IL-6 Modulates CD5 Expression in B Cells from Patients with
Lupus by Regulating DNA Methylation1
Soizic Garaud,2* Christelle Le Dantec,2* Sandrine Jousse-Joulin,†Catherine Hanrotel-Saliou,†
Alain Saraux,*†Rizgar A. Mageed,‡Pierre Youinou,3*†and Yves Renaudineau*†
B lymphocytes from patients with systemic lupus erythematosus (SLE) are characterized by reduced expression levels of mem-
brane CD5. Recent studies from our laboratory have revealed that the level of membrane CD5 is determined by the relative level
of two alternative CD5 isoforms; CD5-E1A, which is expressed on the membrane, and CD5-E1B, which is retained in the cyto-
plasm. Using bisulfite sequencing and methylation-sensitive endonuclease assays we show that the promoter for the alternative
CD5-E1B isoform is demethylated in B cells from patients with SLE but not in healthy controls. We go on to show that differential
methylation is more pronounced following BCR engagement. As a result of this demethylation, CD5-E1B mRNA is transcribed
at the expense of CD5-E1A mRNA transcription. We provide further evidence that production of high IL-6 levels by SLE B cells
abrogates the ability of SLE B cells to induce DNA methyl transferase (DNMT1) and then to methylate DNA, an effect that is
reversed in the presence of a blocking Ab to the IL-6 receptor. The pattern of demethylation of CpG islands in the CD5-E1B
promoter in SLE B cells is similar to those in B cells from healthy controls stimulated in the presence of IL-6, or treated with
the methylation inhibitor PD98059. The study reveals that engagement of the BCR with constitutive IL-6 down-regulates the
level of membrane CD5, which negatively regulates BCR signaling, in SLE B cells. This altered signaling could, in turn,
promote the activation and expansion of autoreactive B cells in SLE patients.
lymphocyte proliferation, and the production of pathogenic Abs to
self-Ags. B cell abnormalities in SLE also include excess cytokine
production, autoantigen presentation to T cells and modulation of
the function of other immune cells (2). Thus, SLE is generally
considered a B cell disease, a theme strengthened by the efficacy of
therapies targeting B cells. For example, therapeutic approaches
using depleting anti-CD20 has proved to be highly beneficial in
treatment of SLE (3). Further, neutralization of cytokines that pro-
mote B cell responses such as IL-6, or interruption of cognate T
cell and B cell interactions has been successful in early clinical
trials (4, 5). All this provides the rationale for further investigation
The Journal of Immunology, 2009, 182:
ystemic lupus erythematosus (SLE)4is associated with di-
verse clinical manifestations (1). The main features of au-
toimmunity in SLE are B cell hyperactivity, spontaneous
of mechanisms of B cell involvement in driving autoimmunity and
to develop more selective therapeutic targets.
The central role played by B cells in immunity necessitates that
its responses are tightly regulated. B cell responses are initiated by
signaling through the BCR. Signaling initiated following BCR en-
gagement is regulated by coreceptors and by a network of protein
tyrosine kinases and phosphatases. Recent findings suggest that
defects in BCR-mediated signaling can result in lupus autoimmu-
nity. For example, there is an association between SLE autoim-
munity and mutations in a number of genes that encode B cell-
specific signaling molecules including protein tyrosine kinases,
non-receptor phosphatase type 22, B cell scaffold protein with
ankyrin repeats 1 and the inhibitory IgG Fc?RIIb (6). In addition
to direct evidence for the effect of mutations in signaling molecules
on BCR-mediated signaling, the contribution of epigenetic factors
has also been proposed (7). The most commonly observed epige-
netic abnormality implicated in SLE pathology is altered DNA
methylation at the 5-carbon position of cytosines of CpG dinucle-
otides. DNA methylation is regulated by three DNA methyl trans-
ferases (DNMTs), DNMT1, DNMT3a, and DNMT3b. DNMT1
preferentially targets hemi-methylated DNA over non-methylated
DNA. DNMT3a and DNMT3b, in contrast, exhibit de novo activ-
ities. The methyl-CpG-binding domain (MBD) protein-4 is a DNA
glycosylase that acts preferentially on hemi-methylated CpGs and
initiates demethylation by replacing a 5-methylcytosine with an
unmethylated cytosine (8). The action of MBD2 on DNA de-meth-
ylation has been suggested, but remains controversial (9). DNA
de-methylation activates the expression of many genes, such as
CD21 in B cells in patients with rheumatoid arthritis (10), CD40
ligand in T cells from patients with SLE (11), and the expression
of silenced retroviral genes (12). Increased expression of genes
resulting from de-methylation was confirmed using DNA methyl-
ation inhibitors in T cells from patients with SLE. Among those
inhibitors the ras signal blocker PD98059 appears to be the more
*Research Unit EA2216 Immunology and Pathology, IFR148 ScInBioS, Universite ´
de Brest and Universite ´ Europe ´enne de Bretagne;†CHU Brest, Ho ˆpital Morvan and
Ho ˆpital de la Cavale Blanche, Brest, France;‡William Harvey Research Institute,
Barts and the London School of Medicine and Dentistry, Queen Mary University of
London, London, United Kingdom
Received for publication July 30, 2008. Accepted for publication February 18, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the Conseil Regional de Bretagne, the
Conseil Ge ´ne ´ral du Finiste `re, and the French Ministry for Education and Research.
2S.G. and C.L.D. contributed equally to the work.
3Address correspondence and reprint requests to Prof. Pierre Youinou, Laboratory of
Immunology, Brest University Medical School Hospital, BP824, F-29609, Brest,
France. E-mail address: email@example.com
4Abbreviations used in this paper: SLE, systemic lupus erythematosus; ChIP, chro-
matin immunoprecipitation; DNMT, DNA methyl transferase; HERV, human endog-
enous retrovirus; HC, healthy control; MBD, methyl-CpG-binding domain protein;
LTR, long terminal repeat; SLEDAI, SLE disease activity index.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
relevant inhibitor because it induces cellular defects similar to
those observed in SLE (13).
