Effects of conjugated linoleic acid on growth and cytokine expression in Jurkat T cells

Immunology Letters 90 (2003) 195–201

Effects of conjugated linoleic acid on growth and cytokine expression in Jurkat T cells Diomira Luongo a , Paolo Bergamo a,b , Mauro Rossi a,∗ b

a Istituto di Scienze dell’Alimentazione, CNR, via Roma 52, 83100 Avellino, Italy Istituto per il Sistema Produzione Animale in Ambiente Mediterraneo, CNR, via Argine 1085, Naples, Italy

Received 22 August 2003; received in revised form 20 September 2003; accepted 26 September 2003

Abstract Conjugated linoleic acid (CLA) has shown beneficial properties in animal models including anti-cancer, anti-atherogenic and anti-diabetic effects, while contrasting immunological effects were reported. While its anti-inflammatory activity has been associated to inhibition of arachidonic acid biosynthesis and to peroxisome proliferator-activated receptors (PPARs) activity, the molecular pathways underlying its immunoenhancing activity are essentially unknown. The aim of our study was to examine whether CLA showed specific effects in vitro on a T cell model, represented by the Jurkat cell line. CLA was found non toxic for Jurkat in the range 50–200 ␮M, as assessed by LDH release; however, incubation with 50 ␮M CLA was associated to a significant inhibitory effect on cell proliferation. The analysis of IL-2 and IFN-␥ transcript levels, produced in stimulated Jurkat cells, showed an increased expression of both cytokines in CLA-treated cells. Interestingly, the increased induction of IL-2 but not of IFN-␥ mRNA, could be suppressed by co-incubation with Gö 6976, a protein kinase C (PKC) inhibitor. Co-incubation with superoxide dismutase (SOD) or N-acetyl-l-cysteine (NAC) restored the basal levels of RNA synthesis for both cytokines. Taken together, these results suggest a specific role for dietary CLA in the modulation of the immune response in a T cell line model that is mediated, at least in part, by PKC and through the production of oxidative molecules. © 2003 Elsevier B.V. All rights reserved. Keywords: Conjugated linoleic acid; Jurkat cell line; Immunomodulation

1. Introduction Fatty acids are important in the functioning of the immune system because they are structural components of cell membranes determining its fluidity, an important parameter for the expression of cell surface structures such as immune receptors. Moreover, dietary fatty acids represent the precursor of prostaglandins and leukotrienes, which have potent proinflammatory and immunoregolatory properties, as well as of diacylglicerols and ceramides, molecules of intracellular signalling pathways. Most research into how fat in the diet may influence the functioning of the immune system has essentially focused on specific types of fatty acids. Among these ones, conjugated linoleic acid (CLA) has been extensively studied.

∗ Corresponding author. Tel.: +39-0825-299391; fax: +39-0825-299104. E-mail address: [email protected] (M. Rossi).

0165-2478/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2003.09.012

CLA is a mixture of positional (e.g. 7,9; 9,11; 10,12; 11,13) and geometric (cis or trans) isomers of linoleic acid (9 cis, 12 cis octadecadienoic acid); beef and dairy products are considered the greatest dietary sources of CLA [1]. The predominant CLA isomers are generated from ruminally derived vaccenic acid through the activity of specific mammalian desaturases [2,3]. CLA has shown a wide range of beneficial properties in rodent model systems including anti-cancer [4,5], anti-atherogenic [6] and anti-diabetic effects [7], while contrasting immunological effects were reported [8]. According to some authors CLA inhibited inducible inflammatory events while maintaining or enhancing adaptive immune responsiveness [9,10]. The anti-inflammatory activity of CLA seems to be related to the suppression of the biosynthetic pathway of arachidonic acid and to the modulation of the expression of genes regulated by peroxisome proliferator-activated receptors (PPARs) [9]. However, mice receiving CLA-supplemented diet showed significantly increased splenocytes blastogenesis [11] and

