Effect of arachidonic acid on proliferation, cytokines production and pleiotropic genes expression in Jurkat cells—A comparison with oleic acid

Life Sciences 73 (2003) 2939 – 2951 www.elsevier.com/locate/lifescie

Effect of arachidonic acid on proliferation, cytokines production and pleiotropic genes expression in Jurkat cells—A comparison with oleic acid Rozangela Verlengia a,*, Renata Gorja˜o a, Carla Cristine Kanunfre b, Silvana Bordin a, Thais Martins de Lima a, Rui Curi a a

Departamento de Fisiologia e Biofı´sica, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 1524, 05508-900, Butanta˜, Sa˜o Paulo, SP, Brazil b Department of Biology, University of Ponta Grossa, Ponta Grossa, SP, Brazil Received 26 February 2003; accepted 14 April 2003

Abstract The effects of arachidonic (AA) and oleic acids (OA) on proliferation, cytokine production and pleiotropic genes expression in Jurkat T cells were investigated. The following parameters were evaluated: cytotoxicity assessed by loss of membrane integrity and DNA fragmentation, cell proliferation as measured by [14C]thymidine incorporation, production of IL-2, IL-4, IL-10, and INF-g, and expression of pleiotropic genes as determined by macroarray technique (83 genes in total). AA was more toxic for Jurkat cells than OA. However, the inhibiting effect of OA on Jurkat cells proliferation was more pronounced than that of AA. The reduction in the production of IL-2 and INF-g was more intense by OA (50 AM) than by AA (5 AM). The percentage of genes changed by the fatty acids was: 20.5% (17 genes) for AA (5 AM) and only 2.4% (2 genes) for OA (50 AM). AA markedly affected the expression of genes clustered as: signal transduction pathways, transcription factors and related genes, cell cycle, defense and repair, apoptosis, DNA synthesis, cell adhesion, cytoskeleton and related genes. In particular, AA induced marked changes in cell cycle, signal transduction, and anti-apoptosis genes expression. Therefore, the effect of AA on T-lymphocyte function does involve regulation of expression of important genes, whereas oleic acid did not markedly affect gene expression of Jurkat cells. D 2003 Elsevier Inc. All rights reserved. Keywords: Jurkat cells; Gene expression; Macroarray; Arachidonic acid; Oleic acid; Proliferation; Cytokines and cytotoxicity

* Corresponding author. Tel.: +55-11-3091-7245; fax: +55-11-3091-7285. E-mail address: [email protected] (R. Verlengia). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.04.003

2940

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

Introduction T lymphocyte is an important cell for the body defense. This cell plays a critical role in defining the type and magnitude of the immune response. Regulation of immune function by the T lymphocyte is partially mediated by the type of cytokines it produces. The mechanism leading to the propagation of T lymphocytes involves their activation, which leads to interleukin-2 (IL-2) production and subsequent proliferation referred to as clonal expansion. Depending on the mode of activation, T lymphocytes differentiates into either a helper Th-1 or Th-2 cells (CD4+ T lymphocytes), or become a cytotoxic T lymphocyte (CD8 + cells). The Th-1 cell produces primarily IL-2, interferon-g (INF-g), and tumor necrosis factor-a (TNF-a), resulting in enhanced cell-mediated or cytotoxic responses. The Th-2 cell produces IL-4,-5,-6, and -10, generating a humoral or antibody-mediated immune response. Several studies have demonstrated that nonesterified fatty acids modulate T lymphocyte proliferation and cytokines production (Calder et al., 1995; Jenski et al., 1998; Peterson et al., 1998). Arachidonic acid inhibits the proliferation of cultured lymphocytes and decreases the production of IL-2 (Calder et al., 2002). This fatty acid is precursor of eicosanoids such as prostaglandins, leukotrienes and thromboxanes. Eicosanoids (particularly PGE2 and leukotriene B4) modulate the intensity and duration of inflammatory and immune responses (Tilley et al., 2001). The pro-inflammatory effects of PGE2 include inducing fever, increasing vascular permeability and vasodilatation. PGE2 suppresses lymphocyte proliferation and inhibits the production of interleukins such as IL-2 and INF-g. LTB4 increases vascular permeability and enhances blood flow. This leukotriene inhibits lymphocyte proliferation and increases the production of cytokines such as IL-2 and INF-g (Calder and Field, 2002). Thus, the effects of AA on inflammatory and immune responses are partially due to eicosanoids production. However, arachidonic acid may affect lymphocyte function by eicosanoid-independent mechanisms including actions on intracellular signalling pathways and transcription factor activity (McMurray et al., 2000). It has become conventional to use monounsaturated fatty acid (such as oleic acid that can be found in olive oil and cashew seed oil) as the baseline or neutral fatty acid. By this is meant that oleic acid neither raises nor lowers total cholesterol concentrations (Mata et al., 1997). Yaqoob et al. (1998) and Yaqoob (2002) using an oleic acid enriched diet did not observe changes in lymphocyte proliferation and cytokines production. This issue however still remains controversial. Mata et al. (1997) showed that patients receiving oleic acid-rich diets present low levels of oxidized LDL in plasma. Experiments in isolated lymphocytes show that oleic acid in sufficient amounts can partially inhibit proliferation and IL2 production (Calder et al., 1995; Pompe´ia et al., 2000). In fact, oleic acid raises CD44 and CD45 expression in lymphocytes (Peck et al., 1996). The information above led us to investigate the effect of arachidonic acid, a well-known modulator of T cell function; on a T lymphocyte cell model (Jurkat cells) and its effects were compared with those of oleic acid that is considered by several authors as a neutral fatty acid. The parameters evaluated were: cytotoxicity assessed by Flow Cytometric Analysis (FACS) analysis for cell membrane integrity and DNA fragmentation, Jurkat cells proliferation by the measurement of [14C]-thymidine incorporation, production of IL-2, IL-4, IL-10 and INF-g by ELISA and expression of pleiotropic genes by macroarray technique.