B cells that express the CD5 protein, also known as B1 cells,
primarily express BCRs that are autoreactive, have a reduced ca-
pacity to enter the cell cycle, and have a longer lifespan. One
model for the role of CD5 in intracellular signaling suggests that
surface CD5 modulates signaling from the BCR and thereby con-
trols autoreactivity (14). According to this model, it is necessary
that the level of membrane CD5 is maintained to control the
threshold of BCR-mediated signaling. In humans, three mecha-
nisms have been shown to be involved in regulating the level of
membrane CD5. These are shedding (15), internalization of the
protein (16), and transcriptional regulation through altered level of
expression of two alternative CD5 isoforms (17). The first isoform,
designated CD5-E1A, corresponds to the full-length CD5 protein
which is synthesized and translocated to the cell membrane. The
second isoform derived from an alternative promoter, designated
CD5-E1B, encodes a truncated form of the protein that lacks a
leader peptide and is retained intracellularly. Indeed, CD5-E1B is
a fusion transcript from a new exon (4) that incorporates the 5?
long terminal repeat (LTR) of a human endogenous retrovirus
(HERV) sequence integrated at the time of old and new world
monkeys divergence estimated at ?25–30 million years ago (18).
We recently observed that transcription of mRNA for this isoform
inversely correlates with the level of DNMT1 mRNA (19).
Here, we provide evidence that relative to B cells from healthy
controls (HCs), the level of DNA methylation in BCR-mediated B
cell activation in patients with SLE is reduced. A consequence of
this hypomethylation is increased expression of CD5-E1B. Excess
production of IL-6 augments CD5-E1B transcription. Based on
this observation, we propose that modulation of B cell autoreac-
tivity in SLE could be achieved by targeting IL-6.
Materials and Methods
B lymphocyte isolation
PBMCs were isolated from the blood of 25 patients with SLE and 25 HCs
by centrifugation on Ficoll-Hypaque. All patients fulfilled the 1982 Amer-
ican College of Rheumatology criteria for SLE (20). SLE activity was
assessed by the SLE disease activity index (SLEDAI), and those with
SLEDAI of ?5 were considered active. The characteristics of SLE patients
and HCs are shown in Table I. The cells were stained with FITC-anti-CD19
and PE-anti-CD5 Abs, and CD5?CD19?B cells sorted on an Epics Elite
FACS (Beckman-Coulter). All sorted cells were ?98% CD19?. Informed
consent was obtained from the patients before taking blood, and the study
protocol approved by the Institutional Review Board at Brest University.
The Daudi human B cell line was purchased from American Type Culture
FITC-anti-CD19 (clone J4-119) and PE-anti-CD5 (clone BL1a) were ob-
tained from Beckman-Coulter, whereas anti-DNMT1 and anti-p27kip1were
obtained from Abcam. Intracellular staining was performed after perme-
abilization of the cells with 70% methanol. Binding of primary unconju-
gated Abs was revealed with FITC-conjugated anti-mouse Abs (Jackson
In pilot experiments, the number of CD5 molecules per cell was quan-
tified by determining the amount of Ab binding to the cells (ABC) at
saturating concentrations, using the Quantum Simply Cellular kit (Flow
Cytometry Standards). Arbitrary ABC value was then determined from a
standard ABC curve generated from the mean fluorescence intensity ob-
tained from the FACS analysis of 50 ?l calibrated microspheres stained
with 20 ?l of the same anti-CD5 Ab.
FACS-sorted B cells were suspended in RPMI 1640 supplemented with
10% heat-inactivated FCS, 2 mM L-glutamine, 200 U/ml penicillin and 100
?g/ml streptomycin. B lymphocytes were seeded at 2 ? 105cells per well,
and incubated with 1 ?g/ml anti-IgM Ab-coated Sepharose beads (BioRad)
and 10 U/ml IL-2, in the presence or absence of 10–40 ng/ml anti-IL-
6RAb (R&D Systems), or 100 ng/ml rhIL-6 (Immuno Tools). Repression
of DNMTs was achieved by incubating the cells with 50 ?M of the ras
signal blocker PD98059. IL-6 and IFN-? were detected in sera and IL-6 in
the supernatant of cultured cells using ELISA kits according to the man-
ufacturer’s instructions (Beckman Coulter).
mRNA extraction and quantitative RT-PCR
Total mRNA was extracted using the RNAble method (Eurobio), and
cDNA synthesized by reverse transcription in 20 ?l volume with Super-
script II RNase H-RT (Invitrogen Corporation). Quantitative RT-PCR was
conducted in 20 ?l mixtures containing 50 ng template cDNA, 1X Sybr
Green PCR Master mix (Applied Biosystems), and 500 nM of each primer
(Table II). As described in detail elsewhere (17), CD5 promoter usage was
evaluated using two sets of primers. All assays included a negative control
which consisted of the reaction mixture with no template, and a positive
control which consisted of the mixture with 18S rRNA. Comparison of
cycle thresholds was completed with the 2???ctmethod using 18S as an
The 5? transcript ends were amplified from mRNA using SMART-RACE
kit (Clontech). As described previously (17), the first strand of cDNA was
synthesized using the sense UPM primer and the gene-specific antisense
primer CD5 E5 (Table II). The PCR protocol included an initial denatur-
ation step at 94°C for 5 min, starting 5 touchdown-PCR cycles of dena-
turation at 94°C for 30 s and annealing at 72°C for 3 min. These cycles
were followed by another 5 cycles of 94°C for 30 s, 70°C for 30 s, and
72°C for 3 min, then with a decreasing temperature for 35 cycles of 94°C
for 30 s, 68°C for 30 s, and 72°C for 3 min. A nested PCR was performed
using the sense NUP primer and the gene-specific antisense primer CD5
E3. The second PCR round was for 40 cycles of 30 s at 94°C, 1 min at
56°C, and 1 min at 72°C with a final extension at 72°C for 10 min.
This assay is based on the inability of some restriction enzymes to digest
a methylated 5?-CmG-3? site. Genomic DNA was purified using QIAmp 96
DNA blood kit (Qiagen) and digested with 20 U of the methylation-sen-
sitive restriction enzymes HpaII, HaeII, FauI, HgaI, or the methylation-
insensitive restriction enzyme MspI for 3 h at 37°C. Undigested genomic
DNA was used as positive control. The PCR primers were positioned up-
stream and downstream of the recognition site in the promoters of E1A and
E1B of the cd5, cd19, cd70, Pax5, Syk, and HRES-1 genes (11, 19, 21, 22).
