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Originally published online as doi:10.1189/jlb.1004604 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1339-1346.)
© 2005 by Society for Leukocyte Biology

Different signaling pathways inhibit DNA methylation activity and up-regulate IFN-{gamma} in human lymphocytes

Victoria Bonilla-Henao, Raquel Martínez, Francisco Sobrino and Elizabeth Pintado1

Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina y Hospital Universitario Virgen Macarena, Universidad de Sevilla, Spain

1 Correspondence: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Avda. Sanchez Pizjuán, 4, E-41009, Sevilla, Spain. E-mail: elizabet{at}us.es


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ABSTRACT
 
DNA methylation is recognized increasingly for its prominent role in controlling diverse immune processes. In this study, we show that in Jurkat T cells and fresh peripheral lymphocytes, short-time incubation with protein kinase C activators or phosphatase inhibitors down-regulate DNA methylation activity in a dose-dependent manner. This inhibition correlates with the induction of the interferon-{gamma} (IFN-{gamma}) gene, which contains several CG sequences in its promoter. The expression of mRNA and protein of the different DNA methyltransferases did not decrease after the treatment. In addition, sulfydryl reagents have a strong inhibitory effect on DNA methylation activity and also induce IFN-{gamma} gene expression, thus suggesting a link between both effects.

Key Words: DNA methyltransferases • PKC • phosphatase inhibitors • sulfhydryl-blocking reagents


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INTRODUCTION
 
It is well established that DNA methylation plays an important role in the silencing of gene expression, which is critical in X chromosome inactivation, parental imprinting, cancer, and development [1 2 3 4 5 ]. DNA methylation is also increasingly recognized for its prominent role in controlling diverse immune processes [6 7 8 ]. Methylation has been implicated as a main mechanism during immune development, controlling VDJ recombination, lineage-specific expression of cell-surface antigens, and transcriptional regulation of cytokine during immune response [9 ]. Maintenance of the methylation patterns is sensitive to exogenous and endogenous influences; however, signal pathways involved in DNA methylation/demethylation are poorly known [3 , 6 ]. DNA methyltransferases are the enzymes responsible for the transfer of methyl groups from S-adenosylmethionine to CG sequences in DNA [10 11 12 ]. In murine adrenocortical cells, the levels of DNA methyltransferase 1 (DNMT1) increase through members of the Ha-ras pathway, and in Jurkat T cells, nitric oxide increases DNMT1 activity without changing its expression [13 , 14 ]. DNA demethylation has been implicated in the transcriptional regulation of several genes [8 ] and the demethylase enzymes; although more elusive to study than the methyltransferases, its activity has been measured in several preparations [15 16 17 ].

T cell activation via antigen receptor is associated with the production of inositol trisphosphate and diacylglycerol, which increase intracellular calcium and protein kinase C (PKC) activity, respectively [18 , 19 ]. Following activation, T cells respond by inducing the expression of different mediators in the immune system, such as interferon-{gamma} (IFN-{gamma}) [20 ], which is an immunoregulatory gene that plays a major role in promoting specific mechanisms of host defense. This is of crucial importance in nearly all phases of immune and inflammatory responses. The gene structure is remarkably similar among all species, and it is interesting that the promoters are more highly conserved than the exons. In this conserved promoter region, there are a number of potential methylation sites [21 , 22 ]. However, the extent to which DNA methylation contributes to proper regulation of T cell effector function is unclear and in many cases, controversial [23 ]. Increased IFN-{gamma} gene transcription has been correlated with hypomethylation of the proximal promoter region, and hypermethylation has been demonstrated to inhibit nuclear factor binding to the human IFN-{gamma} promoter [24 ]. In addition, treatment of human neonatal naïve T cells with 5-aza-2'-deoxycytidine was shown to markedly up-regulate their capacity to produce IFN-{gamma} [25 ]. It is also of interest to note that human immunodeficiency virus type 1 infection of human T cells inhibits IFN-{gamma} gene expression associated with CpG hypermethylation in its promoter and that treatment of infected cells with an antisense DNA methyltransferase reverses the hypermethylation and increases IFN-{gamma} expression [26 ]. In addition, IFN-{gamma} production in vivo is regulated tightly, and PKC activity seems to be involved in the signal transduction leading to IFN-{gamma} gene transcription [27 , 28 ].