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

2941

Material and methods Determination of the toxic concentrations of AA and OA on Jurkat cells by flow cytometric analysis Jurkat cells were obtained from the Dunn School of Pathology, Oxford University, England. The cells (2  105 per mL) were plated in 24 wells plate and treated for 24 hours with arachidonic (C20:4) and oleic (C18:1cis9) acids. These FA did not show any sign of toxicity up to 50 AM concentration (data not shown). The FA concentrations (50, 100, 150, 200, 300 and 350 AM) were used to carry out membrane integrity and DNA fragmentation analysis. The fatty acids were first dissolved in ethanol before emulsification in the serum proteins containing medium. The percentage of ethanol was always lower than 0.05% of the total volume of culture medium. This concentration of ethanol has shown not to be toxic for the cells as also observed by Siddiqui et al. (2001). At the end of the culture period, 0.5 mL of medium containing cells were used to test the membrane integrity. In this assay, 50 AL of a propidium iodide (PI) solution (100 Ag per mL in saline buffer) were added to the cells (Nicoletti et al., 1991). Propidium iodide is a highly water-soluble fluorescent compound that cannot pass through intact membranes and is generally excluded from viable cells. It binds to DNA by intercalating between the bases with little or no sequence preference. After 5 minutes incubation at room temperature, the cells were evaluated in a FACScalibur flow cytometer equipment (BectonDickinson, California, USA) by using the Cell Quest software. The loss of membrane integrity is noticeable when the PI fluorescence (620 nm) arises. Ten thousand events were evaluated per experiment. The remaining medium containing cells was centrifuged at high speed (12,000  g, 30 seconds) and the pellet was resuspended in 0.2 mL lyse buffer (0.1% sodium citrate and 0.1% Triton X100) containing 50 Ag per mL of PI. The lysed cells were then incubated in the dark, at 4 jC for 24 hours, and used for the DNA fragmentation analysis. PI intercalates between bases in double stranded nucleic acids of exposed nuclei. The DNA fragmentation is observed by the occurrence of low fluorescent particles. This indicates that DNA was cleaved and there are little DNA fragments that, because of their high condensation and small size, cannot be intensively bound by PI. These particles are seen as low fluorescent particles. Proliferation assay Jurkat cells (3.3  105 cells per mL) were plated in 96 well microtiter plates and treated for 48 hours with AA (2.5, 5, 25, 50, 75 and 100 AM) and OA (25, 50, 100, 150, 200, 250 and 300 AM). [14C] Thymidine (1 ACi per mL) was added to the medium at the beginning of the experiment. The plates were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37 jC. The radioactivity was analysed by using a liquid scintillation counter (Packard TRI-CARB 2100 TR counters; Downers Grove, IL, USA). Measurement of cytokines The cells (2  105 cells per mL) were plated in 24 wells plate and treated for 24 hours with AA (5 AM) and OA (50 AM). This concentration is in the plasma physiological range (Painter et al., 1994) and it was the same employed to study pleiotropic genes expression. The cells were then cultured for another period of 24 hours in the presence of the 25 Ag/mL concanavalin A (Con A)—a T lymphocyte mitogen