The PCR protocol included an initial denaturation at 94°C for 5 min, fol-
lowed by 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for
1 min, and primer extension at 72°C for 1 min; PCR cycles were followed
by final extension at 72°C for 10 min. The PCR products were separated on
agarose gel and visualized with 0.5 ?g/ml ethidium bromide.
Table I. Demographic and clinical characteristics of SLE patients and
HCs included in the study
n ? 25n ? 25
Medication usage (%)
47.6 ? 15.1 ?22–74?a
3.7 ? 5.3 ?0–19?
43.3 ? 10.7 ?28–68?
11.1 ? 8.1 ?0–19?
aMean ? SD ?min ? max?.
5624IL-6 CONTROLS DNA METHYLATION AND CD5 IN LUPUS B CELLS
To determine the methylation status of DNA, non-methylated cytosines
were converted to uridines by bisulfite treatment using the EZ-DNA meth-
ylation-Gold kit according to the manufacturer’s instructions (Zymo Re-
search). Unmodified DNA (100 ng) was amplified 40 times at 56°C using
specific primers. The bisulfite-converted DNA was amplified by nested
PCR using two rounds of 40 cycles each at 56°C with primers specific for
methylated cytosines (Table II). PCR products were purified using the high
pure PCR product purification kit (Roche), and directly sequenced with
internal primers by means of the BigDye Terminator Cycle Sequencing kit
using an automated ABI-310 genetic analyzer (Applied Biosystems). The
electrophoregram T and C peaks were quantified and methylation status
determined as [peak (C)/peak (T) ? peak (C)] ? 100. At the same time, the
unmodified DNA was amplified and sequenced using specific primers.
Chromatin immunoprecipitation (ChIP) was conducted using the EpiQuik
kit (Epigentek Group) according to the manufacturer’s instructions to eval-
uate activation of the CD5-E1B promoter. In brief, sonicated DNA (200–
1000 bp) was transferred into strip wells precoated either with mouse anti-
RNA polymerase II, or with a nonspecific mouse IgG, used as a negative
control. After a 90-min incubation at room temperature and extensive
washes, precipitated DNA-protein complexes were treated with 250 ?g/ml
proteinase K in the DNA release buffer for 15 min, and left in the same
buffer for 90 min at 65°C. The DNA samples were collected by the P-spin
columns, washed with ethanol, and eluted. Using purified DNA as a tem-
plate, PCR was performed using GAPDH and CD5-E1B-specific primers
(Table II) and 40 cycles at 56°C. PCR products were separated on agarose
gel, and visualized with 0.5 ?g/ml ethidium bromide.
Computational promoter analysis
Putative transcription factor binding sites were identified by using Alibaba,
version 2.1, the transcription element search system (http://www.cbil.
open.edu/tess/index.html) and Genomatix (http://www.genomatix.de).
The results were expressed as arithmetic means with SD. Comparisons
were made using the Mann-Whitney U test for unpaired data and the Wil-
coxon test for paired data. Correlations were established using Spearman’s
CD5 expression in B cells
The percentage of CD5-expressing B cells was similar in the 25
SLE patients and 25 HCs (Fig. 1A). However, membrane density
of CD5 on B1 cells was lower in the patients (Fig. 1B) compared
with the controls (49,971 ? 15,124 vs 80,703 ? 22,462; p ?
0.001). A representative example is depicted in Fig. 1C.
Table II. Oligonucleotide primers used in the studya
CD5 promoter-1B sense
CD5 promoter-1A sense
CD5 promoter-1B sense
CD5 promoter-1B sense
CD5 promoter-1A sense
CD5 promoter-1A sense
CD19 promoter sense
CD19 promoter antisense
CD70 promoter sense
CD70 promoter antisense
Pax5 promoter sense
Pax5 promoter antisense
Syk promoter sense
Syk promoter antisense
HRES-1 promoter sense
HRES-1 promoter antisense
aE1A, exon 1A; E1B, exon 1B; DNMT, DNA methyltransferase; MBD, methyl-CpG binding domain protein.
5625The Journal of Immunology
Expression of CD5-E1B, which is retained in the cytoplasm,
reduces membrane expression of CD5-E1A (17, 19). This suggests
that CD5-expressing B cells may, under certain physiological con-
ditions (such as activation), preferentially up-regulate CD5-E1B
relative to CD5-E1A. To test this proposition, levels of CD5-E1A
and CD5-E1B mRNA were determined using a 5? specific real
time PCR following 24h anti-IgM stimulation of FACS-sorted
CD5-negative B cells from the blood of 10 SLE patients and 15
HCs (Fig. 1D). BCR engagement of the cells raised the level of
CD5-E1B mRNA in B cells from the patients (54.4 ? 68.1-fold)
but not in those from the controls (0.5 ? 0.2-fold). In contrast,
after BCR engagement expression of CD5-E1A is increased both
in SLE patients and in HCs (7.7 ? 4.9-fold and 5.5 ? 5.6-fold,
NS). The 5?-RACE analysis of CD5 cDNA using B cells from HCs
in resting B cells. A, Schematic representation of the affected promoters. The CD5-E1B promoter arises from a LTR element subdivided into U3, R, and
U5 regions. The two splice donors (SD) are indicated, and positions of DNA-specific primers shown. The 1177-bp CD5-E1B amplicon contains six
HpaII/MspI motifs (5?-CCGG-3?) at positions ?8736, ?8726, ?8522, ?8268, ?8114, and ?7795 bp, and one HaeII motif (5?(A/G)GCGC(T/C)3?) at
position ?8393bp according to numbering from the established ATG initiation site in exon 1A (22). The 783-bp CD5-E1A amplicon contains one HgaI
motif (5?-GACGC(N)5-3?) at position ?151, one FauI motif (5?-CCCGC(N)4-3?) at position ?96, and two HpaII/MspI motifs at positions ?65 and ?87.
