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Immunology. Jul 2008; 124(3): 339–347.
PMCID: PMC2440828

Transcript levels of DNA methyltransferases DNMT1, DNMT3A and DNMT3B in CD4+ T cells from patients with systemic lupus erythematosus

Abstract

Global DNA hypomethylation in CD4+ T cells has been detected in systemic lupus erythematosus (SLE) and it seems to be linked to its pathogenesis. We investigated the relationship between overall DNA methylation and the expression of three DNA (cytosine-5) methyltransferases involved in the DNA methylation process. The DNA deoxymethylcytosine (dmC) content of purified CD4+ T cells from 29 SLE patients and 30 healthy controls was measured by means of an enzyme-linked immunosorbent assay (ELISA). The transcript levels of DNA cytosine-5-methyltransferase 1 (DNMT1), DNA cytosine-5-methyltransferase 3A (DNMT3A) and DNA cytosine-5-methyltransferase 3B (DNMT3B) were quantified by real-time reverse transcription–polymerase chain reaction (RT-PCR). Association studies were also carried out with several laboratory parameters, as well as with the patients’ clinical manifestations. SLE patients had a significantly lower CD4+ T-cell DNA dmC content than controls (0·802 ± 0·134 versus 0·901 ± 0·133) (P = 0·007). No differences in transcript levels were observed for DNMT1, DNMT3A and DNMT3B between patients and controls. The simultaneous association of low complement counts with lymphopenia, high titres of anti-double-stranded DNA (anti-dsDNA), or an SLE disease activity index (SLEDAI) of > 5, resulted in the increase of at least one of the three DNA methyltransferases. It is possible that patients were reacting indirectly to an underlying DNA hypomethylation status by increasing the mRNA levels of DNA methyltransferases when the disease was being definitely active.

Keywords: DNA methylation, epigenetics, lupus

In mammals, DNA methylation only occurs at cytosine residues found within cytosine–phosphate–guanosine (CpG) dinucleotides and it involves methylation in the fifth carbon of the pyrimidine ring, leading to the formation of 5-methylcytosine (5-mC). The majority of CpG sites (70–80%) in human DNA are methylated and many of the non-methylated sites are found in the so-called CpG islands, which are normally on functioning promoters. Several studies report a strong correlation between DNA methylation and genetic inactivity.1 On the other hand, DNA methylation inhibitors [5-azacytidine (5-aza-C)] are able to re-activate genes that have been previously methylated and silenced.2 Thus, DNA methylation is an epigenetic process linked to the regulation of several biological events, including embryonic development,3 transcriptional regulation of gene expression, X-chromosome inactivation, genomic ‘imprinting’, chromatin modification and the silencing of endogenous retroviruses.47 Altered DNA-methylation patterns have been detected and widely studied in tumorigenic events.8

The enzymes that methylate DNA are known as DNA cytosine-5-methyltransferases (DNMTs), the most studied among them being DNMT1. DNMT1 prefers hemimethylated DNA as a substrate and therefore will methylate newly replicated DNA only when the template nucleotides are methylated. DNMT1 is constitutively expressed and is required to maintain global methylation after DNA replication has taken place. Recently, other enzymes with the ability to methylate DNA have been identified, including DNMT3A and DNMT3B, which appear to be involved in de novo methylation, that is, methylation which involves the addition of methyl groups to sites not previously methylated. Both DNMT3A and DNMT3B play important roles in embryonic development.9 They are also able to carry out a maintenance methylation activity similar to that performed by DNMT1.10

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology. SLE patients suffer from several clinical manifestations that are often associated with the presence of anti-nuclear antibodies, mainly anti-double-stranded DNA (anti-dsDNA). During the course of the disease, tissue injuries develop as a result of the deposition of antibodies and immunocomplexes, which leads to the lesions observed on the skin and mucous membranes, and in kidneys, joints, the nervous system, lungs and the heart.