2942

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

(Boyum, 1968). Afterwards, cell culture supernantant fluid was harvested to carry out the determinations of secreted cytokines. The production of IL-2, IL-10, IL-4 and INF-g was evaluated by enzyme-linked immunosorbent assay (ELISA) using Kit OptEIAk from Pharmingen (San Diego, CA-USA). The detection limit of IL2, IL-10 and IL-4 was 7.8 pg/mL and of INF-g was 4.7 pg/mL according to the manufacturer. Treatment of Jurkat cells with the fatty acids to evaluate pleotropic genes expression Cells were resuspended at a density of 2  105 cells per mL in 25 cm3 flask containing RPMI 1640 medium and 10% fetal calf serum. The cells were treated for 24 hours with AA and OA at 5 AM and 50 AM concentrations, respectively. Total RNA extraction Total RNA was obtained from 0.5–1  107 cells using Trizol reagent (Life Technologies Inc., Rockville, MD, USA). Briefly, the cells were lysed using 1 mL Trizol reagent (Life Technologies, Rockville, MD, USA). After 5 min incubation at room temperature, 200 AL chloroform were added to the tubes and centrifuged at 12,000  g. The aqueous phase was transferred to another tube and the RNA was precipitated by centrifugation (12,000  g) with cold ethanol and dried in air. RNA pellets were eluated in RNase-free water and treated with DNase I. RNAs were then stored at 70 jC until to the time of the experiment. The RNA was quantified by measuring absorbance at 260 nm. The purity of the RNAs was assessed by the 260/280 nm ratio and on a 1% agarose gel stained with ethidium bromide at 5 Ag/mL (Sambrook and Russell, 2001). These samples were used for macroarray and RT-PCR analysis. Synthesis of cDNA probes The cDNA probes were synthetized using the pure total RNA labeling system Atlas Kitk according to manufacturer’s recommendations (Clontech Laboratories, California, USA). Briefly, 10 Ag of total RNA and 2 AL of primers mix ‘‘CDs’’ were heated at 70 jC for 5 min in a Techne-Genius Thermal cycler (Oxford, UK). The temperature was decreased to 50 jC and 13.5 AL of the following reagents were added: 4 AL reaction buffer 5  , 0.5 AL 100 mM DTT, 2 AL 10  dNTP mix (dCTP, dGTP, dTTP), 5 AL of [a-33P] ATP (at 10 ACi/AL) and 2 AL of reverse superscript enzyme (Life Technologies Inc., Rockville, MD, USA). The reaction was incubated for 25 min at 50 jC and stopped by using 2 AL Termination Mix. The 33P-labeled probe was purified from unincorporated nucleotides by passing the reaction mixture through a push column (NucleoSpin Extraction Spin Column-Clontech Laboratories). Experiments using 20 Ag total RNA were also performed and the results indicated saturation of the hybridization reactions (data not shown). Macroarrays hibridization All solutions for hybridization were obtained from the Clontech Laboratories. The membrane was pre-hybridized for 30 min at 68 jC in Express Hyb containing 50 Ag freshly denaturated salmon sperm DNA. Subsequently the membrane was hybridized during 18 h at 68 jC with 2  106 cpm/mL 33Plabeled denaturated probe. The membrane was washed twice at 68jC with 1  SSC, 0.1% SDS