B, Analysis of CD5-E1B promoter methylation by amplification of genomic DNA digested with methylation-sensitive HaeII, HpaII, or methylation-
insensitive MspI enzymes. C, Analysis of the CD5-E1A promoter region using HgaI, FauI, HpaII, and MspI enzymes. Undigested genomic DNA is
amplified by PCR, separated on agarose gel, and visualized with ethidium bromide.
Amplification of methylation-sensitive, endonuclease-digested genomic DNA reveals methylation status of the alternative promoters of CD5
gram depicting percentage of CD5-expressing B cells in
25 SLE patients and 25 HCs. B, The number of CD5
molecules per cell is given in the scattergram. The num-
bers are expressed as the amount of anti-CD5 Ab bound
to the cell membrane. C, Representative FACS profile
for cell surface expression of CD5 in blood B cells from
one SLE patient (bold line) and one HC (thin line). D,
Quantitative RT-PCR results presenting as histograms
revealing that a 24-h stimulation of B cells with anti-
IgM increased CD5-E1B transcription in B cells from
SLE patients but not HCs. Incubation of anti-
IgM-activated B cells from HCs with PD98059 resulted
in up-regulation of CD5-E1B. Mean and SD of data
from 10 SLE patients and 15 HCs are shown. E, CD5
5?-RACE analysis of cDNA revealing that CD5-E1B
(639 bp) and CD5-E1A (259 bp) are induced when B
cells from the HCs were stimulated with anti-IgM in the
presence of PD98059, whereas only CD5-E1A (259 bp)
is induced in the presence of anti-IgM. These data in-
dicate that MAPK/Erk has a role in regulating CD5-
CD5 expression in B cells. A, A scatter-
5626IL-6 CONTROLS DNA METHYLATION AND CD5 IN LUPUS B CELLS
confirmed that BCR engagement induce CD5-E1A but did not in-
duce CD5-E1B transcripts (Fig. 1E).
To test the hypothesis that epigenetic changes in the cd5 gene
contribute to the generation of alternative transcripts in activated B
cells from patients with SLE, BCR engagement was re-evaluated
in the presence of PD98059 which decreases DNA methylation. In
these conditions, an up-regulation of CD5-E1B upon anti-IgM/
PD98059 stimulation was observed in B cells from HCs using
real-time PCR (61.5 ? 11.9-fold) and 5?-RACE analysis (Fig. 1, D
The CD5-E1B promoter is hypomethylated in resting SLE
To determine whether epigenetic changes result in increased CD5-
E1B in SLE B cells, genomic DNA from six randomly selected
patients and seven HCs were analyzed for the level of DNA meth-
ylation restriction enzyme treatment of the DNA followed by PCR
(Fig. 2A). The protocol is based on the inability of methylation-
sensitive restriction enzymes to cut methylated DNA. The results
of treatment with HaeII enzyme revealed that the CD5-E1B pro-
moter was de-methylated in B cells from all 6 SLE patients but in
none of the 7 HCs (Fig. 2B). Treatment with HpaII confirmed
demethylation in two of six SLE patients but in none of the HCs.
The positive control was the DNA methylation of Daudi cell line
cells (23). The negative control was the HpaII isoschizomer, MpsI,
that cut the CpG sequences so that there were no PCR products.
In addition to this data, the methylation status of the CD5-E1A
promoter was sought using HgaII, FauI and HpaII (Fig. 2C). The
results showed that the CpG motifs were de-methylated, or hemi-
methylated in B cells from both the SLE patients and HCs.
The U3 region in CD5-E1B is hypomethylated in B cells from
The presence of HaeII site (site 4) in the U3 region of the 5?-LTR
element of HERV-CD5, and three HpaII sites upstream U3-LTR
(site 1) or in the R region of the 5?-LTR (sites 7 and 13) raises the
possibility that the HERV U3-R-U5 regulatory elements may be
de-methylated in B cells from patients with SLE (Fig. 3A). To
address this issue, bisulfite-treated genomic DNA were amplified
Bisulfite C3T transition in SLE CD5-E1B amplicons were an-
alyzed and quantified as shown in Fig. 3B and supplemental Fig.
1.5Among 26 CpG sites studied in the 1439-bp amplicon of CD5-
EIB, five CpG sites are hemi- or de-methylated. These CpGs (sites
2 to 6) are located in the U3-LTR at positions ?8466 from the
5The online version of this article contains supplemental material.
TATA box location within the U3 region. Circles and boxes identify the CpG and U3-R-U5 regions. The HpaII and HaeII sites are also indicated. B, The
level of CpG methylation was determined by bisulfite sequencing using genomic DNA obtained from six SLE patients (white) and seven HCs (black). CpG
sites are numbered as in A. C, Correlation between CD5 cell surface expression and methylation status for CpG sites 3 to 6. White circle, SLE patients;
black circle, HC subjects. ?, p ? 0.05; p ? 0.001.
The U3-LTR HERV-CD5 region is de-methylated in B cells from SLE patients. A, Regulatory motifs for transcription factor binding and
5627The Journal of Immunology
known ATG1 initiation site (24), ?8396, ?8393 (HaeII), ?8347,
and ?8363, respectively. De-methylated CpG motifs contain, or
are located near, the binding sites of E2/Rb family proteins (con-
sensus TTT(C/G)(C/G)CGC, position ?8394 to ?8391), E-box
family proteins (CA(N)3TG, ?8391 to ?8388), and STAT family
proteins (inverted consensus TT(N)4AA, ?8353 to ?8344).
When comparing SLE patients to HCs, the cytosine residues
near the E2/Rb binding sites 3 and 4, plus the CpG at site 5, were
significantly less affected by the bisulfite treatment in the SLE
patients (Fig. 3B, p ? 0.05). Furthermore, the level of methylation
of CpG sites 3 to 5, are inversely correlated with CD5 cell surface
expression in CD5?B cells (Fig. 3C, p ? 0.01). However, de-
methylation of the CD5-E1B promoter identified in B cells from
the SLE patients could be due to gene polymorphisms. Such a
proposition was discounted because only one association between
a methylated CpG motif at position ?8001 and a SNP site was
observed (supplemental Fig. 2).
In contrast to CD5-E1B promoter, the 791bp amplicon for CD5-
E1A which contains 12 CpG was equally de-methylated in B cells
from the SLE patients and HCs (Fig. 2C and supplemental Fig. 1).