Some authors postulate that exposure to some environmental agents could induce SLE in predisposed people. The mechanisms by which such agents could interact with the immune system to trigger this pathology have not been discerned. Some medications that cause drug-induced lupus (procainamide, hydralazine), as well as ultraviolet light (which triggers lupus flares), can inhibit DNA methylation in cloned T-cell lines and can induce self-reactivity.11 Such agents induce overexpression of the lymphocyte function-associated antigen 1 (LFA-1), which confers an autoreactive status to T cells.12,13 CD4+ T cells from patients with active lupus have hypomethylated DNA14 and overexpress LFA-1 on an autoreactive subset, which spontaneously lyses autologous macrophages.15,16 Methylation levels in the thymus and lymphatic nodules of a murine model of lupus (MRL/lpr) are lower than those found in the MRL/+ strain.17 Finally, CD4+ T cells of mice treated with methylation inhibitors (5-aza-C or procainamide) and transferred to syngeneic mice induce a glomerulonephritis mediated by immunocomplexes, as well as anti-DNA and anti-histone IgG.18 This all supports the hypothesis that methylation inhibition is sufficient to cause a lupus-like illness. Furthermore, DNMT1 activity is decreased in SLE CD4+ T cells, and the levels of DNMT1 mRNA seem to be low in SLE patients with active disease.14,19

The identification of DNA methyltransferases means that cells have the capacity to modify their methylation patterns. The role they may have on the hypomethylation associated with some pathological conditions is still unknown. As stated before, genome-wide hypomethylation in SLE CD4+ T cells has already been described. However, studies of other DNA methyltransferases (other than DNMT1) in SLE patients are still lacking. In the present work we investigated, for the first time, the simultaneous gene expression of DNMT1, DNMT3A and DNMT3B in patients suffering from SLE. We suggest that an increase of these enzymes may be taking place when the disease is highly active.

Subjects and methods

Subjects

Data were collected from 29 Spanish individuals (seven men and 22 women; mean age 33·62 years, range: 20–50 years) who suffered from SLE. An ethnically matched random healthy control population (blood donors) was also included in the study (n = 30, 17 men, 13 women; mean age 36·83 years, range: 21–66 years). The subjects’ written consent was obtained according to the Declaration of Helsinki,20 and the design of the work conformed to standards currently applied in Spain. All the SLE patients fulfilled at least four of the American College of Rheumatology criteria.21 Complete medical histories were obtained and physical examinations and laboratory tests were conducted for patients at the time of sample withdrawal. Laboratory parameters were evaluated as previously described.22 Clinical manifestations were defined according to the American Rheumatism Association glossary committee.23 A flare was defined as any clinical event directly attributable to disease activity that required a change in treatment. SLE activity was assessed by the SLE disease activity index (SLEDAI),24 and those with an SLEDAI of > 5 were considered to have active disease. The type of clinical flare, serological variables and medications taken by the patients are detailed in Table 1.

Table 1
Patients’ distribution according to their clinical and serological features and the medications taken until the clinical flare was manifested

Isolation of peripheral blood mononuclear cells and CD4+ T cells

A total of 20 ml of ethylenediaminetetraacetic acid (EDTA)-K3-preserved venous peripheral blood was withdrawn from both patients and controls. Peripheral blood mononuclear cells (PBMCs) were obtained by Histopaque-1077 (Sigma, Madrid, Spain) density-gradient centrifugation. CD4+ T cells were isolated by negative selection using the CD4+ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) using a cocktail of biotin-conjugated monoclonal antibodies (mAbs) against CD8, CD14, CD16, CD19, CD36, CD56, CD123, T-cell receptor-γδ (TCR-γδ) and glycophorin A, and magnetic microbeads conjugated to a monoclonal anti-biotin antibody. Thus, following the instructions provided by the manufacturer, magnetically labelled non-CD4+ T cells were depleted by retaining them on a MACS LS Column in the magnetic field of a MACS Separator (Miltenyi Biotec), while the unlabelled T helper cells passed through the column and were collected. After removal of the column from the magnetic field, the flow-through non-CD4+ T cells were also collected by flushing them out using a plunger. The purity of both the enriched CD4+ T cells and the non-CD4+ T cells was evaluated by flow cytometry in a FACSCalibur flow cytometer (Beckton Dickinson, Mountain View, CA) after incubating an aliquot of the cell fractions (5 × 104 cells) with 2 μl of anti-CD4–fluorescein isothiocyanate (FITC) (Miltenyi Biotec) for 10 min at 4°. The purity of CD4+ T cells and non-CD4+ T cells was generally higher than 90 and 95%, respectively.