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

2943

followed by two washings in 1  SSC, 1% SDS and then exposed to phosphor screen for 48 hours and scanned in the Storm 840 (Molecular Dynamics, Sunnyvale, CA, USA). Analysis of macroarray results Changes in the genes expression induced by the fatty acids were analysed by comparison with untreated cells using the software Array-Prok Analyzer, version 4 (Media CyberneticsR, Silver Spring, MD, USA). Normalization was performed by housekeeping gene h-actin present in the membrane. Duplicate hybridizations using separate sets of nylon membranes were performed for all conditions. Only signals that differed from the control by at least 2-fold in the two independent experiments were considered as significant. Similar procedure was used by Yamazak et al. (2002). RT-PCR RT-PCR using specific primers was performed to confirm the differential expression of the mRNAs detected with the macroarray analysis. The sequences of the primers were designed using information contained public database in GeneBank of the National Center for Biotechnology Information (NCBI). The RT-PCR was performed using parameters described by Innis and Gelfand (1990). The number of cycles used was selected to allow linear amplification of the cDNA under study. For semi-quantitative PCR analysis, the housekeeping h-actin gene was used as reference. The primer sequences and their respective PCR fragment lengths are shown in the Table 1. Published guidelines were followed to guard against bacterial and nucleic acid contamination (Kwok and Higuchi, 1989). Analysis of the PCR products The analysis of PCR amplification products was performed in 1,5% agarose gels containing 0.5 Ag/ mL ethidium bromide and electrophoresed for 1 h at 100 V. The gels were photographed using a DC120 Table 1 The standardized conditions for RT-PCR analysis. Sequences of the primers, PCR fragment lengths, temperature and number of cycles are shown for each gene under study Genes

Sense primer

Antisense primer

Anneling temperature (jC)

PCR fragment lengths (bp)

Number of cycles

PLA2

5V-AGCCCGTAGG TCATCTTGG-3V 5V-CCATCAAATGC TCCCTCAACCC-3V 5V-CGTACAATGCC TC CATCACGC-3V 5V-GCAAAGCATTT AGCCAGCCTTC-3V

5V-GCTTCAGCTTC GTCTCCTTGG-3V 5V-TTGCCAAAGCT GCCTTTCCC-3V 5V-ACTTCTTGCA CCAAGCCACTG-3V 5V-TCTCTCCAGTG TGCATCCTCG-3V

56

559

30

57

424

35

56

436

30

56

313

40

PKC-beta TOP1 Kruppel related zinc finger protein*

For all genes 1.5 mM MgCl2 was used. * For kruppel related zinc finger protein RT-PCR, formamide was used at 2.5% concentration. PLA2, phospholipase A2; PKC-beta, Protein Kinase C type beta I; TOP1, DNA Topoisomerase 1.

2944

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

Zoom Digital Camera System from Kodak (Life Technologies, Inc., Rockville, MD, USA). The images were processed and analyzed in the software Kodak Digital Science 1D Image Analysis (Life Technologies). PCR band intensities were expressed as OD normalized for h-actin expression. The data are presented as the ratio with the respective controls, which received an arbitrary value of 1 in each experiment. Statistical analysis The results of toxicity and proliferation assay and measurement of the production of cytokines were expressed as mean F SEM. Comparisons between groups were performed by analysis of variance (ANOVA). Significant differences were found by using the Turkey-Kramer’s method (INStat-Graph Pad Software, Inc., San Diego, CA, USA) for comparison with control (ethanol).

Fig. 1. Determination of the toxicity of AA and OA on Jurkat cells. (A) The percentage of cells with intact membrane after treatment for 24 hours with arachidonic acid (AA) at 50, 100 and 150 AM and oleic acid (OA) at 50, 100, 150, 200, 300 and 350 AM. (B) Percentage of cells with fragmented DNA after the same treatment. The values are presented as mean of six determinations of two different experiments. SEM was always lower than 10% of the mean. (*) indicate P < 0.05 as compared with 50 AM concentration. The values of 50 AM were not different of the corresponding controls (not treated with fatty acids).

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

2945

Results Determination of the toxic concentrations of AA and OA acids to Jurkat cells Initially the comparative toxicity of arachidonic and oleic acids on Jurkat cells was determined. Toxicity was assessed by either loss of cell membrane integrity or DNA fragmentation using flow cytometric analysis. There were remarkable differences in the toxicity of both fatty acids on Jurkat cells (Fig. 1). The proportion (%) of control cells with integral membrane was 95.5 F 3.4 and with integral DNA was 94.3 F 3.2 after treatment. Oleic acid did not cause loss of membrane integrity up to 200 AM concentration (94% of viable cells) and DNA fragmentation up to 300 AM (29% of cells with fragmented DNA). In contrast, AA led to loss of membrane integrity already at 100 AM (65% of viable cells). The

Fig. 2. Effect of arachidonic (A) and oleic acid (B) on Jurkat cells proliferation. The cells (3.3  105 cells per mL) were cultured in various fatty acids concentrations for 48 h. Cells were pulsed with 0.2 ACi/200 AL [14C]-thymidine, then harvested and the radioactivity of the thymidine incorporated into DNA was determined by using a liquid scintillation counter. The incorportion of [14C]-thymidine is expressed as total counts per min. The values are presented as mean F SEM of six determinations from three experiments. (*) indicate P < 0.05 as compared with the corresponding controls (no fatty acids added).