De-methylated CpGs were located at positions: ?148, ?123, ?102,
?73 and ?64. These regions contain, or are located nearby to con-
sensus binding sites for AP-1 (?151), Sp-1 (?120, ?96), E-box fam-
ily proteins (?50) and the INR transcriptional start site (?61).
Activation modifies methylation of the CD5-E1B promoter
To assess the influence of BCR engagement on methylation of the
CD5 locus, FACS-sorted CD5-negative B cells from six SLE pa-
tients and six HCs were stimulated with anti-IgM for 24 h. Meth-
ylation patterns were then compared in these activated cells with
the pattern of methylation in the cells before activation (Fig. 4). In
the SLE patients, engagement of the BCR did not modify CD5
promoter methylation status, whereas in the HCs, it resulted in
methylation of the CD5-E1B promoter at positions 3 and 4 (Fig.
4D), but not that of CD5-E1A (not shown). Interestingly, when B
cells from the HCs were stimulated with anti-IgM in the presence
of PD98059, CpG sites 3, 4, and 5 were prone to demethylation
similar to what was seen in the SLE B cells. All these results are
consistent with previous findings that the U3-LTR regions in
HERV elements are prone to demethylation (25).
Analysis of DNA methylation enzymes
To gain insights into the cause(s) of de-methylation of the CD5-
E1B promoter in B cells from SLE patients, levels of mRNA for
DNMTs and MBDs were determined in B cells from ten SLE
patients and 15 HCs. Levels of DNMT1, DNMT3a, DNMT3b,
MBD2, and MBD4 were not different in B cells from the patients
and the controls (Table III). With regard to SLE activity, we failed
to find correlations between patients with SLEDAI ? 5 (n ? 3)
and changes in DNMTs or MBDs activity. On the basis of our
observation that B cell activation through the BCR influences U3-
LTR methylation, we measured the level of mRNA for DNMT1
and MBDs by real-time PCR in B cells stimulated for 24 h with
anti-IgM from ten SLE patients and 15 HCs (Fig. 5A). The ex-
pression of DNMT1 was increased by 2.3 ? 0.2-fold in B cells
from the patients and by 16.6 ? 13.4-fold in the controls (p ?
0.005). Importantly, the expression of MBDs was not affected by
methylation. A, Methylation of CD5-E1B promoter an-
alyzed by restriction enzymes and bisulfite sequencing.
Enzymes and symbols used are the same as in the leg-
end to Fig. 3. B, Effect of restriction enzymes on CD5-
E1A. C, The level of CpG methylation measured by
bisulfite sequencing with (white) or without (black)
BCR engagement in six SLE patients. D, CpG methyl-
ation in B cells from six HCs stimulated with anti-IgM
in the presence (gray) or absence (white) of PD98059.
?, p ? 0.05.
Effect of BCR engagement on cd5 gene
Table III. Level of DNMTs and MBDs mRNA in resting B cells from
10 SLE patients and 15 HCsa
14.8 ? 8.4
1.03 ? 0.52
44.2 ? 22.9
34.2 ? 21.5
15.4 ? 10.4
0.61 ? 0.76
37.1 ? 19.4
23.5 ? 12.5
acDNA were subjected to quantitative RT-PCR (primers indicated in Table I).
Their relative expression was adjusted to 18S levels (?10?6).
5628 IL-6 CONTROLS DNA METHYLATION AND CD5 IN LUPUS B CELLS
stimulation of the BCR or by the addition of the DNMTs inhibitor
FACS analyses verified that BCR engagement modulated DNMT1
expression. Results from four of ten independent experiments are de-
picted in Fig. 5B. Interestingly, DNMT1 staining showed two peaks,
cells could be divided into 76.1 ? 7.9% DNMT1dimand 23.8 ? 7.9%
DNMT1brightcells in the patients and into 74.7 ? 12.3% DNMT1dim
and 25.3 ? 12.3% DNMT1brightcells in the controls (p ? NS). A
24-h culture of B cells with anti-IgM increased the proportion of the
DNMT1brightpopulation to 86.1 ? 12.2% in the HCs compared with
46.1 ? 7.8% in the SLE patients (p ? 0.05). Thus, these results
indicate that induction of DNMT1 following BCR engagement is re-
duced in patients with SLE.
The role of IL-6 in de-methylation and CD5-E1B expression
Based on our previous observations that IL-6 over-expression con-
trols the cell cycle in BCR-activated in B cells from SLE patients,
we predicted that IL-6 by arresting the cell cycle at late G1phase
may control the expression of DNMT1 and its capacity to meth-
ylate DNA and subsequently CD5 cell surface expression (26–28).
To test whether IL-6 acts on its own, or requires engagement of
the BCR, FACS-sorted B cells from six HCs were stimulated for
48 h with rhIL-6 in the presence or absence of anti-IgM. Expres-
sion of CD5-E1B increased by 4.1 ? 3.13-fold in B cells cultured
with rhIL-6, and by 54.8 ? 11.3-fold in B cells cultured with
rhIL-6 and anti-IgM (Fig. 6A, p ? 0.05). The induction of CD5-
E1B upon IgM/rhIL-6 stimulation was also demonstrated by ChIP
expression. A, Quantitative PCR measurement of
DNMT1, MBD2, and MBD4 mRNA levels in ten SLE
patients and 15 HCs following BCR engagement. Role
of activation of MAPK/Erk pathway in DNMT1 induc-
tion was examined in HCs using the PD98059 inhibitor.