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higher splenocyte IL-2 than those fed the control diet [12]. Moreover, immunoglobulin and cytokine production could be differentially modulated by different isomers [13] strengthening the hypothesis that specific immune functions can be associated with specific CLA isomers. The mechanisms of action underlying the immunoenhancing activity of CLA are not clearly understood, although its association with CLA pro-oxidant properties has been demonstrated in cancer cells [14]. In T lymphocytes, cell activation is linked to the intracellular redox state; in fact, antioxidant compounds can inhibit lymphocyte proliferation [15]; in particular, the formation of reactive oxygen species can regulate the signal transduction pathways by acting as second messenger [16]. Considering the potential therapeutic benefits of CLA, it is important to analyse the effects of this fatty acid on the proliferation and differentiation capacities in different immune cell lines. To more directly address the question of a specific effect of CLA on T cells, we analysed the activity of CLA on the Jurkat cell line. Jurkat cells have long served as a useful human T cell model for different immunological studies. They have proved valuable, particularly for the characterisation of the IL-2 and Ag-receptor structures, as well as molecules of signal transduction. However, in comparison with normal T cells, the ability of some transduction pathway to respond to external stimuli is altered [17]; so, data obtained in Jurkat needs careful interpretation before to extrapolate it to the normal T cell biology. In this work we provide evidence that in Jurkat, non cytotoxic concentrations of CLA were able to inhibit cell proliferation. In addition, residual cells showed increased IL-2 and IFN-␥ mRNA levels, as a consequence of a pro-oxidant activity of CLA.

for mycoplasma contamination. The cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated FCS, 2 mM l-glutamine, 1% (v/v) NEAA and antibiotics (100 U/ml penicillin/100 ␮g/ml streptomycin), in a humidified atmosphere of 5% CO2 /95% air at 37 ◦ C. Cells were maintained in the exponential phase of growth by routine passages at 2–3 days intervals. Jurkat cells were cultured in the presence of various concentration of CLA directly diluted into the medium from the stock solution for 72 h. Control cells were cultured by adding the same volume of DMSO.

2. Materials and methods

2.5. Analysis of lymphokine production

2.1. Reagents

The expression of cytokine mRNA was assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Cells were cultured in 75 cm2 flasks (8 × 105 ml−1 ) in complete medium supplemented with 0, 50, 100 or 200 ␮M CLA, for 72 h. In experiments with inhibitors, cells were cultured with 0, 50 or 200 ␮M CLA in the presence/absence of 2.5 ␮M Gö 6976, 5 mM N-acetyl-l-cysteine (NAC) or 50 U/ml superoxide dismutase (SOD). Subsequently cells were plated in 24-well plates for 24 h in the presence/absence of 1 ␮g/l phorbol-12-myristate-13-acetate (PMA), 0.5 ␮M ionomycin(I), 1 mg/l anti-CD3 monoclonal antibody (OKT3; Janssen-Cilag, Cologno Monzese, Italy). Total RNA was recovered from cells using a single step guanidine isothiocianate–phenol–chloroform isolation method [18]. Reverse transcription of 1 ␮g RNA was primed using oligo-(dT)12–18 and different aliquots of cDNA were then

CLA, RPMI 1640 medium, foetal calf serum (FCS), antibiotics, non-essential aminoacids (NEAA), enzymes and other reagents grade biochemicals were from Sigma (St. Louis, MO). Gö 6976 was purchased from Calbiochem (S. Diego, CA). CLA was a mixture of 50% (w/w) 9 cis, 11 trans-octadecadienoic acid and 40% (w/w) 10 trans, 12 cis-octadecadienoic acid, as analysed by the manufacturer. Stock solutions were prepared by dissolving CLA in dimethyl sulphoxide (DMSO) to the final concentration of 1 M. 2.2. Cell culture Jurkat cells were obtained from Dr. M. Londei (Kennedy Institute of Rheumatology, London) and tested negative

2.3. Analysis of CLA toxicity The cells were diluted to 2 × 105 cells/ml in 24-well plates in complete medium with 0, 50, 100 or 200 ␮M CLA. Cytotoxicity was evaluated by using the CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega Bioscience Inc., San Luis Obispo, CA) to assess the activity of the intracellular lactate dehydrogenase (LDH) released into the medium. Media were collected after 72 h to measure the reduction of tetrazolium salt into formazan at an absorbance of 492 nm. Data were expressed as percentage of LDH release following treatment of control cells with 1% Triton X-100. 2.4. Cell proliferation studies The cells were cultured at 8 × 105 /ml in 75 cm2 flasks in complete medium supplemented with 0 or 50 ␮M CLA for 48 h; subsequently viable cells were counted using Nigrosin solution (Sigma) and seeded in 96-well plates at different cell dilutions in the presence/absence of CLA. Eighteen hours prior to harvesting, cells were pulsed with 1 ␮Ci/well [3 H]-thymidine. Results were expressed as counts per minutes (cpm).