In this work, we show for the first time that short periods of incubation with PKC activators down-regulate DNA methylation activity, which correlates tightly with the expression of inducible CpG-containing genes such as IFN-{gamma}, suggesting a link between both effects. In addition, okadaic acid, a protein phosphatase (PP) inhibitor, and thimerosal, a sulfydryl reagent, also decrease DNA methylation activity and induce IFN-{gamma} expression. These results further support the view that DNA methylation is a dynamic process that can be affected by different signaling pathways, probably involving phosphorylation, and could regulate key aspects of the peripheral immune system.


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MATERIALS AND METHODS
 
Materials
RPMI 1640, L-glutamine, fetal bovine serum (FBS), and other tissue culture reagents were purchased from BioWhittaker (Verviers, Belgium). Restriction enzymes and DNMT1-specific antibodies were from New England Biolabs (Hertfordshire, UK). Cold and 3H-labeled S-adenosylmethionine, Ficoll-Paque, Hybond-N+, polyvinylidene difluoride (PVDF) membrane, and the chemiluminescence detection system were from Amersham Bioscience (Uppsala, Sweden). Taq polymerase, poly dI-dC, cell proliferation kit I [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)], RNA isolation kit, and specific primers were from Roche (Mannheim, Germany). DNMT3A and DNMT3B antibodies were from Santa Cruz Biotechnology (CA). Mezerein, phorbol 12-myristate 13-acetate, ionomycin, okadaic acid, bisindolymaleimide, and protease inhibitors were obtained from Sigma Chemical Co. (Madrid, Spain). The DNA modification kit was from Chemicon International (Temecula, CA). Antibodies against human cell-surface antigens CD14, CD20, and CD56 and coated magnetic anti-immunoglobulin G (IgG) were from Coulter-IZASA (Barcelona, Spain). All others reagents were of the best commercially available quality.

Fresh peripheral lymphocytes, T lymphocytes, and Jurkat T cell culture
Human peripheral lymphocytes were isolated from fresh heparinized blood of healthy human donors, after informed consent, by Ficoll-Paque gradient centrifugation, followed by hypotonic lysis of residual erythrocytes as described [29 ]. After elimination of adherent cells by incubation on plastic dishes for 45 min at 37°C, lymphocytes were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin and maintained at 37°C in an atmosphere of 5% CO2. Purified T cells were obtained by the magnetic cell system (magnetic cell sorter) with a mixture of mouse anti-human CD14, CD20, and CD56 monoclonal antibodies (4 µg/ml each antibody), followed by the addition of goat anti-mouse IgG-coated magnetic beads [30 ]. The purity of the population, detected by flow cytometry, was always greater than 95% CD3+ cells. Jurkat T cells were grown in the same medium plus 2.5 µg/ml amphotericin B under 5% CO2 at 37°C as described [14 ]. Following incubation with the different agents, cells were harvested, and DNA, RNA, or nuclear proteins were extracted. Cell proliferation in control and treated cells after 5 h incubation was determined by a colorimetric assay (cell proliferation kit, MTT).

DNA methylation activity assay
DNA methylation activity was determined in nuclear protein extracts as previously reported [14 ] with minor modifications. Cells were lysed in buffer containing 20 mM Tris- HCl, pH 8, 137 mM NaCl, 5 mM MgCl2, 5 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 100 µg/ml RNase. After centrifugation, nuclear extracts were prepared by suspension of the crude nuclei in a high salt buffer. Proteins (10–20 µg) were incubated for 2 h at 37°C with 4 µg poly (dI-dC) as template and 3 µCi 3H-labeled S-adenosylmethionine as methyl donor. Reactions were stopped, proteins were extracted, and DNA template was recovered by ethanol precipitation. RNA was removed by suspension of the precipitates in NAOH. DNA was spotted on square pieces of Hybond-N+ membranes, cross-linkage with ultraviolet (UV), and washed by drastic agitation in cold phosphate buffer for 20 min, three times, and another 20 min in ethanol. The membranes were placed in a scintillation mixture and 3H counted using a Wallac ß-counter (Perkin Elmer, Zaventem, Belgium). Results were expressed as pmoles [3H]-CH3 per mg protein or in percentage of the control. All the experiments were performed in duplicate and repeated at least three times. Background levels were determined in assays where poly (dI-dC) was omitted.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was extracted from Jurkat T cells or fresh peripheral lymphocytes using the RNA isolation kit following the manufacturer’s protocols. RNA (1 µg) was reverse-transcribed using random hexamers, and the cDNA was amplified using specific primers. IFN-{gamma} gives a product size of 510 base pairs (bp) using the primers described by Shulzhenko et al. [31 ]. Hodge et al. [32 ] described the primers used for the amplification of DNMT1 and yielded two bands of 267 bp and 315 bp, respectively. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was used as control [14 ]. PCR aliquots were electrophoresed on 1% agarose gels, visualized on ethidium bromide-stained agarose gels, and photographed under UV light.