B, Cytoplasmic staining of DNMT1 in methanol-per-
meabilized non-activated, or B cells activated with
Involvement of DNMTs on CD5-E1B
sion and promoter methylation. A, FACS-sorted B cells
from HCs were incubated with IL-6 in the presence, or
absence of anti-IgM, or anti-IL-6R Abs. Quantitative
PCR measurement of CD5-E1B (white boxes) and
DNMT1 (black boxes) in six HCs. B, CD5-E1B pro-
moter ChIP analysis using a nonspecific mouse IgG as
negative control (C?), or a mouse anti-RNA polymer-
ase as positive control (C?). C, Cytoplasmic staining of
DNMT1 and p27kip1as a marker of cell cycle arrest in
the G1phase in methanol-permeabilized B cells from
the HCs incubated with IL-6 in the presence or absence
The effect of IL-6 on CD5-E1B expres-
5629 The Journal of Immunology
analysis. This experiment showed that RNA polymerase II was
recruited to the CD5-E1B promoter upon anti-IgM/rhIL-6 stimu-
lation (Fig. 6B). Thus, rhIL-6 induces CD5-E1B expression and
this effect is more pronounced when B cells are activated through
The induction of DNMT1 mRNA by anti-IgM was negated in
the presence of rhIL-6 (Fig. 6A). The experiments were re-
peated in the presence of anti-IL-6R Ab to inhibit the activity of
IL-6. DNMT1 induction was restored (10.0 ? 3.6 vs 0.63 ?
0.25-fold without anti-IL-6R Ab, p ? 0.05) and CD5-E1B was
reduced (54.8 ? 11.3 vs 35.6 ? 5.2-fold without anti-IL-6R Ab,
p ? 0.05). Moreover, FACS analyses showed that the number
of DNMT1brightcells was reduced after anti-IgM stimulation in
the presence of rhIL-6 (86.1 ? 12.2% vs 33.5 ? 3.7%, p ?
0.05). A representative example of two experiments is shown in
Fig. 6C. We also studied whether these differences could be
attributed to a cell cycle blockade. As suspected, the cyclin-
dependent kinases inhibitor p27kip1was over-expressed in anti-
IgM/rhIL-6-stimulated B cells compared with rhIL-6 or anti-
IgM stimulation alone (Fig. 6C). Overall, IL-6 appears to
control CpG methylation in SLE B cells resulting, probably,
from its effect on arresting cells at the late G1phase of the cell
Effects of IgM/IL-6 stimulation on promoters of other genes
To determine the extent of IL-6 effect on methylation, we deter-
mined the methylation status of other promoters (CD19, CD70,
Pax 5, Syk, and HRES-1) that are known to be regulated by meth-
ylation (11, 21, 22). In the six HCs studied (Fig. 7), the promoters
were hypomethylated in resting cells. Stimulation of B cells with
anti-IgM only increased the methylation of the HRES-1. This ef-
fect by anti-IgM was reversed in the presence of IL-6.
Effect of IL-6 on SLE B cells
To validate the role of IL-6 in the altered methylation status of
CD5-E1B in B cells in SLE patients, membrane CD5 expression
levels were compared between patients whose sera were IL-6 pos-
itive and negative. The presence of IL-6 in serum was associated
with decreased membrane CD5 expression (Fig. 8A, p ? 0.01). In
CD70, Pax5, Syk, and HRES-1. A, B cells from HCs were incubated with
anti-IgM in the presence or absence of anti-IL-6R Ab. The methylation
status of all five promoters was determined by PCR using predigested
genomic DNA with methylation-sensitive HpaII, or methylation-insensi-
tive MspI. B, The methylation of the HRES-1 promoter was quantified by
calculating the ratio of HpaII-digested to undigested bands in six HCs.
?, p ? 0.05.
Anti-IgM-induced methylation of promoters for CD19,
ation in SLE B cells. CD5 cell surface expression in
relation to the detection level of IL-6 (A) or IFN-? (B)
in the serum of 18 SLE patients. The detection limit
used corresponds to the sensitivity limit of the ELISA.
C, FACS-sorted B cells from six SLE patients were cul-
tured with anti-IgM in the presence or absence of 40
ng/ml anti-IL-6R Ab. All SLE patients, except one,
were inactive (SLEDAI ? 5). Dose effect response at 10
and 20 ng/ml anti-IL-6R were performed on three SLE
patients. Quantitative PCR measurement of CD5-E1B
(white boxes) and DNMT1 (black boxes). D, Effect of
anti-IgM/anti-IL-6R (gray) on CD5-E1B promoter
IL-6-dependent modulation of methyl-
5630IL-6 CONTROLS DNA METHYLATION AND CD5 IN LUPUS B CELLS
contrast, changes in membrane CD5 expression were not associ-
ated with serum IFN-? (Fig. 8B). CD5 cell surface expression was
reduced in patients who had SLEDAI ? 5 (39,789 ? 11,450 vs
51,294 ? 15,215), but this was not significant (data not shown).
Finally, the effect of a blocking anti-IL-6R Ab was studied be-
cause we previously observed that anti-IgM stimulation of B cells
from SLE patients produced higher levels of IL-6 than matched
HCs (514.1 ? 159.7 pg/ml vs 99.0 ? 116.0 pg/ml, data not shown)
(26). Blocking the autocrine loop of IL-6 increased DNMT1 ex-
pression (58.9 ? 13.5-fold with 40 ng/ml anti-IL-6R) and contrib-
uted to the methylation of the U3-LTR sites 3 to 5 (Fig. 8, C and
D). In addition, expression of CD5-E1B was reduced (17.2 ? 3.5-
fold with 40 ng/ml anti-IL-6R vs 54.4 ? 68.1-fold without, p ?
0.05). Further, this effect was dose-dependent.
This study provides evidence for the notion that reduction in the
level of membrane CD5 on circulating CD5?B cells SLE is due
to increased expression of the cytoplasmic isoform of CD5, CD5-
E1B. As a consequence of reduced membrane CD5, increased
CD5-E1B limits the negative regulatory effects of CD5 on BCR-
mediated signaling. The available evidence indicates that mem-
brane CD5 increases the threshold of BCR-mediated responses
(14). The implication of such data is that regulation of CD5-E1B,
which in turn regulates expression of CD5, is involved in main-
taining the anergy status of autoreactive B cells. Results generated
from our previous studies indicated that the modulatory effects of
CD5 protein on BCR signaling are attributed to the CD5 isoform
which contains E1A, the previously documented exon 1. Introduc-
tion of cDNA for the recently identified CD5-E1B isoform into
CD5?T and B cells reduced membrane expression of CD5 protein
(17, 19). Coexpression experiments have also shown that in the
presence of CD5-E1B, CD5-E1A was not translocated to the mem-
brane but associated with CD5-E1B in cytoplasmic aggregates
(19). The exact dynamics of the interaction between CD5-E1B and
CD5-E1A remain unknown. However, we have established that
the stretch of amino acids from positions 286–400 in CD5-E1B is
crucial for reducing membrane CD5-E1A translocation (19). Func-
tionally, these data suggest that up-regulated CD5-E1B expression
in B cells could reduce the BCR activation threshold by Ags and
thus, promote autoimmunity.