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used for PCR. The temperature profile of the amplification consisted of 25–35 cycles of 1 min denaturation at 95 ◦ C, 1 min annealing at 58 ◦ C (␤-actin) or 60 ◦ C (IL-2 and IFN-␥) and 1 min extension at 72 ◦ C. Negative control were performed by omitting RNA from the cDNA synthesis and specific PCR amplification. The oligonucleotides used for amplification were obtained from Dr. Salvati (Department of Pediatrics, University of Naples, Naples); their sequences and sizes of the corresponding PCR products have been previously described [19]. PCR products were analysed on a 1.5% agarose gel stained with VISTRA Green (Amersham International plc, Buckinghamshire, UK). Fluorescence scanning and quantitative analysis of detected bands were carried out on STORM 860 system by IMAGEQUANT software (Molecular Dynamics Inc., Sunnyvale, CA). Results were expressed as cytokine/␤-actin mRNA ratio. 2.6. Statistical analysis Student’s t-test was used to compare proliferation responses. Results were expressed as mean ± S.D. Statistical significance was reached at P < 0.05.

3. Results 3.1. Prolonged administration of CLA was not cytotoxic To determine a possible effect of CLA on the Jurkat cells viability, intracellular lactic dehydrogenase (LDH) release was evaluated as perturbation of the cell membrane, following 72 h incubation of 2 × 105 cells with different doses of CLA. As shown in Fig. 1, there was very little increase of released LDH at all the examined concentrations, suggesting absence of significant necrotic effects mediated by CLA. On the basis of these data, 50 ␮M was used as standard CLA concentration to evaluate possible effects on cell proliferation.

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3.2. Effect of CLA on cell proliferation The effect of CLA on the proliferation of Jurkat cells was next examined by assessing their DNA synthesis. To exclude possible effects of cell density on [3 H]-thymidine incorporation, cells were previously cultured in flask (8 × 105 ml−1 ) for 48 h supplemented with 0 or 50 ␮M CLA; cells were then collected and seeded into a 96-well plate at different cell numbers for further 24 h with fresh medium in the presence/absence of 50 ␮M CLA. As shown in Fig. 2, CLA had a significant inhibitory effect at all the various examined cell densities. 3.3. Effect of CLA on mitogen-induced cytokine expression Next, to assess if CLA influenced other functional parameters in Jurkat cells, IL-2 and IFN-␥ levels were determined following incubation with CLA. Cytokine production was assessed by a semiquantitative RT-PCR. Cells were pretreated with CLA for 72 h before to be transferred in fresh medium without CLA; cytokine production was stimulated by overnight incubation with ionomycin, PMA and anti-CD3 antibody. Following incubation, CLA-treated cells showed an increased expression of IL-2 at all the examined concentrations of CLA, while IFN-␥ transcripts levels resulted higher than in control cells only at 200 ␮M CLA (Fig. 3A). The densitometric analysis of the fluorescent bands, after normalisation to the ␤-actin mRNA content, confirmed these observations; in particular, IL-2 transcripts levels showed a dose-dependent increase in CLA-treated cells (Fig. 3B). 3.4. Suppression of CLA activity by PKC inhibition and antioxidant molecules To analyse the molecular mechanisms involved in CLA-induced cytokine transcription, cells were incubated 200000 150000

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Fig. 1. Assessment of CLA cytoxicity in Jurkat cells. Cells (2 × 105 cells/well) were incubated in complete medium with 0, 50, 100 or 200 ␮M CLA. Cytotoxicity was evaluated by assessing the LDH released into the medium after 72 h. Results represent one of three independent experiments and are expressed as percentage of LDH released from control cells treated with 1% Triton X-100 (mean value ± S.D.).

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Fig. 2. CLA-mediated inhibition of cell proliferation. Jurkat cells were cultured at different cell densities in the absence (䊉) or presence (䊏) of 50 ␮M CLA. DNA synthesis was measured by [3 H]-thymidine incorporation and expressed in cpm. Each value represents the mean ± S.D. of three experiments. Counts in the two experimental conditions were found significantly different at all the examined points (P < 0.05).