2946

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

concentrations then selected to examine the effect of these FA on cytokines production and pleiotropic genes expression were much lower than those that caused toxic effect: 5 AM for AA and 50 AM for oleic acid. Therefore, the changes observed were due to the effect of the FA per se and did not result from induction of apoptosis or necrosis. Proliferation assay As compared with controls (no fatty acid added), [14C]-thymidine incorporation by Jurkat cells was markedly reduced by OA already at 50 AM (12%) and 100 AM (42%) concentrations and reached maximal inhibition at 200 AM (99%). The decrease of Jurkat cells proliferation by AA was of 63% at 75 AM and of 80% at 100 AM (Fig. 2), as compared with controls. Therefore, oleic acid presented a stronger inhibiting effect on Jurkat cells proliferation than AA. Measurement of production of cytokines AA (5 AM) and OA (50 AM) were able to inhibit the cytokines production in 24 h cultured Jurkat cells, except for AA on IL-4 (Fig. 3). AA (at 5 AM) reduced the production of IL-2 (50%), IL-10 (by 32%), and INF-g (36%), as compared with control (no fatty acid added). Oleic acid (at 50 AM) caused a marked reduction of the four cytokines production: IL-2 (66%), IL-4 (48%), IL-10 (56%) and INF-g (61%); the values were compared with those of control.

Fig. 3. Effect of the fatty acids on cytokines production by Jurkat cells. The cells were cultured in the presence of AA (5 AM) and OA (50 AM) for 24 hours. The cells were then cultured for another period of 24 hours in the presence of 25 Ag per mL concanavalin A (Con A) and afterwards the supernatant was used for determination of the cytokines by ELISA as described in Material and Methods. Values represent mean F SEM of six determinations from three experiments. (*) indicate P < 0.05 as compared with the corresponding controls (no fatty acids added).

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

2947

Pleiotropic genes expression The comparative effect of AA (5 AM) and OA (50 AM) on expression of genes involved with several aspects of T lymphocyte function is shown in the Table 2. To validate the results of the macroarray analysis, four genes were selected for confirmation by RT-PCR (Fig. 4). Although the magnitude was not the same, Table 2 Modifications in Jurkat cells genes expression after treatment with arachidonic (AA) and oleic (OA) acids Cluster/GAN*

Gene name

1. Signal transduction pathways X06318 Protein Kinase C beta I (PKC-beta) M35203 Janus tyrosine-protein Kinase I (JAK-1) U39657 MAP Kinase Kinase 6 (MAPKK6) M86400 Phospholipase A2 (PLA2) 2. Transcription factors and related genes X12794 v-erbA homology-like 2 L11672 Kruppel related zinc finger protein M15400 Retinoblastoma-associated protein 1 (RB1); phosphoprotein 110 (pp110) 3. Cell cycle M81933 X51688 L27211 M25753

Cell division cycle 25 homolog A (CDC25A) G2/mitotic-specific cyclin A (CCNA) Cyclin-dependent kinase 4 inhibitor 2 (CDK4I) G2/mitotic-specific cyclin B1 (CCNB1)

4. Defense and repair X08058 M25627

Glutathione S-transferase pi (GSTP1) Glutathione S-transferase A1 (GSTA1)

5. Apoptosis U66879 U43746

Bcl2-binding component 6 (bbc6) Breast cancer type 2 susceptibility protein (BRCA2)

6. DNA synthesis J03250 L04088

DNA Topoisomerase I (TOP1) DNA topoisomerase II alpha (TOP2A)

