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Originally published online as doi:10.1189/jlb.0107064 on March 14, 2007

Published online before print March 14, 2007
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(Journal of Leukocyte Biology. 2007;81:1609-1616.)
© 2007 by Society for Leukocyte Biology

Transcript overexpression of the MBD2 and MBD4 genes in CD4+ T cells from systemic lupus erythematosus patients

Eva Balada, Josep Ordi-Ros1, Silvia Serrano-Acedo, Luis Martinez-Lostao and Miquel Vilardell-Tarrés

Research Unit in Systemic Autoimmune Diseases, Vall d’Hebron Research Institute, Hospital Vall d’Hebron, Barcelona, Spain

1 Correspondence: Research Unit in Systemic Autoimmune Diseases, Vall d’Hebron Research Institute, Hospital Vall d’Hebron, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain. E-mail: jordi{at}vhebron.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 two methyl CpG-binding domain (MBD) proteins. DNA deoxymethylcytosine (dmC) content of purified CD4+ T cells from 29 SLE patients and 30 healthy controls was measured by means of an ELISA. Transcript levels of two methyl CpG-binding proteins (MBD2 and MBD4) were quantified by real-time RT-PCR. Association studies were also carried out with several laboratory parameters, as well as with the patients’ clinical manifestations. SLE patients had significantly less CD4+ T cell DNA dmC content than controls (0.802±0.134 vs. 0.901±0.133; P=0.007). MBD2 and MBD4 mRNA levels were considerably higher in the patients’ group: 0.975 ± 0683 versus 0.604 ± 0.614 (P=0.004) and 0.359 ± 0.330 versus 0.092 ± 0.169, respectively (P<0.0005). It is interesting that SLE patients showed a negative correlation between methylation indices and MBD2 (r=–0.609, P<0.0005) and MBD4 (r=–0.395, P=0.034) transcript levels. MBD2 and MBD4 transcript overexpression and inverse correlations with DNA methylation indices indicate that both enzymes may really have a direct and active role on the genome-wide DNA hypomethylation observed in CD4+ T cells from SLE patients.

Key Words: DNA methylation • autoimmunity • epigenetics


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown etiology. SLE patients suffer from several clinical manifestations, which are often associated with the presence of antinuclear antibodies, mainly anti-dsDNA. During the course of the disease, tissue injuries develop as a result of the deposition of antibodies and immunocomplexes, which lead 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 [1 ]. Such agents induce overexpression of the LFA-1, which confers an autoreactive status to T cells [2 , 3 ]. CD4+ T cells from patients with active lupus have hypomethylated DNA and overexpress LFA-1 on an autoreactive subset, which lyses autologous macrophages spontaneously [4 5 6 ]. Methylation levels in thymus and lymphatic nodules of a murine model of lupus (MRL/lpr) were lower than those found in the MRL/+ strain [7 ]. Finally, CD4+ T cells of mice treated with methylation inhibitors (5-azacitidina or procainamide) and transferred to syngenic mice induce a glomerulonephritis mediated by immunocomplexes, as well as IgG anti-DNA and antihistone antibodies [8 ]. This all supports the hypothesis that methylation inhibition is sufficient to cause a lupus-like illness.

In mammals, DNA methylation only occurs at cytosine residues found within CpG dinucleotides, and it involves methylation in the fifth carbon of the pyrimidine ring, leading to the formation of 5-methylcytosine (5-mC). It is an epigenetic process linked to the regulation of several biological events including embryonic development, transcriptional regulation of gene expression, X chromosome inactivation, genomic "imprinting", chromatin modification, and silencing endogenous retroviruses [9 10 11 12 13 ]. DNA methylation-altered patterns have been detected and studied widely in tumorigenic events [14 ].

DNA methylation has been postulated to be irreversible, as breaking the bridge between the methyl group and the carbon at the fifth position of cytosine requires a high activation energy. Nevertheless, when using an assay that calculated the cytosine conversion of methyl-cytosines from a polymer made of double chains of methyl-CpG, a demethylase was purified, which was able to perform such a reaction [15 ]. This enzyme [methyl CpG-binding domain 2 (MBD2)] is a member of a conserved family of MBD proteins [16 ]. Another member, which seems to exert a catalytic action when bound to methyl-CpG sites, is MBD4, which can remove thymine or uracil from a mismatched CpG site in vitro, suggesting that it may function to minimize mutation at these sites [17 ]. Furthermore, 5-mC DNA glycosylate activity is also present in human MBD4 [18 ].