Genomic DNA extraction and measurement of DNA deoxymethylcytosine content by enzyme-linked immunosorbent assay

DNA was extracted from cells using the QIAamp DNA Blood Midi kit (Qiagen, Izasa, Spain). The DNA concentration and 260:280 nm absorbance ratios were calculated using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Montchanin, DE). The DNA deoxymethylcytosine (dmC) content was measured by means of an enzyme-linked immunosorbent assay (ELISA) developed by us.25

RNA isolation and reverse transcription–polymerase chain reaction (RT-PCR)

Total RNA from CD4+ T cells was isolated using the Ultraspec RNA isolation system (Biotecx Laboratories, Inc., Houston, TX). Reverse transcription was carried out using the QuantiTect Reverse Transcription Kit (Qiagen), according to the manufacturer's instructions. Two microlitres of cDNA were taken for PCR amplification by the QuantiTect Multiplex PCR Kit (Qiagen) in a total volume of 50 μl, which included 25 μl of ×2 QuantiTect Multiplex PCR master mix, 0·4 μm of each forward primer, 0·4 μm of each reverse primer, 0·2 μm of each probe and 0·5 units of uracyl-N-glycosylase (UNG) (Sigma). Primers for β-actin were purchased from Invitrogen Life Technologies (Paisley, UK), whereas those for the other genes were obtained from Operon Biotechnologies GmbH (Cologne, Germany). Taqman probes were used in all cases [the β-actin probe was from Applied Biosystems (Cheshire, UK) and all the others were from Operon Biotechnologies GmbH], and all reactions were run in duplicate in MicroAmp optical 96-well plates sealed with optical adhesive covers (Applied Biosystems) on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). Taqman dual probes were labelled with a reporter dye [6-carboxy-fluorescein (6-FAM) or VIC® (Applied Biosystems)] and either a fluorescent quencher [6-carboxy-tetramethylrhodamine (TAMRA)] or a non-fluorescent quencher (NFQ). Forward primers, reverse primers and probe sequences were as follows. β-actin: 5′-TCACCCACACTGTGCCCATCTACGA-3′, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′ and 5′-VIC-ATGCCCTCCCCCATGCCATCCTGCGT-NFQ-3′; DNMT1: 5′-CGGTTCTTCCTCCTGGAGAATGTCA-3′, 5′-CACTGATAGCCCATGCGGACCA-3′ and 5′-6-FAM-AACTTTGTCTCCTTCAAGCGCTCCATGGTC-TAMRA-3′; DNMT3A: 5′-CAATGACCTCTCCATCGTCAAC-3′, 5′-CATGCAGGAGGCGGTAGAA-3′ and 5′-6-FAM-AGCCGGCCAGTGCCCTCGTAG-TAMRA-3′; DNMT3B: 5′-CCATGAAGGTTGGCGACAA-3′, 5′-TGGCATCAATCATCACTGGATT-3′ and 5′-6-FAM-CACTCCAGGAACCGTGAGATGTCCCT-TAMRA-3′. Thermocycling parameters were: 2 min at 50° for UNG activation, 15 min at 95° for HotStarTaq DNA polymerase activation and 50 cycles consisting of 1 min at 94° for denaturation and 1 min at 60° for annealing/extension. Negative controls (in which water instead of cDNA was added) were also run in each plate. Furthermore, contamination of the RNA samples by genomic DNA was excluded by an analysis without prior cDNA conversion [i.e. by excluding reverse transcriptase from the reverse transcription–polymerase chain reaction (RT-PCR)].

Reactions for determining the expression of the gene of interest and the reference gene (β-actin) were carried out either as duplex PCR reactions (i.e. amplification of both genes in the same well) (DNMT1 and DNMT3A) or as separate PCR reactions (DNMT3B). Each assay included a standard curve for both genes. The standard curve was constructed with serial dilutions of reverse transcription products corresponding to different concentrations of total RNA from a reference cell line (HeLa). Expression was compared with the standard curve and reported in equivalent quantity of total RNA from the reference cell line. Normalization of RNA amounts was performed using β-actin expression analysed with the same procedure. Finally, expression ratios between the gene of interest and β-actin were calculated.

Statistical analysis

Either the Mann–Whitney U-test or the independent-samples t-test for equality of means (along with the Levene's test for equality of variances) was used to compare values. Spearman's rank correlation was used to examine the relationship between two continuous variables. P-values of <0·05 were considered significant. All analyses were performed using spss, version 10·0 (SPSS Inc., Chicago, IL).