7. Cell adhesion, cytoskeleton, and related genes Z15009 Laminin gamma 2 subunit (LAMC2) X02761 Fibronectin 1 (FN1)

AA

Oleic

2.8 2.3 + 7.4 + 2.4

+ 12.7 4 2.1

2.0 4.2 + 23.8 2.3

2.6 + 2.7

+ 19.8 + 3.8

+ 2.9 2.3

+ 6.1 + 15.1

Jurkat cells were treated for 24 hours with arachidonic (5 AM) and oleic (50 AM) acids. Total RNA was isolated, retrotranscribed, 33P-labelled and hybridized to the cDNA array presenting 83 transcripts of known genes. The signals were then analyzed by pro-analysis software Array-Prok Analyzer, version 4 (Media CyberneticsR, Silver Spring, MD, USA) and expressed as fold of increase or decrease in relation to untreated cells. Data are presented as means of two different experiments. Only signals that differed from untreated cells by at least 2-fold were considered as significant. Changes are indicated as (+) increase and ( ) decrease as compared to untreated cells. * GAN – GeneBank accession number.

2948

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

Fig. 4. Confirmation by RT-PCR of the genes modified by the fatty acids as detected by macroarray analysis (Table 2). Jurkat cells (107 cells per condition) were exposed for 24 h to the following conditions: control (no fatty acids added), AA (5 AM), and OA (50 AM). After this time point, the cells were harvested, mRNA extracted, and RT-PCR performed with the equivalent of 107 cells. PCR band intensities were expressed as OD normalized for h-actin expression. The data are presented as the ratio with the respective controls, which received an arbitrary value of 1 in each experiment. The values are presented as mean FSEM of four experiments. PLA2, phospholipase A2; PKC-beta, Protein kinase C type beta I; TOP1, DNA Topoisomerase 1; Kruppel-related zinc finger protein. *P < 0.05 compared with the corresponding controls (no fatty acids).

the changes induced by the FA were observed by both macroarray and RT-PCR. Therefore, macroarray analysis performed in duplicate using pooled cells from two experiments provides reliable observations. In fact, the cDNA array has been used for diagnose of disease such as diabetes (Cardozo et al., 2001). Of the surveyed genes (83 in total), 19 were modified by at least one of the fatty acids tested. The proportions of genes changed by the fatty acids were 20.5% for arachidonic acid, and only 2.4% for oleic acid. The treatment with AA caused an increase in expression of 10 genes and a decrease of 7. Oleic acid reduced the expression of two genes only; Kruppel related zinc finger protein and RB1 or phosphoprotein 110 (pp110). AA had a marked effect (more than 10-fold increase) on the expression of v-erbA homology-like 2, CDK4I, bbc6 and FN1.

Discussion AA was clearly much more toxic for Jurkat cells than oleic acid. However, the inhibiting effect of OA on Jurkat cells proliferation was more pronounced than that of AA. This indicates that the inhibition of cell proliferation observed was not due to cell death. The effect of fatty acids including OA on lymphocyte proliferation has been shown by others (Calder et al., 1995; Yaqoob, 2002).

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

2949

AA reduced the production of IL-2, IL-4, IL-10 and INF-g. Similar observations have been obtained by others (Calder et al., 1995). OA at 10-fold higher concentrations than that of AA caused marked reduction in the production of the four cytokines. Therefore, OA at high concentration is also able to decrease cytokines production. These results confirm the findings of Calder et al. (1995) and Pompe´ia et al. (2000). The effect of both fatty acids on pleiotropic genes expression of Jurkat cells was then examined. The fatty acids responsive genes were clustered according to main putative biological functions of their encoded protein. AA markedly affected the expression of genes clustered as: signal transduction pathways, transcription factors, cell cycle, defense and repair, apoptosis, DNA synthesis, cell adhesion, cytoskeleton and related genes. AA increased the expression of 58.8% of the genes changed, whereas OA decreased the expression of two genes only. The mechanism of action of OA on T lymphocytes functions remains to be investigated. Oleic acid contributes to enhance cellular antioxidant defenses against mitochondria oxidative stress induced by glutathione peroxidase (Duval et al., 2002). This fatty acid is also involved in regulation of cell cycle (Vacaresse et al., 1999; Zugaza et al., 1995). E-6 Fatty acids are widely recognized as pro-inflammatory agents (Calder, 2001; Pompe´ia et al., 2000). AA does lead to production of inflammatory mediators by macrophages (Grundy, 1997). The results presented herein suggest that the effect of AA is not restricted to macrophages. Several genes are under control of this fatty acid in T lymphocytes. AA decreased the expression of signal transduction proteins (PKC-beta and JAK-1), cell cycle genes (CDC25A, CCNA, and CCNB1) and of GSTP1 (defense and repair). AA raised the expression of MAPKK6, PLA2, v-erbA homology-like 2, CDK4I, GSTA1, bbc6, BRCA2, Topoisomerase 1, LAMC2, and FN1. In particular, AA induced marked changes in cell cycle, signal transduction, and anti apoptosis genes expression. This is a novel finding and led us to postulate that the pro-inflammatory action of AA may also result from activation and/or inhibition of several genes in T lymphocytes. However, it remains unknown if these effects were caused by the fatty acid itself or by the eicosanoids produced through AA metabolism (Kelley et al., 1998). There is substantial evidence that AA may affect lymphocyte function eicosanoid-independent mechanisms (McMurray et al., 2000). The results presented herein show important effects of AA and OA on a T lymphocytes cell line. Evidence exists that macrophages are able to produce, export and transfer mainly arachidonic acid, but also oleic acid to lymphocytes in co-culture (Peres et al., 1999). This process has been found to regulate the proliferation activity and cytokines production by the acceptor cells (Nishiyama et al., 2000). Therefore, AA derived from macrophages may modulate lymphocytes function by regulating genes expression.