The identification of these enzymes means that cells have the capacity to modify their DNA methylation patterns. The role they may have on the hypomethylation associated to some pathological conditions is still unknown. As stated before, genome-wide hypomethylation in SLE CD4+ T cells has been described already. However, studies of enzymes [other than the DNA cytosine-5-methyltransferase 1 (DNMT1) enzyme] in this autoimmune disease are still lacking. In the present work, we have investigated for the first time the simultaneous gene expression of MBD2 and MBD4 in SLE patients. We suggest some alternative hypotheses, which could help to understand the causes of the global DNA hypomethylation observed in the CD4+ T cells of these patients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Data were collected from 29 Spanish individuals (7 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). Subjects’ written consent was obtained according to the Declaration of Helsinki, and the design of the work conformed to standards currently applied in Spain [19 ]. All the SLE patients fulfilled at least four of the American College of Rheumatology criteria [20 ]. 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 described previously [21 ]. Clinical manifestations were defined according to the American Rheumatism Association Glossary Committee [22 ]. A flare was defined as any clinical event directly attributable to disease activity, which required a change in treatment. SLE activity was assessed by the SLE disease activity index (SLEDAI), and those with a SLEDAI equal to or higher than 5 were considered to have active disease [23 ]. Type of clinical flare, serological variables, and medications taken by the patients are detailed in Table 1 .


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  		Table 1. Patients’ Distribution According to Their Clinical and Serological Features and the Medications Taken until the Clinical Flare Was Manifested

 
Isolation of PBMC and CD4+ T cells
A total of 20 ml EDTA-K3-preserved, venous peripheral blood was withdrawn from patients and controls. PBMC were obtained by Hystopaque-1077 (Sigma Chemical Co., Madrid, Spain) density gradient centrifugation. CD4+ T cells were isolated by negative selection with the CD4+ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). After removing the column from the magnetic field, the flow-through non-CD4+ T cells were also collected by flushing them out by a plunger. The purity of the enriched CD4+ and the non-CD4+ T cells was evaluated by flow cytometry. Purity of CD4+ T and non-CD4+ T cells was generally higher than 90% and 95%, respectively.

Genomic DNA extraction and measurement of DNA deoxymethylcytosine (dmC) content by ELISA
Cell DNA extraction was carried out with the QIAamp DNA blood midi kit (Qiagen, Izasa, Spain). DNA concentration and 260:280 absorbance ratios were calculated with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA).

DNA dmC content was measured by means of an ELISA using a mouse IgG anti-5-mC mAb (5-MeCyd; Oncogene Research Products, San Diego, CA, USA) according to the following procedure developed by us. To get the DNA attached, wells of high-binding polystyrene plates (Costar, Corning, NY, USA) were filled with 50 µL 0.01% poly-L-lysine solution and then incubated for 90 min at room temperature. DNA samples were diluted in 10 mM Tris-Cl, 1 mM EDTA (TE buffer), pH 7.5, denatured at 95°C for 5 min, and kept on ice 5 more min. After washing twice with PBS, 50 µL diluted DNA was added to quadruplicated wells and then incubated overnight at 4°C. After washing twice with PBS, 200 µL blocking solution (1% BSA prepared in PBS) was added per well and then incubated 1 h at room temperature. After two washes, 50 µL of a 1:2000 dilution of 5-MeCyd diluted in blocking solution was added per well, and plates were incubated 2 h at room temperature. After six washes, 50 µL of a 1:1000 ditution of alkaline phosphatase-labeled goat antimouse IgG prepared in blocking solution was added, and incubation was carried out for 1 h at room temperature. After washing six times with PBS, color was developed with 50 µL of p-nitrophenyl phosphate (Sigma Chemical Co.) prepared in diethylamine buffer (Merck, Darmstadt, Germany); plates were kept for 30 min at 37°C, and O.D. was read at 405 nm in a spectrophotometer (Labsystems iEMS Reader MF, Barcelona, Spain). Nonspecific background O.D. (wells without DNA) was subtracted from the corresponding tested sample.