Real-time PCR
After total RNA extraction, 100 ng was used subsequently for cDNA synthesis as described above. Real-time PCR was performed in an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) using the specific Taqman probe and primers and the thermocycler conditions recommended by the manufacturer. PCR reactions were performed in triplicate in a total volume of 25 µl containing 2 µl of the RT reaction. Each sample was analyzed for GAPDH to normalize for RNA input amounts and perform relative quantifications. Melting curve analysis showed a single sharp peak with the expected temperature melting for all samples. For the relative quantification of gene expression, the comparative threshold cycle (CT) method was used as described in User Bulletin 2 for ABI Prism 7700 sequence detection system. CT represents the PCR cycle, at which an increase in reporter fluorescence above a background signal can first be detected (10x the standard deviation of the baseline). First, internal control (GAPDH) CT values were subtracted from the gene-of-interest CT values to derive a {Delta}CT value. The relative expression of the gene-of-interest was then evaluated using the expression 2–{Delta}{Delta}CT, where the value for {Delta}{Delta}CT was obtained by subtracting the {Delta}CT of the calibrator from each {Delta}CT using the mean of the control (t=0) as the calibrator.

Western blot analysis
Cells were homogenized in cold lysis hypotonic buffer containing 20 mM Hepes, pH 7.9, 10 mM KCl, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2% Nonidet P-40 (NP-40), protease inhibitors (0.01% PMSF, 1 mM Na3VO4, 10 µg/ml aprotinin, 10 µg/ml leupeptin), and 1.5 µg/ml RNase-DNase-free for 10 min at 4°C. The homogenate was centrifuged 5 min at 13,000 g, the pellet was suspended in hypertonic buffer containing 20 mM Hepes, pH 7.9, 10 mM KCl, 420 mM NaCl, 20% glycerol, 1 mM EDTA, 1 mM DTT, 0.2% NP-40, and the protease inhibitors are indicated above. Protein concentration was determined with the Bradford protein assay reagent. The lysate (20–100 µg) was loaded and resolved on 5–7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto the PVDF membrane, and subjected to immunodetection using a 1:5000 dilution of primary antibody for DNMT1, 1:1000 for DNMT3A, and 1:500 for DNMT3B. Horseradish peroxidase-conjugated secondary antibodies to rabbit IgG (Promega, Madison, WI) were used as well as enhanced chemiluminescence for detection [33 ].

Methylation rate of the IFN-{gamma} gene by PCR
DNA was extracted from cell lines or fresh peripheral T cells by using the salt-out procedure as described previously [14 ]. Genomic DNA (100 ng) was digested by the restriction enzyme SnaBI at 37°C overnight in 20 µl-specific restriction buffer. Cytosine methylation in CpG dinucleotide at a sensitive site (bp –52) in the 5'-flanking region was measured by PCR. The-20 µl PCR reaction mixture contained 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM deoxy-unspecified nucleoside 5'-triphosphates (dNTPs), 0.5 µM each primer, 5 µl digested sample, and 1 unit Taq polymerase. The primers used were (–178) 5'-GAC CCA AGG AGT CTA AAG GAA ACT CTA ACT-3' and (+1) 5'-CTG ATC TTC AGA TGA TCA GAA CAA TGT GCT-3'. The temperature profiles for the amplification were 4 min at 94°C, 25 cycles of 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C, followed by a final extension of 7 min at 72°C. The amount of DNA was normalized with a PCR from the GAPDH, which is unaffected by SnaBI digestion. The 20 µl PCR products were loaded on 1% agarose gel, and the results were analyzed by densitometry as described [34 ].