Because CD5-E1B arises from the integration of an HERV el-
ement known to be silenced by DNA methylation (19), we pre-
dicted that epigenetic modifications could be involved in regulat-
ing CD5 isoform expression in B cells. Normally, methylation of
DNA is evident in CpG-poor regions and in regions of repetitive
sequences including LTR in HERV sequences. In contrast, CpG
islands present within gene promoters resist to methylation. In nor-
mal B cells, this paradigm holds true for the cd5 locus, where the
U3-LTR element is methylated while the CD5-E1A promoter is
not. However, in SLE B cells, the rate of de-methylation is higher
in the U3 region of the HERV-CD5 element. Consistent with this
observation is the finding that another HERV element, HRES-1,
was also integrated at the stage of old world primate divergence
(22). Indeed, expression of HRES-1 retroviral proteins, HRES-1/
p28 and HRES-1/Rab4 (29), and our observations on HRES-1 pro-
moter methylation status (Fig. 7) may hint to a global defect in
controlling repetitive elements in SLE. Our preliminary results are
indeed indicative that HRES-1 methylation pattern was not in-
creased in SLE patients upon BCR engagement. In this respect,
global DNA methylation has been reported to be altered in almost
all forms of SLE including drug-induced lupus (30). As a conse-
quence, it was postulated that epigenetic transformation modifies B
cell physiology resulting in polyclonal activation, IgG1 class-
switching (31), V(D)J rearrangement by RAG1/RAG2 enzymes
(32) and a shift in cytokine profiles (33). Altered methylation also
influences T cells in SLE causing up-regulation of T cell costimu-
lating molecules (CD70, CD40 ligand), and a shift in cytokine
profile from Th1 to Th2 (for review, see Ref. 34). We propose that
these alterations, together with our findings on CD5 down-regula-
tion, contribute to promoting autoreactivity.
De-methylation of DNA can be result from the inhibition or lack
of DNMTs or be due to specific enzymatic reactions including
MBDs or a combination of both. Thus, DNA methylation patterns
can be altered owing to a shift in the balance between MBD de-
methylation and DNMT methylation enzymes. Quantitative PCR
assays were performed to evaluate the relative levels of MBD and
DNMT transcripts in resting and in anti-IgM stimulated B cells. At
basal level, B cells from SLE patients and HCs had similar levels
of DNMT1, DNMT3a, MBD2, and MBD4. The amount of
DNMT3b was very low or undetectable in both SLE patients and
HCs as previously described for T cells (35). A recent study re-
ported reductions in DNMT1 in a subgroup of SLE patients (36).
However, this study was conducted using a relatively small cohort
and was not confirmed either in CD4?T cells (35) or in B cells by
the present study. In addition, the recently reported increase in
MBD2 and MBD4 in patients with SLE (37) was also not con-
firmed in our study. These differences may be because we have
examined B cells instead of CD4?T cells, or that the patient
population included in the other study was different. Interestingly,
analysis of stimulated B cells revealed that expression of DNMT1
was reduced following engagement of the BCR in SLE in agree-
ment with findings in stimulated CD4?T cells (13).
Our results, suggesting that DNA hypomethylation in activated
SLE B cells, could be attributed to cell cycle arrest in G1is sur-
prising. The surprising aspect of the observation is based on the
fact that proliferation of lymphocytes did not differ between the
patients and HCs implying that decreased DNMT1 activity in SLE
was not due to a cell cycle arrest (13). However, inhibition of the
MAPK/Erk2 pathway was clearly associated with DNMT1 reduc-
tion in those patients (13, 38). Further, inhibition of this pathway
is likely to elicit cell cycle arrest through p27kip1induction (39,
40). However, inhibition of MEK/Erk2 pathway using PD98059 in
pro-B induces cell growth arrest with p27kip1accumulation (39).
Therefore, in such a setting, it was not surprising that PD98059
decreased methylation in HCs as in stimulated SLE B cells. Hence,
these data indicate that decreased activation of the Erk pathway
could be important for the development of autoimmunity (7). This
was previously confirmed in animal models showing that treating
normal mice with inhibitors of DNA methylation, or with Erk in-
hibitors causes a lupus-like disease (41). Further, Tg mice express-
ing dominant-negative MEK leads to overexpression of methyla-
autoantibodies by B cells (38).
The proposed involvement of IL-6 in promoting autoreactivity
is supported by in vitro and in vivo studies. For example, high
levels of serum IL-6 is correlated with lupus disease activity and
targeting IL-6 with blocking Abs is an effective treatment for SLE
(4). However, the precise mechanism by which IL-6-mediated B
cell proliferation, differentiation, and autoantibody production in
SLE remains to be elucidated. Repression of IL-6 is associated
with hypermethylation of its promoter in HCs whereas its overex-
pression in SLE is associated with promoter hypomethylation (42).
Interestingly, by treating B cells from HCs with IL-6 in the pres-
ence of anti-IgM, we have shown that CD5-E1B expression can be
significantly increased and that this correlates with a cell cycle
arrest in late G1phase. In addition, this effect is associated with an
Ig gene rearrangement following RAG re-expression (26). The loss
5631 The Journal of Immunology
of, or reduction in, the inhibitory effects of CD5 is associated with Download full-text
RAG re-expression in B cells in patients with SLE and these may
synergize to induce autoantibody production.
In conclusion, the finding that IL-6 activates CD5-E1B tran-
scription is relevant for understanding mechanism of action and
therapeutic benefits of anti-IL-6. Thus, treatment of patients with
SLE with anti-IL-6R mAb could inhibit autoreactive B cell expan-
sion by restoring DNA methylation and cell cycle progression. In
addition, the data provide further evidence for the pivotal role of
CD5 in maintaining anergy in autoreactive B cells.