Several assays were run before proceeding with our samples to establish the best conditions for developing a relative quantification method to evaluate global methylation levels. At first, a reference DNA sample (377 ng/µL) was twofold serially diluted in TE (starting at 1:1000 dilution) to observe the kinetics of our ELISA. This way, we could ascertain that the best DNA concentration range, in which it generated a direct and equal proportional correlation with O.D., was 0.025–0.05 ng/µL (Fig. 1 ). Therefore, we diluted our samples accordingly to obtain a DNA concentration which fell within this range. Methylation indices were finally calculated by getting the ratio between O.D. and DNA concentration for each sample. To minimize experimental variability between plates, we included the reference sample in each run, and control and patient methylation indices were corrected by establishing the ratio with the reference methylation index. (To see raw data obtained in a typical experiment, see Supplementary Table 1.)


Figure 1
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  		Figure 1. Kinetics of the ELISA used for determining global DNA methylation levels. Direct and equal proportional correlation with O.D. was seen with DNA concentrations between 0.025 and 0.05 ng/µL.

 
RNA isolation and real-time quantitative RT-PCR
Total RNA from CD4+ T cells was isolated using the Ultraspec RNA isolation system (Biotecx Laboratories, Inc., Houston, TX, USA). RT was carried out with the QuantiTect RT kit (Qiagen), according to the manufacturer’s instructions. cDNA was amplified by using the QuantiTect Multiplex PCR kit (Qiagen). Taqman probes were used in all cases [ß-actin probe from Applied Biosystems (Cheshire, UK), and MBD2 and MBD4 probes from Operon Biotechnologies GmbH (Germany)], and all reactions were run by duplicate in MicroAmp optical 96-well plates sealed with optical adhesive covers (Applied Biosystems) on an ABI PRISM 7000 sequence detection system. Primer and probe sequences are listed in Table 2 . 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., excluding RT from the RT reaction.


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  		Table 2. Primer and Probe Sequences for Quantitative RT-PCR Assays

 
Reactions for determining the expression of the gene of interest and the reference gene (ß-actin) were carried out as duplex PCR. Each assay included a standard curve for both genes. The standard curve was constructed with serial dilutions of RT 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 ß-actin expression analyzed with the same procedure. Finally, expression ratios between the gene of interest and ß-actin were calculated.

Statistical analysis
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 less than 0.05 were considered significant. All analyses were performed with SPSS, Version 10.0 (SPSS Inc., Chicago, IL, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Global DNA methylation in CD4+ T cells and non-CD4+ T cells
To determine whether global DNA 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 3 . 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 vs. 0.833±0.159, respectively). When comparison was established for CD4+ T cells, a decreased mean methylation index was observed in the patients’ group (0.802±0.134) with respect to the one observed in the control group (0.901±0.133), and it was in fact statistically significant (P=0.007; Fig. 2 ).


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  		Table 3. DNA Methylation Indices in CD4+ and Non-CD4+ T Cells and Normalized Gene Expression Levels in Healthy Controls and SLE Patients

 

Figure 2
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  		Figure 2. Comparison of methylation indices (A) and levels of normalized MBD2 (B) and MBD4 (C) between healthy controls and SLE patients. Data are presented as box plots, where the lines inside the boxes represent the medians, the boxes represent the 25th and the 75th percentiles, and the lines outside the boxes indicate the highest and lowest value, excluding the outliers (which are defined as those values within 1.5- and 3-box lengths from the upper or lower edge of the box).

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

Other authors [24 ] have reported age-related changes in 5-mC content. In our case though, no correlation was found between the methylation value and the patient’s age when non-CD4+ T cells were considered or when CD4+ T cells were evaluated.

mRNA levels of MBD2 and MBD4 in CD4+ T cells
Once the methylation status was found to be decreased in SLE CD4+ T cells, quantitative real-time PCR assays were carried out to evaluate the mRNA levels of two methyl-CpG-binding proteins in this cell population. ß-Actin was chosen as a control to normalize mRNA levels, as its expression does not change in lupus T cells [25 ].