Conclusion The findings presented herein led us to conclude that AA and OA present remarkable differences on functional parameters and gene expression of Jurkat cells. AA was more toxic than OA, but its inhibiting effect on proliferation and cytokine production (mainly IL-2 and INF-g) was less pronounced than that of OA. The changes in genes expression induced by AA were much different than those caused by OA. AA enhanced the expression of most genes and suppressed several others, whereas OA decreased the expression of two genes only. AA induced changes mainly in cell cycle, signal transduction and antiapoptosis genes expression.

2950

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

Acknowledgements The authors are grateful to the technical assistance of J.R. Mendoncßa, G. de Souza, and E. Portioli and to Dr. C. K. Miyasaka. FAPESP, Pronex, CNPq, CAPES, and The British Council support this research.

References Boyum, A., 1968. Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation 21, 77 – 78. Calder, P.C., 2001. Polyunsaturated fatty acids, inflammation, and immunity. Lipids 36, 1007 – 1024. Calder, P.C., Field, C.J., 2002. Fatty acids, inflammation and immunity. In: Calder, P.C., Field, C.J., Gill, H.S. (Eds.), Nutrition and Immune Function, 57 – 92. Calder, P.C., Costa-Rosa, L.F., Curi, R., 1995. Effects of feeding lipids of different fatty acid compositions upon rat lymphocyte proliferation. Life Sciences 56, 455 – 463. Calder, P.C., Yaqoob, P., Thies, F., Wallace, F.A., Miles, E.A., 2002. Fatty acids and lymphocyte functions. British Journal of Nutrition 87 (1), S31 – S48. Cardozo, A.K., Kruhøffer, M., Leeman, R., Ørntoft, T., Eizirik, D.L., 2001. Identification of novel cytokine-induced genes in pancreatic h-cells by high-density oligonucleotide arrays. Diabetes 50, 909 – 920. Duval, C., Auge, N., Frisach, M.F., Casteilla, L., Salvayre, R., Negre-Salvayre, A., 2002. Mitochondrial oxidative stress is modulated by oleic acid via an epidermal growth factor receptor-dependent activation of glutathione peroxidase. Biochemical Journal 367 (3), 889 – 894. Grundy, S.M., 1997. What is the desirable ratio of saturated, polyunsaturated, and monounsaturated fatty acids in the diet? The American Journal of Clinical Nutrition 66, 988S – 990S. Innis, M.A., Gelfand, D.H., 1990. Optimization of PCRs. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocols: A Guide to Methods and Applications, 1st ed. Academic Press, San Diego, CA, pp. 3 – 12. Jenski, L.J., Scherer, J.M., Caldwell, L.D., Ney, V.A., Stillwell, W., 1998. The triggering signal dictates the effect of docosahexaenoic acid on lymphocyte function in vitro. Lipids 33, 869 – 878. Kelley, D.S., Taylor, P.C., Nelson, G.J., Mackey, B.E., 1998. Arachidonic acid supplementation enhances synthesis of eicosanoids without suppressing immune functions in young healthy men. Lipids 33, 125 – 130. Kwok, S., Higuchi, R., 1989. Avoiding false positives with PCR. Nature 339, 237 – 238. Mata, P., Varela, O., Alonso, R., Lahoz, C., de Oya, M., Badimon, L., 1997. Monounsaturated and polyunsaturated n-6 fatty acid-enriched diets modify LDL oxidation and decrease human coronary smooth muscle cell DNA synthesis. Arteriosclerosis, Thrombosis, and Vascular Biology 17, 2088 – 2095. McMurray, D.N., Jolly, C.A., Chapkin, R.S., 2000. Effects of dietary n-3 fatty acids on T cell activation and T cell receptormediated signalling in a murine model. The Journal Infectious Diseases 182, S103 – S107. Nicoletti, I., Migliorati, G., Pagliacci, M.C., Grignani, F., Riccardi, C., 1991. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. Journal of Immunological Methods 139, 271 – 279. Nishiyama, A., Cavaglieri, C.R., Curi, R., Calder, P.C., 2000. Arachidonic acid-containing phosphatidylcholine inhibits lymphocyte proliferation and decreases interleukin-2 and interferon-g production from concanavalin A-stimulated rat lymphocytes. Biochimica et Biophysica Acta 1487, 50 – 60. Painter, P.C., Cope, J.Y., Smith, J.L., 1994. In: Burtis, C.A, Ashwood, E.R. (Eds.), Tietz Textbook of Clinical Chemistry. W.B. Saunders, Philadelphia, PA, pp. 2161 – 2217. Appendix. Peck, M.D., Li, Z., Han, T., Wang, W., Jy, W., Ahn, Y.S., Ziboh, V.A., Chu, A.J., Bourguignon, L.Y., 1996. Fatty acid unsaturation increases expression and capping of Murine lymphocyte CD44 and CD45. Nutrition 12, 616 – 622. Peres, C.M., Procopio, J., Costa, M., Curi, R., 1999. Thioglycolate-elicited rat macrophages exhibit alterations in incorporation and oxidation of fatty acids. Lipids 34, 1193 – 1197. Peterson, L.D., Jeffery, N.M., Thies, F., Sanderson, P., Newsholme, E.A., Calder, P.C., 1998. Eicosapentaenoic and docosahexaenoic acids alter rat spleen leukocyte fatty acid composition and prostaglandin E2 production but have different effects on lymphocyte functions and cell-mediated immunity. Lipids 33, 171 – 180.