Methylation-specific PCR (MSP)
The DNA methylation pattern in the IFN-{gamma} promoter was also determined by MSP [35 ]. Briefly, 1 µg genomic DNA was treated with sodium bisulfite by using the CpGenome DNA modification kit. After bisulfite treatment, unmethylated cytosine residues are changed into uracil residues, whereas methylated cytosine remains unmodified. The differentiation between methylated and unmethylated sequences can then be made by amplification using specific primers that target the uracil or the cytosine nucleotide. The primers used in MSP were designed based on the IFN-{gamma} promoter sequence (AF330164) by MethPrimer Design for Methylation PCR (www.urogene.org/methprimer/index1.html): unmethylated reaction, 5'- GTG ATA ATG GGT TTG TTT TAT T-3' (sense) and 5'-CCT AAT TAA AAT CTC CTA AAA ATT ACA TA-3' (antisense), and methylated reaction, 5'-GTG GGT ATA ATG GGT TTG TTT TAT C-3' (sense) and 5'-AAT TAA AAT CTC CTA AAA ATT ACG TA-3' (antisense). The 25-µl PCR reaction mixture contained 1x PCR buffer, 2.5 mM MgCl2, 500 µM dNTPs, 1 µM each primer, and 1 unit Taq polymerase (AmplyTaq Gold; PE Applied Biosystems, Foster City, CA). Temperature profiles for amplification were 12 min at 95°C, 10 cycles of 2 min at 94°C, 1 min at 50°C, and 3 min at 68°C, followed by 25 cycles of 15 s at 94°C, 15 s at 50°C, and 3 min and 20 s at 68°C, increasing the time of extension in each cycle by 20 s. As the first PCR is not productive, we performed a similar PCR with 5 µl from the first one. PCR products were analyzed on a 2% agarose gel stained with ethidium bromide. All reactions were repeated three times to ensure reproducibility of results.

Statistical analysis
Data were presented as mean ± SEM. A Student’s t-test was used to make comparisons between groups. P < 0.05 was considered statistically significant.


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RESULTS
 
Concentration-dependent effect of mezerein, okadaic acid, and thimerosal on DNA methylation activity
The effect of a phosphorylation-mediated signaling pathway on DNA methylation activity was examined. Figure 1A shows the concentration-dependent inhibition of DNA methylation activity in Jurkat T cells after 5 h incubation with mezerein plus a constant concentration of ionomycin (500 nM). Okadaic acid, a specific inhibitor of PP1 and PP2A, which can mimic PKC activation to regulate various cell functions, also inhibited DNA methylation activity in a dose-dependent manner. The time course of DNA methylation activity inhibition with mezerein (100 nM) plus ionomycin (500 nM) in fresh peripheral lymphocytes is shown in Figure 1B . These results indicate that DNA methylation activity can be down-regulated by signaling pathways involving phosphorylation. In addition, thimerosal, a sulfhydryl-blocking reagent, strongly inhibits DNA methylation activity in a dose-dependent manner, directly applied on the nuclear extracts or to the cell culture (Fig. 1C) . DNA methylation activity, measured as the net incorporation of H3-CH3 into the DNA, is considered to be the result of the activity of the different DNA methyltransferases and demethylases also present in the protein nuclear extract.



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  		Figure 1. DNA methylation activity in cells treated with PKC activators, phosphatase inhibitors, and sulfydryl reagents. (A) Jurkart T cells were treated with the indicated concentrations of mezerein (plus 500 nM ionomycin) or okadaic acid for 5 h. DNA methylation activity was measured in the nuclear protein extracts as described in Materials and Methods. Data are expressed as percentage of control values ± SEM of three different experiments, each point performed in duplicate. (B) Time dependence of DNA methylation activity in fresh peripheral lymphocytes treated with mezerein (100 nM) plus ionomycin (500 nM). Data are expressed in counts per minute (c.p.m.) ± SEM of three different experiments, each point performed in duplicate (**, P<0.01). (C) Jurkat T cells were treated with different concentrations of thimerosal for 5 h (in vivo) and for 2 h when applied directly to the nuclear extracts (in vitro).