MBD2- and MBD4-normalized values were considerably higher in the patients’ group: 0.975 ± 0.683 versus 0.604 ± 0.614 (for MBD2) and 0.359 ± 0.330 versus 0.092 ± 0.169 (for MBD4; Table 3 ). These findings were statistically significant (P=0.004 and P<0.0005, respectively; see Fig. 2 ). Overall, both enzymes reflected a wide range of variability among individuals.

Men and women had similar levels of MBD2 and MBD4 mRNA. When considering just the control group, and when age was taken into account, Spearman’s rank negative-correlation coefficients were obtained with respect to MBD4 (r=–0.499, P=0.005).

There seemed to be a tight transcription regulation between both enzymes. Thus, MBD2 mRNA levels correlated directly with MBD4 mRNA levels in patients (r=0.464, P=0.011) and controls (r=0.610, P<0.0005).

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 behavior was identical in controls and SLE patients. Positive correlations with methylation indices were detected for each enzyme in the control group, and statistically significant values were observed for both of them (r=0.466, P=0.010 for MBD2; r=0.441, P=0.015 for MBD4; Fig. 3 ). On the contrary, a statistically significant, negative correlation was observed with methylation indices for MBD2 and MBD4 in the patients’ group (r=–0.609, P<0.0005; r=–0.395, P=0.034, respectively).


Figure 3
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  		Figure 3. Correlation between methylation indices and normalized levels of MBD2 and MBD4 in healthy controls (A) and SLE patients (B). *, Statistically significant values.

 
Treatment effect on methylation indices and enzyme transcript levels
Medications could 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 those who were receiving nothing (n=7). No statistically significant differences with respect to methylation values or mRNA levels were observed between both groups. As when all SLE patients were taken as a whole, nontreated patients also had low global methylation indices (0.795±0.117) and high MBD2 and MBD4 mRNA levels (0.979±0.603 and 0.230±0.208, respectively) in comparison with controls. When we focused on the seven individuals who had been receiving only prednisone, we found that both methylation indices and MBD2 and MBD4 mRNA levels were similar to the ones found in the nontreated group.

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 antibody titers 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 titers (>15 IU/ml) and those with low titers (<15 IU/ml; methylation indices: 0.829±0.137 and 0.753±0.121, respectively; P=0.100). In fact, no differences were seen between these two groups regarding the mRNA enzyme levels either.

The other laboratory findings showed no correlation with the methylation indices or the mRNA enzyme levels.

Disease activity, clinical flare, and methylation status
When all patients were taken as a whole, no correlations were detected between the SLEDAI values and the levels of any of the methylation variables studied. Patients with a SLEDAI <5 and patients with a SLEDAI ≥5 had similar methylation indices as well as mRNA MBD2 and MBD4 levels.

Finally, we wanted to ascertain whether the type of clinical flare could be associated to any methylation-related variable. Patients whose only clinical feature was asthenia also showed low methylation indices (0.730±0.067) and high mRNA MBD2 and MBD4 levels (1.494±0.212 and 0.285±0.139, respectively). Of note, the three asymptomatic SLE patients showed higher methylation indices (0.955±0.065) and lower mRNA MBD2 and MBD4 levels (0.398±0.130 and 0.045±0.044, respectively) than all the other patients. The only patient who just had a cutaneous flare showed the highest mRNA MBD2 levels (2.667), whereas those who were diagnosed as suffering from only a renal flare had the highest mRNA MBD4 values (0.626±0.383).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
DNA extracted from T cells of SLE patients is hypomethylated when compared with DNA from normal T cells [6 ]. In this work, we have corroborated such a finding by using a new technique for assessing global methylation. Methylcytosine concentration in genomic DNA is usually quantified by high-performance separation means (HPLC and high-performance capillary electrophoresis) or by enzymatic/chemical approaches [26 ]. These methods are neither simple nor rapid; besides, several of these techniques require high amounts of DNA. Immunological analyses have also been carried out by using mAb raised against 5-mC. In these cases, antibodies have been used mainly to detect cellular chromosomes (by immunofluorescence microscopy) or DNA immobilized on nitrocellulose [27 ]. We have used an anti-5-MeCyd and have developed an ELISA to detect overall methylation in genomic DNA. In our hands, our method was highly reproducible and easy to perform. Furthermore, small amounts of DNA were necessary to perform the test.