Results

Global DNA methylation in CD4+ T cells and in non-CD4+ T cells

To determine whether global methylation was decreased in SLE patients, CD4+ T cells and non-CD4+ T cells were isolated from 29 patients and 30 normal controls. DNA methylation indices are summarized in Table 2. No statistically significant differences were found between patients and controls when the methylation status of non-CD4+ T cells was assessed (0·818 ± 0·175 versus 0·833 ± 0·159, respectively). When comparison was established for CD4+ T cells, a decreased mean methylation index was observed in the group of patients (0·802 ± 0·134) with respect to the methylation index observed in the control group (0·901 ± 0·133) and it was, in fact, statistically significant (P = 0·007).

Table 2
DNA methylation indices in CD4+ and non-CD4+ T cells, and normalized gene expression levels in healthy controls and in patients with systemic lupus erythematosus (SLE)

Although women had slightly higher methylation indices compared with men in both CD4+ and non-CD4+ T cells, no statistically significant differences were detected between men and women in either the group of patients or in the control group.

Age-related changes in 5-mC content have been reported by other authors.26 In our case, however, no correlation was found between the methylation value and the patient's age, either when non-CD4+ T cells were considered or when CD4+ T cells were evaluated.

mRNA levels of DNA (cytosine-5) methyltransferases (DNMT1, DNMT3A and DNMT3B)

Once the methylation status was found to be decreased in SLE CD4+ T cells, quantitative real-time PCR assays were carried out in order to evaluate the mRNA levels of three DNA (cytosine-5) methyltransferases. β-Actin was chosen as a control to normalize mRNA levels because its expression does not change in lupus T cells.27 We were able to detect considerable amounts of each transcript, except for DNMT3B, whose degree of transcription was very low; in fact, some individuals (three controls and 15 SLE patients) were not taken into account when their mean DNMT3B levels were calculated because their DNMT3B mRNA levels were too low to obtain similar and reliable duplicate values. DNMT3A was the enzyme that showed the highest mRNA levels, followed by DNMT1 and DNMT3B.

Patients and controls showed similar mRNA levels of DNMT1, DNMT3A and DMT3B (see Table 2). Overall, all enzymes reflected a wide range of variability among individuals, especially DNMT1 and DNMT3A, for which one patient in particular (patient 1) had very high levels (5·58 and 4·40, respectively).

When considering just the control group, and when age was taken into account, Spearman's rank negative correlation coefficients were obtained with respect to all the enzymes: r = −0·607, P < 0·0005 (DNMT1); r = −0·577, P = 0·001 (DNMT3A); and r = −0·490, P = 0·009 (DNMT3B). In the group of patients, just one negative correlation was observed, for DNMT3A (r = −0·414, P = 0·025).

Men and women had similar levels of DNMT1 and DNMT3B mRNA. Nevertheless, it should be pointed out that healthy women had significantly higher levels of DNMT3A mRNA (0·607 ± 0·389) than healthy men (0·319 ± 0·248) (P = 0·013). In fact, although not statistically significant, this tendency was also observed in the group of patients: 0·624 ± 0·908 in women versus 0·397 ± 0·169 in men.

Apparently, there seemed to be a tight transcription regulation between DNMT3A and DNMT3B in healthy controls (r = 0·760, P < 0·0005) and SLE patients (r = 0·578, P = 0·030). The DNMT1 mRNA levels also correlated with the DNMT3A mRNA levels (r = 0·420, P = 0·021 in the control group, and r = 0·688, P < 0·0005 in the group of patients), whereas they did not correlate with the DNMT3B mRNA levels (r = 0·245, P = 0·219 in the control group; and r = 0·486, P = 0·078 in the group of patients).

Correlations between mRNA enzyme levels and methylation indices

We then wanted to ascertain whether a relationship between mRNA enzyme levels and methylation indices existed and if such behaviour was identical both in controls and in SLE patients. Positive correlations with methylation indices were detected for each enzyme in the control group and statistically significant values were observed for DNMT3B (P = 0·018) (see Fig. 1). On the contrary, a statistically significant negative correlation was observed with methylation indices for DNMT3A and DNMT3B in the group of patients (P = 0·028, P = 0·030, respectively). DNMT1 also showed a negative correlation with methylation indices, and such a correlation was statistically significant when patient 1 was withdrawn from the group of patients (r = −0·383, P = 0·044). Interestingly, this negative correlation was clearly apparent in individual SLE patients over time (Fig. 2). Thus, when we studied five patients from whom we could obtain one or two more blood samples, a few or several weeks apart, we observed that when methylation indices increased, a fall of DNMT1 levels followed, and vice versa. In general, the inverse relationship observed between global methylation and mRNA levels of DNMT3A and DNMT3B could also be detected individually in these five patients over time.