Induction of gene expression after inhibition of DNA methylation activity
Next, we explored whether inhibition of DNA methylation activity was correlated with the induction of CpG-containing genes. We studied the expression of IFN-{gamma} in Jurkat T cells and fresh peripheral lymphocytes treated with the agents whose effect was tested previously on DNA methylation. The correlation between inhibition of methylation activity and the expression of IFN-{gamma} in Jurkat T cells is shown in Figure 2A . Incubation with mezerein (100 nM) plus ionomycin (500 nM) for 5 h induced IFN-{gamma} gene expression. However, when these agents were used separately, DNA methylation activity inhibition was not statistically significant, and the induction of IFN-{gamma} was decreased markedly. Moreover, okadaic acid (100 nM), which produced a pronounced inhibition of methylation activity, also elicited a clear up-regulation of IFN-{gamma}. Although fresh lymphocytes had basal methylation activity lower than Jurkat T cells, incubation with mezerein/ionomycin or okadaic acid also induced a drastic inhibition of DNA methylation activity, which correlated with the up-regulation of IFN-{gamma} mRNA (Fig. 2B) . These results suggest that in addition to other factors, such as activation of transcription factors, phosphorylation-mediated signaling pathways may also favor gene expression by inhibiting methylation activity. The important role of DNA methylation is, in addition, supported by the strong induction of IFN-{gamma} mRNA induced by thimerosal (20 µM; see Fig. 2A ).



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  		Figure 2. DNA methylation activity correlates with the expression of IFN-{gamma}. (A) DNA methylation activity and expression of IFN-{gamma} in Jurkat T cells treated during 5 h with mezerein (100 nM) and ionomycin (500 nM), these two separately or together, okadaic acid (100 nM), or thimerosal (20 µM). (B) DNA methylation activity and expression of IFN-{gamma} after 5 h incubation with mezerein (100 nM) plus ionomycin (500 nM) or okadaic acid (100 nM) in fresh peripheral lymphocytes. DNA methylation activity was measured as indicated in Materials and Methods. Results of DNA methylation activity are presented as means ± SEM in which each measurement was performed in duplicate (*, P<0.05; **, P<0.01). Gene expression was assessed by RT-PCR, as indicated in Materials and Methods. In both types of cells, DNMT1 expression was also studied. GAPDH was used as control. Data shown are representative of at least three separate experiments with similar results.

To further investigate the role of phosphorylation pathways in the inhibition of methylation and gene induction, we incubated the cells for 1 h with the PKC inhibitor bisindolymaleimide (1 µM) prior to the addition of the different agonists mentioned above. In Jurkat T cells, inhibition of DNA methylation activity, induced by mezerein/ionomycin or by okadaic acid, was reverted by bisindolymaleimide, which was correlated with the level of induction of IFN-{gamma}. Bisindolymaleimide, however, did not have a statistically significant effect when the cells were incubated with thimerosal (not shown) or without any other treatment (Fig. 3 ). Conventional RT-PCR showed that DNMT1 mRNA did not change in any of the conditions studied (see Figs. 2A and 2B and 3 ). Real-time PCR of DNMT1, DNMT3A, and DNMT3B showed a slight increase in DNMT3A with mezerein/ionomycin (Fig. 4A ). The significance of this is not clear and should be investigated further. Western blots with specific antibodies against DNMT1, DNM3A, and DNMT3B showed that the expression of any of them was inhibited by mezerein/ionomycin or okadaic acid (Fig. 4B) .



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  		Figure 3. Effect of PKC inhibitor on DNA methylation activity and gene expression in stimulated cells. Jurkat T cells were preincubated with 1 µM bisindolymaleimide 1 h before the addition of mezerein (100 nM) plus ionomycin (500 nM) or okadaic acid (100 nM). Thimerosal was used as a positive control, and the effect of bisindolymaleimide alone was also studied. After 5 h incubation with the different agents, DNA methylation activity was measured in the nuclear protein extracts as explained in Materials and Methods (*, P<0.05; **, P<0.01). Gene expression was assessed by RT-PCR, as also indicated in Materials and Methods. Data are representative of similar results of three independent experiments.