Our study demonstrates that a cell population of PBMC (CD4+ T cells) from SLE patients has a low DNA methylcytosine content. As other authors, we found no different methylation indices between SLE patients and controls in non-CD4+ T cells [6 ]. No statistically significant differences were detected in both cell populations regarding sex and age. Other authors have, however, reported an age-dependent decrease of global methylation in T cells [24 , 28 ]. These studies included individuals older than 65 years of age, and comparisons were established with younger people. Thus, the fact by which we did not find such an association is most probably a result of the lower mean age of the subjects we evaluated. Besides, the effect observed by these authors may have been manifested because they covered much wider ranges of age.

Quantitative real-time PCR assays were performed to ascertain the relative transcript levels of two enzymes which bind to methylated DNA. Evidence for interindividual variation of transcript values was observed for both 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 MBD4. Therefore, we may postulate that a decrease on MBD4 may already take place during adulthood. As one of the functions of MBD4 seems to be the removing of thymine or uracil from mismatched CpG sites, its decrease could potentially account for an increased rate of mutations at CpG regions throughout life.

Normalized mRNA values of MBD2 and MBD4 were higher in our patients’ group. Furthermore, negative correlations between methylation indices and mRNA levels for both genes were observed in SLE individuals. It is interesting that 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; that is, MBD2 and MBD4 mRNA levels increased as methylation indices rose. MBD2 is an enzyme capable of actively demethylating DNA [15 ]. Other authors have, however, reported that MBD2 acts as a transcriptional repressor [29 ]. As reported by Detich et al. [30 ], both features may indeed coexist in the same enzyme to coordinate the expression of some promoters and the inactivation of others. In our hands, it seems that MBD2 may really be functioning as a demethylase. In the control group, MBD2 transcript levels were high when the methylation status was also elevated. This may reflect the existence of a compensatory mechanism for avoiding an excessive incorporation of 5-mC into DNA in normal cells. Conversely, MBD2 transcripts in SLE patients were high when methylation indices were low. If we assume that mRNA really translated into an enzyme with a true catalytic activity, it seems that the overexpression of MBD2 may actually be the cause for the global demethylation observed in SLE CD4+ T cells. In accordance with this hypothesis, it has been shown that breast cancer cell lines express high levels of demethylase mRNA relative to control cells and that the level of demethylase correlates with the level of global hypomethylation [31 ].

It is worth mentioning that non-CD4+ T cells also showed a negative correlation between their methylation indices and the levels of MBD2 mRNA expressed in CD4+ T cells (data not shown). We did not evaluate transcript expression in non-CD4+ T cells; nevertheless, there is the possibility that cells other than CD4+ lymphocytes express abnormal levels of MBD2 too; if that were the case, its effect on DNA demethylation probably would have been diluted as a result of their under-representation in the pool of non-CD4+ T cells.

Although throughout a different mechanism, MBD4 also has the capability of demethylating DNA by means of its 5-mC DNA glycosylate activity [18 ]. We found high MBD4 mRNA levels in our SLE patients. Thus, our results are in line with those of Mandel et al. [32 ], who also observed an overexpression of this gene when applying a gene microarray approach to PBMC of SLE patients. As this enzyme was overexpressed in our SLE patients and as its levels correlated negatively with DNA methylation indices, we could hypothesize that MBD4 may (as MBD2) have an active role in the global demethylation observed in such a population. On the contrary, it would have a passive role on the healthy population—its levels increasing as a result of an overall genome methylation to counteract an excessive DNA methylation. Alternatively, its paralleled increased levels with methylation indices in the control group may somehow reflect the other function this enzyme possesses, i.e., to minimize mutations at methyl-CpG sites [17 ].

DNA hypomethylation and transcript levels could not be accounted for by type of medication. Actually, patients who were not taking any drug also showed low global methylation indices and high MBD2 and MBD4 mRNA values. Of note, Mandel el al. [32 ] also found increased MBD4 mRNA levels in their nontreated SLE patients. As similar results were obtained in nontreated patients and patients who were only taking steroids, our results are in line with those of Dr. Richardson’s group [6 ], which state that changes in methylation status in SLE patients are unlikely to be a result of corticosteroid treatment.