R. Verlengia et al. / Life Sciences 73 (2003) 2939–2951

2951

Pompe´ia, C., Lopes, L.R., Miyasaka, C.K., Procopio, J., Sannomiya, P., Curi, R., 2000. Effect of fatty acids on leukocyte function. Brazilian Journal of Medical and Biological Research 33, 1255 – 1268. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. A8.21. Siddiqui, R.A., Jenski, L.J., Neff, K., Harvey, K., Kovacs, R.J., Stilwell, W., 2001. Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process. Biochimica et Biophysica Acta 1499, 265 – 275. Tilley, S.L., Coffman, T.M., Koller, B.H., 2001. Mixed messages: modulation of inflammation and immune responses by prostaglandins and Thromboxanes. Journal of Clinical Investigation 108, 15 – 23. Vacaresse, N., Lajoie-Mazenc, I., Auge, N., Suc, I., Frisach, M.F., Salvayre, R., Negre-Salvayre, A., 1999. Activation of epithelial growth factor receptor pathway by unsaturated fatty acids. Circulation Research 85 (10), 892 – 899. Yamazaki, K., Kuromitsu, J., Tanaka, I., 2002. Microarray analysis of gene expression changes in mouse liver induced by peroxisome proliferator activated receptor a agonists. Biochemical and Biophysical Research Communications 290, 1114 – 1122. Yaqoob, P., 2002. Monounsaturated fatty acids and immune function. European Journal of Clinical Nutrition 56 (Suppl 3), S9 – S13. Yaqoob, P., Knapper, J.A., Webb, D.H., Williams, C.M., Newsholme, E.A., Calder, P.C., 1998. Effect of olive oil on immune function in middle-aged men. American Journal Clinical Nutrition 67, 129 – 135. Zugaza, J.L., Casabiell, X.A., Bokser, L., Eiras, A., Beiras, A., Casanueva, F.F., 1995. Pretreatment with oleic acid accelerates the entrance into the mitotic cycle of EGF-stimulated fibroblasts. Experimental Cell Research 219 (1), 54 – 63.