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  		Figure 4. mRNA and protein expression of DNMT1, DNMT3A, and DNMT3B in Jurkat T cells. (A) Jurkat T cells were incubated with mezerein (100 nM) plus ionomycin (500 nM) or okadaic acid (100 nM), and mRNA of DNA methyltransferases was measured by real-time PCR as described in Materials and Method. GADPH was used as control. We show the average fold induction ± SEM from three separate measurements. (B) Jurkat T cells were incubated with the same agents, and protein expression was detected by Western blotting analysis with specific antibodies. To verify even protein loading, the blots were subsequently stripped and reprobed with anti-GAPDH antibody or directly stained with Ponceau-red. Data are representative of three independent experiments, which yielded similar results.

Methylation status of IFN-{gamma} promoter after treatment of the cells with mezerein/ionomycin, okadaic acid, and thimerosal
It has been shown that the IFN-{gamma} promoter is highly conserved among species and that it contains a CpG site (at bp –52), important in the control of expression by methylation. We have studied the methylation status of this site using a methylation-sensitive restriction enzyme in fresh peripheral lymphocytes and in purified T cells. Figure 5A shows that after digestion with SnaBI, PCR was less productive in cells treated with the agents previously known to inhibit methylation, thus suggesting a less-methylated site after treatment. The amount of DNA was normalized with the PCR product of the GAPDH after digestion with SnaBI. The same results were obtained with purified peripheral T lymphocytes (Fig. 5B) . We have also studied if mezerein alone could have some effect on IFN-{gamma} promoter methylation in Jurkat T cells. Figure 5C shows that mezerein alone decreases the level of methylation, although with less potency than when cells were treated with mezerein plus ionomycin. Densitometry analysis was expressed as relative units (see Fig. 5A 5B 5C ). To further demonstrate that different biochemical pathways may induce IFN-{gamma} mRNA expression by favoring demethylation, we performed MSP of the IFN-{gamma} promoter in fresh peripheral T lymphocytes. Figure 5D shows that with specific primers for methylated sequences, control cells have a more productive PCR than treated cells, and the opposite was observed with specific primers for unmethylated sequences.



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  		Figure 5. PCR analysis of the degree of CpG methylation in the IFN-{gamma} promoter region. Jurkat T cells (A) or T cells from peripheral blood (B) were treated with mezerein (100 nM) plus ionomycin (500 nM), okadaic acid (100 nM), or thimerosal (10 µM) for 5 h at 37°C. Genomic DNA from T cells was extracted, and 100 ng was digested with SnaBI (10 U/µg DNA) overnight at 37°C. Digestion mix (5 µl) was used for detection of CpG methylation status by PCR, as explained in Materials and Methods. Three different experiments were performed with similar results. The level of methylation was analyzed by densitometry on 2% agarose, and the average of relative unit ± SEMis shown. (C) Jurkat T cells were treated with mezerein (100 nM) plus ionomycin (500 nM) or mezerein alone (100 nM) and processed as indicated in A. (D) MSP was performed as explained in Materials and Methods in T cells from peripheral blood treated as indicated in B. P-IFN-{gamma} M and P-IFN-{gamma} U, Methylated and unmethylated primers, respectively.


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DISCUSSION
 
Methylation of specific CG sequences in mammalian DNA is emerging as an important mechanism to regulate gene expression in the immune system, controlling T lymphocyte lineage commitment, T cell effector functions, and memory [6 , 7 , 36 ]. The DNA methylation pattern seems to be established by a complex interplay of different DNA methyltransferases and demethylases, the regulation of which is poorly known [37 ]. The purpose of this study was to explore if known pathways that mediate T cell activation could influence methylation, which would participate in the induction of CpG-containing genes in human lymphocytes.