Published data indicate that T cell DNA from active, but not inactive, SLE patients contains reduced amounts of 5-mC [6 ]. 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 also had a hypomethylated DNA as well as high MBD2 and MBD4 mRNA levels. We must keep in mind that most of our patients presented with clinical flares, so from this point of view, any of them might be considered at least as a relatively SLE-active patient. 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 and lower MBD2 and MBD4 mRNA values. Like us, other authors have also reported that patients in remission generally have normal DNA methylation levels [6 ].

Patients with SLE have elevated levels of circulating plasma DNA, which is reportedly enriched in hypomethylated CpGs, and it seems to be derived from apoptosis of host cellular DNA [33 , 34 ]. Some authors think that this "naked" DNA would exhibit antigenic properties and would act as a trigger for the induction of anti-dsDNA antibodies. In that case, an inverse correlation between anti-dsDNA antibodies and the methylation status of T cells could potentially exist. Quddus et al. [8 ] reported anti-ssDNA but not anti-dsDNA antibodies when murine CD4+ T cells treated with DNA methylation inhibitors were transferred to syngeneic mice. In this line, we did not find any negative relationship between methylation indices and anti-dsDNA levels either. In fact, we observed a tendency toward the opposite: DNA methylation indices of CD4+ T cells slightly correlated positively with anti-dsDNA levels. Nevertheless, no statistically significant differences were appreciated when DNA methylation indices were compared between patients with high and low titers of anti-dsDNA.

Global T cell DNA hypomethylation may alter the expression of some genes, and if not properly regulated, it may induce autoreactivity. The T cell genes affected by DNA methylation include several cytokines (for a review, see ref. [35 ]). Dr. Richardson’s group [36 37 38 ] have demonstrated that transcription of >100 known genes increases following treatment with DNA methylation inhibitors, including genes of possible relevance to lupus, such as ITGAL (CD11a), perforin, and CD70. The same authors also confirmed the overexpression of these molecules in T cells from SLE patients. However, the underlaying mechanism by which SLE patients show a hypomethylated DNA remains controversial. The aforementioned authors suggest that a defect in DNMT1 may be the primary reason. We have not found decreased levels of this or of other methyltransferases (E. Balada, unpublished results). Conversely, MBD2 and MBD4 mRNA transcript levels were indeed elevated, and it is most important that a negative correlation was observed with methylation indices. A complete, different behavior was seen in the controls group. Therefore, based on our findings, we could infer that MBD2 and MBD4 seem to have a direct and active role by demethylating CD4+ T cell DNA of SLE patients who present with some kind of clinical flare. Of course, more research is needed to truly ascertain such a hypothesis.

Besides the possible DNA-demethylating action MBD2 and MBD4 may exert, we have to consider other features of both enzymes, which could help us to understand the effects of their overexpression on SLE. It has been reported recently that naïve T cells, which lack MBD2, produce increased amounts of IFN-{gamma} and exhibit a TH1 bias in vivo [39 ]. These findings suggest that one effect of MBD2 on T cell cytokine production is the repression of IFN-{gamma}. Consequently, an overexpression of MBD2 could lead to a lower expression of IFN-{gamma}, and it could explain the bias toward the TH2 response so characteristic of SLE. With regard to MBD4, its deficiency seems to reduce the apoptotic response [40 ]. In fact, MBD4 can participate directly on this process by interacting with the Fas-associated death domain protein [41 ]. Therefore, an increase on MBD4 expression could induce an abnormal rate of cell death, which would account for the increased cell apoptosis and the impaired clearance of dying cells so frequently found in SLE patients.

Our work reinforces the idea that epigenetic alterations exist on SLE and opens the door for studying in more detail the effect that some enzymes involved on DNA methylation may have on the pathogenesis of SLE. A more exhaustive work in this direction will perhaps provide us with a better comprehension about the etiology of this disease. Future experiments will also determine whether inhibition of the overexpressed genes may indeed be considered as a therapeutical approach.


   ACKNOWLEDGEMENTS
 
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. We thank Dr. Philip E. Pellett for critical reading of the manuscript.

Received January 25, 2007; revised February 8, 2007; accepted February 9, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Cornacchia, E., Golbus, J., Maybaum, J., Strahler, J., Hanash, S., Richardson, B. (1988) Hydralazine and procainamide inhibit T cell DNA methylation and induce autoreactivity J. Immunol. 140,2197-2200[Abstract]
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