Figure 1
Correlation between methylation indices and normalized levels of DNA cytosine-5-methyltransferase 1 (DNMT1), DNA cytosine-5-methyltransferase 3A (DNMT3A) and DNA cytosine-5-methyltransferase 3B (DNMT3B) in healthy controls (a) and in patients with systemic ...
Figure 2
Inverse correlation between methylation indices and the levels of normalized DNA cytosine-5-methyltransferase 1 (DNMT1) in five patients from whom one or two more samples were obtained after the moment (indicated by number 1) in which they were included ...

Treatment effect on methylation indices and enzyme transcript levels

Medications can also be responsible for CD4+ T-cell DNA hypomethylation. Thus, we evaluated methylation indices and enzyme transcript levels in those patients receiving some kind of treatment (n = 22) and in those who were receiving no treatment (n = 7). No statistically significant differences with respect to either methylation values or mRNA levels were observed between the groups. Similarly to the situation when all SLE patients were analysed together, non-treated patients also had low global methylation indices (0·795 ± 0·117) in comparison with controls.

As some authors28 have reported that the expression of DNMT1 mRNA may be increased after steroid treatment, we focused on the seven individuals who had been receiving prednisone only. The methylation indices were similar to those found in the non-treated group (0·786 ± 0·092) and although a tendency towards increased mRNA expression was seen for DNMT1 and the other enzymes, it did not reach statistical significance.

Relationship between laboratory parameters and global methylation

To determine whether any of the methylation variables we had studied were correlated with the laboratory parameters defined in Table 1, we performed several Spearman's correlation tests. A slight statistically significant positive correlation was observed between anti-dsDNA titres and methylation indices (r = 0·386, P = 0·047). Nevertheless, when patients were grouped according to their immunological status for such antibodies, we found no statistically significant differences between the mean values of those with high titres (> 15 IU/ml) and those with low titres (≤ 15 IU/ml) (methylation indices: 0·829 ± 0·137 and 0·753 ± 0·121, respectively, P = 0·100). Moreover, no differences were seen between these two groups regarding the mRNA enzyme levels.

As for the other laboratory findings, only C3 complement levels were found to be negatively correlated with DNMT1 mRNA levels (r = −0·427, P = 0·021); but when comparison was established between those patients with low titres (< 85 mg/dl) and those with higher titres (≥ 85 mg/dl) no statistical differences were observed (0·696 ± 1·32 and 0·247 ± 0·081, P = 0·054). Patient no. 1 seemed to be responsible for obtaining such an elevated mRNA DNMT1 mean value in the first group (when she was withdrawn, the mean value and standard deviation were 0·370 ± 0·227).

Interestingly, when considering just the 12 patients who had lymphocyte counts lower than 1·2 × 109 cells/l, we realized that a strong negative correlation existed between C3 complement levels and the mRNA levels of DNMT1 (r = −0·726, P = 0·007), DNMT3A (r = −0·851, P <0·0005) and DNMT3B (r = −0·886, P = 0·019). None of these correlations were detected in the 17 patients who showed normal lymphocyte counts. Furthermore, those patients with lymphopenia and low C3 complement levels (n = 8) had higher levels of DNMT3A than those with lymphopenia and normal C3 complement levels (n = 4) (0·663 ± 0·418 and 0·323 ± 0·0517, respectively, P = 0·042). A negative correlation was also detected in these 12 patients between C4 complement levels and mRNA DNMT3A values (r = −0·722, P = 0·008) (a summary of the results is presented in Table 3).

Table 3
Relationship between complement values and normalized mRNA enzyme levels in patients with low lymphocyte counts, in those with anti-double-stranded DNA (dsDNA) titres higher than 15 IU/ml and in those with active disease [systemic lupus erythematosus ...

Finally, when we focused only on those patients with high anti-dsDNA titres (n = 19), inverse correlations with C3 complement levels and either DNMT1 (r = −0·579, P = 0·009) or DNMT3A (r = −0·657, P = 0·002) were observed (Table 3). None of these correlations were detected among the 10 patients with normal anti-dsDNA titres.