We show that PKC activators down-regulate DNA methylation activity parallel to the induction of IFN-{gamma}, suggesting that both events are related. Similar results were obtained with okadaic acid, an inhibitor of PP1 and PP2A, indicating that regulation of methylation could rely on the concerted action of protein kinases and phosphatases. In addition, thimerosal, a sulfydryl-blocking agent extensively used for studying the regulatory mechanism of different processes in cells [38 ], strongly inhibits DNA methylation activity and also induces a potent expression of IFN-{gamma}. However, the role that DNA methylation plays in proper regulation of T cell effector function is unclear and in many cases, controversial [23 ]. Widespread demethylation of the IFN-{gamma} promoter in T CD8+ clones expressing high levels of IFN-{gamma} has been shown and conversely, the majority of clones expressing low or undetectable levels of IFN-{gamma} mRNA exhibited methylation at the CpG sites of the promoter [39 ]. Furthermore, in naïve CD4+ T cells, the IFN-{gamma} locus is generally regarded as being in a repressed state associated with methylation of CpG motifs [40 ]. Nevertheless, it has also been published that in human T cells, methylation is important to inhibit IFN-{gamma} gene expression during in vitro differentiation of a T helper cell type 2 (Th2) population [24 ]. In addition, in murine CD4+ T cells, it has been reported that regardless of Th1 or Th2 culture conditions, all the cells were hypomethylated on a specific CpG site of the IFN-{gamma} promoter [41 ]. In our preparation, control cells (Jurkat T cells and T peripheral lymphocytes) did not express IFN-{gamma} mRNA and displayed at least some degree of methylation at position –52, supported by the efficient PCR after digestion with a methylation-sensitive restriction enzyme and by MSP. Moreover, treated cells with mezerein/ionomycin, okadaic acid, or thimerosal expressed IFN-{gamma} mRNA and clearly decreased methylation of the IFN-{gamma} promoter, thus suggesting that the different pathways activated by agents used in this research result in a permissive transcription status that could act as a complementary and nonredundant mechanism to the activation of transcription factors. It has been reported recently that demethylation of the interleukin (IL)-2 promoter starts within hours after T cell stimulation, showing that epigenetic regulation of the IL-2 gene in T cell receptor-activated T lymphocytes is a key event in the initiation of immune response [8 ]. These data, in agreement with our results, strongly support the view that T cell activation possibly triggers signal transduction pathways favoring a demethylated status, thus allowing the expression of critical cytokines. As DNA synthesis did not occur during the time-frame used in our experiments (not shown), the data powerfully suggest that demethylation is active, although the precise role played by DNA methylases and demethylases remains to be clarified.

An increase in the activity and expression of DNMT1 has been reported in Jurkat T cells stimulated with phytohemagglutinin. However, this effect was observed after a minimum incubation of 24 h [42 ], a much longer time than in our experiments. It has also been shown that incubation with demethylating agents for several days provokes an increase in the expression and activity of DNMT1, probably as a compensatory mechanism [43 ]. The slight increase of DNMT3A mRNA with mezerein/ionomycin observed in our experiments could also be explained as a negative feedback regulatory mechanism, although it should be investigated further. As expected, the effect of mezerein/ionomycin on DNA methylation activity and gene expression was reverted by previous incubation of the cells with bisindolymaleimide, an inhibitor of several PKC isoforms [44 ]. PKC inhibitors also prevented the effect of okadaic acid on methylation and IFN-{gamma} expression, as observed in another process regulated by phosphorylation [45 ].

There are several recent reports, which reveal that aberrant levels and patterns of DNA methylation stimulate T cell autoreactivity and autoimmunity [46 ]. Treatment with procainamide or hydralazine, which inhibit DNMT activity, results in a lupus-like disease in animals, which mirrors many key aspects of idiopathic lupus in humans [47 , 48 ]. Moreover, recently, a new model of maturation of IFN-{gamma}-producing cells, associated with atopic disorders in which IFN-{gamma} levels are elevated in addition to the expected high levels of type 2 cytokines, has been reported [49 ]. The possibility that repetitive activation of T cells may contribute to an aberrant immunological response by methylation inhibition and induction of cytokines and other mediators should be taken into consideration.


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ACKNOWLEDGEMENTS
 
This work was supported by Grants 01/1132 from Instituto de Salud Carlos III and 80/02 from Servicio Andaluz de Salud, Spain. V. B-H. received a predoctoral fellowship from the University Hospital Virgen Macarena (Seville, Spain). R. M. was supported by a predoctoral fellowship from the Servicio Andaluz de Salud. Special thanks go to Michelle Sigrid Kremser for her help in improving the text. We are grateful to Dr. López-Barneo (Laboratorio de Investigaciones Biomédicas, Seville, Spain) for helpful comments on the manuscript.

Received October 21, 2004; revised June 28, 2005; accepted June 30, 2005.


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