20 (c9,t11-CLA:t10,c12-CLA) blends of CLA for 8 weeks had no significant effect on LPS or PHA-induced proliferation (Albers et al, 2003). Although the isomeric mixes of CLA are similar to those used in our study, the amount of CLA supplemented was lower than in the current study. In addition, there were differences in experimental procedures between the proliferation assays. Thus, the higher dose and/or different experimental conditions may explain the differences between studies.

IL-2 promotes T-cell proliferation and differentiation (Goldsmith & Greene, 1996), B-cell differentiation (Waldmann et al, 1984), monocytic and natural killer cell (NKC) activity and cytokine expression (Hillman & Haas, 1995). Three animal feeding trials showed that CLA isomeric blends significantly increased Con A- and PHA-induced IL-2 production in mice (Wong et al, 1997; Hayek et al, 1999; Yang & Cook, 2003), whereas two studies reported no effect (Kelley et al, 2002; Yamasaki et al, 2003). Indeed, Kelley et al (2002) showed that feeding C57Bl/6N mice diets enriched with the purified c9,t11-CLA or t10,c12-CLA did not affect Con A-stimulated IL-2 production. The effects of CLA on TNFα production in animals are also controversial. TNFα is a potent proinflammatory cytokine that induces activation of monocytes and macrophages, and other proinflammatory cytokines (Tracey & Cerami, 1994; Hillman & Haas, 1995). Dietary supplementation with CLA blends decreased plasma TNFα concentrations and basal macrophage TNFα production but had no effect on LPS-induced TNFα secretion in rat or murine macrophages (Turek et al, 1998; Yang & Cook, 2003). Conversely, studies investigating the effects of the purified isomers have yielded equivocal results. In the study of Kelley et al (2002) both the c9,t11-CLA and t10,c12-CLA significantly enhanced LPS-stimulated TNFα secretion in C57BL/6 mice, while Yamasaki et al (2003) reported a significant increase in Con A-induced TNFα production following dietary supplementation with the c9,t11-CLA alone. In contrast, in vitro studies with Raw264.7 macrophages showed that the c9,t11-CLA was associated with a dose-dependent decrease in LPS-induced TNFα production, with smaller decreases associated with the t10,c12-CLA isomer and a CLA isomeric mix (Yang & Cook, 2003). Recent human studies demonstrate that supplementation with CLA blends had no significant effect on ex vivo PHA-induced IL-2 secretion or LPS-stimulated TNFα production (Kelley et al, 2000, 2001; Albers et al, 2003). In our study, PHA-induced IL-2 and TNFα production was enhanced by CLA, which agrees with some of the animal studies; however, this also occurred in the control group. Therefore, within the context of the immuno-modulatory effects of n-6 PUFA (Calder, 1998), our results show that both CLA and linoleic acid enhanced the capacity of PHA-stimulated PBMCs to secrete IL-2 and TNFα, which is suggestive of enhanced cell-mediated immunity.

IL-4 production is indicative of Th2-like phenotype or humoral immune response. IL-4 promotes the growth and differentiation of B- and T cells, antibody production and mediates the regulatory effects on macrophages (Puri & Siegel, 1993). Our study showed that CLA supplementation had no significant effect on PHA-induced IL-4. This finding is in agreement with Albers et al (2003). Intercellular adhesion molecule-1 (ICAM-1, CD54), a transmembrane adhesion molecule expressed on endothelial cells, polymorphonuclear cells and fibroblasts (Kevil & Bullard, 1999). sICAM-1 is the product of proteolytic cleavage of the extracellular region of leukocyte ICAM-1 (Ghaisas et al, 1997) and levels are elevated in patients with coronary vascular disease (CVD) (Ridker et al, 1998). CLA supplementation had no effect on circulating sICAM-1 concentrations in this study group who were young, normolipaemic nonsmokers and had no history of inflammatory conditions. Nevertheless, the effect of CLA and sICAM-1 expression should be reassessed in patients at high risk of CVD.

PGE2 and LTB4 are arachidonic acid (C20:4 n-6; AA) derived metabolites involved in inflammation (Goodwin & Cueppens, 1983). In rats CLA lowered serum PGE2 and ex vivo splenic LTB4 expression (Sugano et al, 1998). In contrast, in humans CLA supplementation did not affect ex vivo LPS-stimulated secretion of PGE2, LTB4 (Kelley et al, 2001; Albers et al, 2003). Our study confirms that CLA has no significant effect on plasma PGE2 and LTB4 concentrations. CLA supplementation did not alter plasma arachidonic acid levels. While PBMC composition was not determined, we can only propose that no alteration of PBMC arachidonic acid accounts for this null effect.

Plasma fatty acid composition was used in this study to check compliance to the CLA supplement. Total plasma lipid c9,t11-CLA levels were significantly increased by 87–90% by CLA supplementation, which confirms compliance. T10,c12-CLA levels were detected but these were relatively lower than c9,t11-CLA. These minimal detection levels are similar to that published by other groups (Mougios et al, 2001). In addition, the level of t10,c12-CLA enrichment was very variable between subjects whereby the standard deviation was almost equal to the mean values as has been demonstrated in a recent publication (Burdge et al, 2004).

In conclusion, this study highlighted that CLA had minimal effects on markers of immune response. The effects of CLA on cytokine expression did not differ dramatically from linoleic acid. Further research is required to determine the effects of the individual CLA isomers in order to evaluate any therapeutic potential of CLA as an immuno-modulatory substance.

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Correspondence to H M Roche.

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Guarantors: AP Nugent and HM Roche.

Contributors: APN analysed the data and wrote the paper. HMR, EJN, AL, DKK and MJG provided consultation on the interpretation of the results and commented on the paper.

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Nugent, A., Roche, H., Noone, E. et al. The effects of conjugated linoleic acid supplementation on immune function in healthy volunteers. Eur J Clin Nutr 59, 742–750 (2005). https://doi.org/10.1038/sj.ejcn.1602132

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Keywords

  • conjugated linoleic acid (CLA)
  • immune function
  • cytokine

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