Disease activity, clinical flare and methylation status

When all patients were taken together, no correlations were detected between the SLEDAI values and the levels of any of the methylation variables studied. Patients with an SLEDAI of < 5 and patients with an SLEDAI of ≥ 5 had similar methylation indices. Transcript levels of DNMT1 and DNMT3A were higher in those patients who had an SLEDAI of ≥ 5; nevertheless, it was not statistically significant.

When just the 18 patients with an SLEDAI of ≥ 5 were taken into account, C3 complement levels and mRNA DNMT1 were inversely correlated (r = −0·473, P = 0·048) (Table 3).

Finally, we wanted to ascertain whether the type of clinical flare (as defined in Table 1) could be associated with any methylation-related variable. The three asymptomatic SLE patients showed higher methylation indices (0·955 ± 0·065) than all the other patients. No type of flare was linked to either methylation indices or mRNA enzyme levels. Of note, patients whose only clinical feature was asthenia also showed low methylation indices (0·730 ± 0·067).

Discussion

In this work we demonstrated that CD4+ T cells from SLE patients had a low DNA methylcytosine content. Quantitative real-time PCR assays were performed in order to ascertain the relative transcript levels of three enzymes involved on the DNA methylation process. We were able to optimize the reactions and to perform duplex amplifications for DNMT1 and DNMT3A, but not for DNMT3B. This gene seems to be transcribed at low levels in CD4+ T cells,29 whereas the levels of β-actin are considerably high. Thus, β-actin was overtaking DNMT3B amplification when a duplex PCR was used and it made the efficiency of the assay low for DNMT3B. Therefore, we had to carry out independent PCR reactions for β-actin and DNMT3B in order to overcome this problem.

Evidence for interindividual variation of transcript values was observed for all the genes. At least to some extent such a variation could be influenced by the age of the person. In fact, an inverse correlation was observed between gene transcription and age in the control population for all the genes. Therefore, we may postulate that a decrease of the DNA methyltransferases DNMT1, DNMT3A and DNMT3B may already take place during adulthood, although its effect on DNA methylation levels will not be detected until a more advanced age. Zhang et al. also found that the levels of DNMT1 and DNMT3A decreased with aging30 and that this decrease correlated with the hypomethylation of sequences flanking the ITGAL promoter, a gene which encodes CD11a, a subunit of LFA-1 (a molecule that confers autoreactivity to T cells);13 it translated into the overexpression of the gene, which leads to the hypothesis that hypomethylation may be one mechanism contributing to increased T-cell gene expression and the development of autoimmunity with aging.

The transcript levels of DNMT1 and DNMT3B were similar in men and women. On the contrary, DNMT3A was expressed at a higher degree in women. Inactivation of the X chromosome in women is a phenomenon that is known to be mediated by DNA hypermethylation. Our finding raises the possibility that DNMT3A is expressed at higher levels in women to maintain this hypermethylation state of the inactive chromosome. Against this hypothesis is the fact that de novo methylation associated with DNMT3A and DNMT3B seems to be dispensable for the initiation and propagation of X-chromosome inactivation in mouse embryos.31 There is, however, the possibility that a different mechanism may be involved in human T cells.

The underlying mechanism by which SLE patients show hypomethylated DNA remains controversial. Some authors suggest that a defect in DNMT1 may be the primary reason.3234 In our study we did not find decreased levels either of DNMT1 or of the other methyltransferases (i.e. patients and controls showed similar transcript levels of DNMT1, DNMT3A and DNMT3B). Other authors19,28 have, however, found that T cells from patients with active lupus have diminished DNMT1 mRNA levels. Besides the fact that the number of patients included in these studies was lower than in this study (seven patients with active lupus in Deng's study19 and nine SLE patients in Ogasawara's study28), it should be pointed out that these authors used PBMCs or total T cells instead of purified CD4+ T cells, and Dr Richardson's group19 was also activating the cells with phytohaemagglutinin (PHA) and expanding them with interleukin-2 (IL-2). It is possible that cells other than CD4+ T cells are those which express such a low amount of DNMT1 mRNA in SLE patients. We used only CD4+ T cells and did not stimulate them; therefore, our findings may reflect more accurately the real expression of these genes in vivo on this SLE cell population. Of note, we have recently found that the transcript levels of other enzymes involved in the DNA methylation process [methyl CpG-binding proteins 2 and 4 (MBD2 and MBD4)] are indeed overexpressed in SLE patients.25