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Fig. 3. Induction of cytokine expression. (A) IL-2 and IFN-␥ mRNA productions of Jurkat cells, treated with different doses of CLA for 72 h and subsequently stimulated (+) or not stimulated (−) for 24 h with the agonists (PMA/I/␣CD3), were assessed by a semiquantitative RT-PCR; products were analysed on a 1.5% agarose gel. (B) Densitometric analysis of cytokine mRNA; after electrophoresis, a densitometric scanning was carried out and results were expressed as cytokine/␤-actin mRNA ratio. Results represent one of three independent experiments.

with 50 ␮M (IL-2) or 200 ␮M CLA (IFN-␥) together with known inhibitors of lymphocyte activation. Interestingly, the increased induction of IL-2, but not of IFN-␥ mRNA, could be partially reverted by co-incubation with Gö 6976, a protein kinase C (PKC) inhibitor (Fig. 4A). Moreover, co-incubation of CLA with the anti-oxidant enzyme superoxide dismutase or N-acetyl-l-cysteine, a reducing agent, restored the basal levels of RNA synthesis for both cytokines (Fig. 4), so suggesting that CLA stimulated cytokine transcription through the production of oxidative molecules. No significant differences with the control were observed when Gö 6976 and antioxidant molecules were incubated in the absence of CLA (data not shown).

4. Discussion Previous data indicated that synthetic CLA mixture, containing 9 cis, 11 trans and 10 trans, 12 cis CLA isomers (50% cis9 trans11 , 40% trans10 cis12 ), induced a more marked induction of apoptosis in Jurkat T cell than individual CLA isomers [20]. In the present study we showed that incubation of non cytotoxic concentration of synthetic CLA mixture,

inhibited proliferation of Jurkat cells and stimulated their cytokine synthesis. In general, the effects of fatty acids on the immune function are at least partially due to lymphocyte death. For this reason we evaluated the LDH release after 72 h treatment with different concentration of CLA and found no signs of toxicity in Jurkat cells (Fig. 1). On the contrary, the CLA isomer linoleic acid has been reported to cause both loss of membrane integrity and DNA fragmentation at a dose of 200 ␮M [21]. From this point of view, CLA resembled monounsaturated fatty acids with similar carbon chain length, shown to be well tolerated by Jurkat cells [21]. However, we also found inhibition of proliferation following CLA treatment, as measured by [3 H]-thymidine incorporation (Fig. 2). Taken together these results seemed to confirm our findings of apoptosis triggered by CLA [20]. A series of data from studies on animal models indicated that CLA possessed immune activities [9,10] that occurred at different points of the immune system [22]. In particular, CLA appeared to modulate thymocyte lineage commitment, favouring a predominance of CD8+ cells, an important step for the down-regulation of the CD4+ -mediated inflammatory response [23]. Moreover, in peripheral lymph nodes,

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Fig. 4. Effect of PKC inhibitor and anti-oxidant molecules on CLA-induced cytokine expression. Cells were cultured with 0 or 50 and 0 or 200 ␮M CLA, for IL-2 and IFN-␥ assessment respectively, in the presence/absence of 2.5 ␮M Gö 6976, 5 mM N-acetyl-l-cysteine (NAC) or 50 U/ml superoxide dismutase (SOD). Cells were subsequently treated with agonists as described in Fig. 3; data were expressed as cytokine/␤-actin mRNA ratio. Results shown are representative of three independent experiments.

CLA was reported to regulate antigen-specific proliferation of T cells and the cytokine pattern: a specific inhibition of Th2 response has been described, even if not associated with an enhanced Th1 cytokine profile [22]. These modulatory activities could reduce inflammation in an experimental model of colitis [22]. Recently, some of the molecular mechanisms involved in the anti-inflammatory activity of CLA were identified: suppression of both arachidonic acid formation and eicosanoid synthesis have been reported [9], as well as activation of a group of steroid hormone receptors, the PPAR-␣ [24] and PPAR-␥ [7,25], transcriptional factors that induce a decrease in the expression of pro-inflammatory cytokines by antagonising the activities of NFkB and STAT1 [26]. However, an immunoenhancing activity of CLA has been also described very recently, as a higher number of blood monocytes occurred in rats fed enriched CLA diets [8]. These differences can be partially explained by the heterogeneous nature of dietary CLA: different CLA preparations can potentially stimulate

differently the immune system. In recent in vivo studies splenocytes, isolated from mice fed the isomer 10 trans, 12 cis CLA produced more IgA and IgM but not IgG and less TNF-␣ than splenocytes from the group treated with 9 cis, 11 trans CLA [13]. Nevertheless, in another study using the very same isomers, different results were reported; in particular both CLA isomers increased TNF-␣ and IL-6 production and decreased IL-4 secretion [27]. The discrepancies among the results from different in vivo studies highlighted the need of performing further in vitro studies on different cellular models to better analyse the mechanisms involved. To address this issue we stimulated the leukaemic T cell line Jurkat, following incubation with 50 ␮M CLA. Stimulation with ionomycin and PMA could mimic the effect of T cell receptor-induced activation of phospholipase C (PLC) and protein kinase C, respectively, However, it has been shown in Jurkat that activation with combined receptor/intracellular agonists (␣CD3/PMA/ionomycin) induced much higher IL-2 expression than PMA/ionomicyn alone [28]; so we