An inverse correlation was observed between methylation and DNMT1, DNMT3A and DNMT3B mRNA levels in the group of patients. Furthermore, we clearly observed such a relationship between methylation and DNMT1 in the five patients from whom we could obtain more than one blood sample over time. Global hypomethylation associated with high levels of these enzymes has been observed in cancer cells.35 Moreover, previously published data supports the hypothesis that cells respond to pharmacological or dietetical inhibition of methylation by induction of methyltransferase activity.36,37 This suggests that this enzymatic activity is itself regulated, in part, by DNA methylation status, which represents a feedback mechanism. Of note, Slack et al.38 found a regulatory region in murine dnmt1 that seems to act as a sensor of the DNA-methylation capacity of the cell.

Interestingly, this relationship between methylation and gene expression levels was completely different in the control group. Thus, healthy individuals showed a positive correlation between mRNA levels and methylation indices. As DNMT1 has a five- to 30-fold preference for hemimethylated substrates,39 it has been defined as a maintenance DNA methyltransferase that restores DNA methylation patterns of the genome shortly after DNA replication. DNMT3A and DNMT3B methylate hemimethylated and unmethylated DNA with equal efficiencies in vitro10 and are therefore considered as de novo DNA methyltransferases. The fact that our healthy individuals showed a positive correlation with the methylation status may indeed be reflecting the function of all these three enzymes.

A direct correlation of mRNA levels was found between DNMT1 and DNMT3A as well as between DNMT3A and DNMT3B. Thus, when the mRNA levels of one enzyme increased, so did the mRNA levels of the other. Robertson et al.40 also found coordinate expression of DNMT1, DNMT3A and DNMT3B in most normal tissues. Therefore, these enzymes seem to be perfectly orchestrated in the DNA methylation pathway.

Neither DNA hypomethylation nor transcript levels were accounted for by type of medication. Ogasawara et al.28 found that the expression of DNMT1 mRNA by PBMCs from SLE patients was increased after steroid treatment. In our five patients followed-up longitudinally, we were able to ascertain that just two patients followed this rule. One had not been receiving anything (before the first blood sample was taken), whereas the other had received non-steroidal anti-inflammatory drugs (NSAIDs). The remaining three patients had been taking prednisone plus mycophenolate and all showed decreased DNMT1 mRNA levels after a new treatment with more steroids and/or mycophenolate was established. Therefore, we cannot rule out the possibility that DNMT1 mRNA levels increase after steroid treatment when the patient has not received any medication, or just NSAIDs, previously. Nevertheless, because similar results were obtained in non-treated patients and in patients who were taking steroids only, our results are more in line with those of other authors,14,19 which state that changes in methylation status in SLE patients are unlikely to be the result of corticosteroid treatment.

Published data indicate that T-cell DNA from active, but not from inactive, SLE patients contains reduced amounts of 5-mC.14 We did not find any difference when patients were grouped according to SLEDAI. We found that DNA methylation indices were low independently of disease activity (at least as measured by SLEDAI). In fact, patients who only suffered from asthenia (which is not even considered in the SLEDAI score calculation) also had a hypomethylated DNA. We must bear in mind that most of our patients presented with clinical flares (renal, cutaneous, arthritis or asthenia), so, from this point of view, any of them might be considered at least as a relatively SLE active patient, independently of their intrinsic SLEDAI value. For us, the clinical status was what marked the DNA methylation alterations. Actually, the three asymptomatic patients (and therefore, truly inactive patients) included in our study were the only ones who showed higher methylation indices. Like us, other authors14 have also reported that patients in remission generally have normal DNA methylation levels.

Complement values were inversely correlated with the mRNA levels of one or more DNA methyltransferases. This association was independently observed in patients with lymphopenia, in those with high titres of anti-dsDNA and in those with an SLEDAI of ≥ 5. Simultaneous association of any of these three conditions with low complement counts resulted in an increase of the transcript levels of at least one of these enzymes. From these results we could hypothesize that patients were indirectly reacting to an underlying DNA hypomethylation status by increasing the mRNA levels of the enzymes when the disease was being (either serologically or clinically) definitely active. It undoubtedly leads to the conclusion that epigenetics seems to play an important role in the course of regulation of SLE.

Acknowledgments

Grant support: this work was supported by funds provided by the Spanish Public Health Service grant FISS 02/0532 (from the ‘Fondo de Investigación Sanitaria’) and by funds provided by MOTEMA S.A. Company.

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