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applied this protocol to activate Jurkat and assess the effect of CLA on the cytokine levels. When we pre-treated Jurkat cells with CLA, a dose-dependent increased induction of IL-2 mRNA levels was detected (Fig. 3). IFN-␥ transcript levels were also induced, but only following a 72 h treatment with high doses of CLA (Fig. 3). Taken together, these data confirmed the immuno-enhancing activity of CLA [8]. However, CLA was shown to decrease the IFN-␥-induced expression of a series of pro-inflammatory cytokines mediators in murine macrophage RAW cells, by acting through PPAR␥ [27]. Interestingly, Jurkat T cells do not express PPAR␥ [29] and this finding can partially explain our data: the lack of this crucial mechanism can give CLA the opportunity, when introduced into the cell membranes, to overcome the anti-inflammatory pathway previously reported on macrophages. Possible alternative explanations may be found in difference in the activation pathways in Jurkat at the level of other transduction mechanisms or of the phospholipid pools solicited during the activation process. The above reported inhibition of proliferation, as a consequence of CLA incubation, occurred despite the fact that 50 ␮M CLA induced a two-fold increase of IL-2 expression on residual cells (Fig. 3). Of course, Jurkat cells are malignant transformed [30] and they proliferate without stimulation; thus the relationship between IL-2 production and proliferation could not be evaluated in this cell line. Moreover, a similar differential effect has been described for the antioxidant ␣-lipoic acid on peripheral blood lymphocytes [31]; these findings suggest that T cell development might be sensitive to CLA and other lipid mediators at least at two different and independent steps, one being the control of DNA synthesis and the other one the modulation of the complex mechanism regulating the IL-2 expression. PKC activation has been implicated in the development of a Th1 helper subset via activation of IL-2 and repression of IL-4 gene transcription [32]. We found that selective inhibition of PKC isozymes by the indolocarbazole Gö 6976 [33] completely abolished the CLA-induced IL-2 expression, indicating that the effect of CLA occurred through PKC activation (Fig. 4). Interestingly, IFN-␥ mRNA levels were not reduced by Gö 6976 treatment, suggesting that CLA induced IFN-␥ transcription was achieved through PKC-independent mechanisms. A similar effect on the IFN-␥ expression has been described for bryostatin-1, a potent ligand and modulator of PKC [34]; in particular, it was shown that the induction was mediated through a p38 mitogen-activated protein kinase-dependent process. If this route is activated in Jurkat cells following CLA incubation, it remains to be demonstrated. Oxidative stress (OS) has been reported to have contrasting effects on cytokine productions during immune responses. OS via endogenous and exogenous agents has been considered an important factor in induction of autoimmunity [35]. Moreover OS found in cancer patients [36] and in immunoinflammatory diseases [37] was paralleled by increased levels of pro-inflammatory cytokines. Recently OS

has been reported to stimulates cytokine production in mast cells [38] but also to induce hyporesponsiveness in normal T lymphocytes [39]. In our system CLA-induced expression of both cytokines was reduced by co-incubation with the anti-oxidant N-acetyl-l-cysteine, that can scavenge several oxidant species including H2 O2 [40] or addition of the enzyme SOD to the medium, strengthening the concept of a pro-oxidant activity for CLA mediating cytokine expression in Jurkat. These data were in agreement with our recent findings of reduced intracellular levels of the antioxidant glutathione, a hallmark of OS [41], following CLA treatment [21] and confirmed previous observations on a pro-oxidant activity demonstrated in other cancer cells [14]. In conclusion, our results confirmed that in the peculiar Jurkat T cell line, CLA was able to increase the IL-2 and IFN-␥ transcription via modulation of PKC activity and production of oxidant species. This effect was paralleled by specific inhibition of cell proliferation, an important finding in the perspective of defining a specific role for dietary CLA in the treatment of leukaemic cells.

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