We are grateful to the Conseil Re ´gional de Bretagne, the Conseil Ge ´ne ´ral
du Finiste `re, the College Doctoral International, and the Universite ´ Europ-
e ´enne de Bretagne for support. Thanks are also due to Cindy Se ´ne ´ and
Simone Forest for excellent secretarial assistance.
The authors have no financial conflict of interest.
1. Rahman, A., and D. A. Isenberg. 2008. Systemic lupus erythematosus. N. Engl.
J. Med. 358: 929–939.
2. Renaudineau, Y., P. O. Pers, B. Bendaoud, C. Jamin, and P. Youinou. 2004.
Dysfunctional B cells in SLE. Autoimmun. Rev. 3: 516–523.
3. Leandro, M. J., J. C. Edwards, G. Cambridge, M. R. Ehrenstein, and
D. A. Isenberg. 2002. An open study of B lymphocyte depletion in systemic lupus
erythematosus. Arthritis Rheum. 46: 2673–2677.
4. Illei, G., C. Yarboro, Y. Shirota, E. Tackey, L. Lapteva, and T. Fleisher. 2006.
Tocilizumab (humanized anti IL-6 receptor monoclonal antibody) in patients with
systemic lupus erythematosus (SLE): safety, tolerability and preliminary efficacy.
Arthritis Rheum. 54 (Suppl): 4043 (Abstract).
5. Finck, B. K., P. S. Linsley, and D. Wofsy. 1994. Treatment of murine lupus with
CTLA4Ig. Science 265: 1225–1227.
6. Hom, G., R. R. Graham, B. Modrek, K. E. Taylor, W. Ortmann, S. Garnier,
A. T. Lee, S. A. Chung, R. C. Ferreira, P. V. Pant, et al. 2008. Association of
systemic lupus erythematosus with C8orf13-BLK and ITGAM-ITGAX. N. Engl.
J. Med. 358: 900–909.
7. Ballestar, E., M. Esteller, and B. C. Richardson. 2006. The epigenetic face of
systemic lupus erythematosus. J. Immunol. 176: 7143–7147.
8. Zhu, B., Y. Zheng, H. Angliker, S. Schwarz, S. Thiry, M. Siegmann, and
J. P. Jost. 2000. 5-Methylcytosine DNA glycosylase activity is also present in the
human MBD4 (G/T mismatch glycosylase) and in a related avian sequence. Nu-
cleic Acids Res. 28: 4157–4165.
9. Hendrich, B., and A. Bird. 1998. Identification and characterization of a family
of mammalian methyl-CpG binding proteins. Mol. Cell Biol. 18: 6538–6547.
10. Schwab, J., and H. Illges. 2001. Regulation of CD21 expression by DNA meth-
ylation and histone acetylation. Int. Immunol. 13: 705–710.
11. Lu, Q., A. Wu, L. Tesmer, D. Ray, N. Yousif, and B. Richardson. 2007. De-
methylation of CD40LG on the inactive X in T cells from women with lupus.
J. Immunol. 179: 6352–6358.
12. Piotrowski, P. C., S. Duriagin, and P. P. Jagodzinski. 2005. Expression of HERV
clone 4-1 may correlate with blood plasma concentration of anti-U1 RNP and
anti-Sm nuclear antibodies. Clin. Rheumatol. 24: 620–624.
13. Deng, C., M. J. Kaplan, J. Yang, D. Ray, Z. Zhang, W. J. McCune, S. M. Hanash,
and B. C. Richardson. 2001. Decreased ras-mitogen-activated protein kinase sig-
naling may cause DNA hypomethylation in T lymphocytes from lupus patients.
Arthritis Rheum. 44: 397–407.
14. Hippen, K. L., L. E. Tze, and T. W. Behrens. 2000. CD5 maintains tolerance in
anergic B cells. J. Exp. Med. 191: 883–890.
15. Jamin, C., G. Magadur, A. Lamour, L. MacKenzie, P. M. Lydyard, P. Katsikis,
and P. Youinou. 1992. Cell-free CD5 in patients with rheumatic diseases. Im-
munol. Lett. 31: 79–84.
16. Lu, X., R. C. Axtell, J. F. Collawn, A. Gilson, L. B. Justement, and C. Raman.
2002. AP2 adaptor complex-dependent internalization of CD5: differential reg-
ulation in T and B cells. J. Immunol. 168: 5612–5620.
17. Renaudineau, Y., S. Hillion, A. Saraux, R. A. Mageed, and P. Youinou. 2005. An
alternative exon 1 of the CD5 gene regulates CD5 expression in human B lym-
phocyte. Blood 106: 2781–2789.
18. Renaudineau, Y., S. Vallet, C. Le Dantec, S. Hillion, A. Saraux, and P. Youinou.
2005. Characterization of the human CD5 endogenous retrovirus-E in B lym-
phocytes. Gene Immun. 6: 663–671.
19. Garaud, S., C. Le Dantec, C. Berthou, P. M. Lydyard, P. Youinou, and
Y. Renaudineau. 2008. Selection of the alternative exon 1 from the cd5 gene
down-regulates membrane level of the protein in B lymphocytes. J. Immunol.
20. Tan, E. M., A. S. Cohen, J. F. Fries, A. T. Masi, D. J. McShane, N. F. Rothfield,
J. G. Schaller, N. Talal, and R. J. Winchester. 1982. The 1982 revised criteria for
the classification of SLE. Arthritis Rheum. 25: 1271–1277.
21. Ushmorov, A., F. Leitha ¨user, O. Sakk, A. Weinhau ¨sel, S. W. Popov, P. Mo ¨ller,
and T Wirth. 2006. Epigenetic processes play a major role in B-cell-specific gene
silencing in classical Hodgkin lymphoma. Blood 107: 2493–2500.
22. Perl, A., J. D. Rosenblatt, I. S. Chen, J. P. DiVincenzo, R. Bever, B. J. Poiesz, and
G. N. Abraham. 1989. Detection and cloning of new HTLV-related endogenous
sequences in man. Nucleic Acids Res. 17: 6841–6854.
23. Ruchusatsawat, K., J. Wongpiyabovorn, S. Shuangshoti, N. Hirankarn, and
A. Mutirangura. 2006. SHP-1 promoter 2 methylation in normal epithelial tissues
and demethylation in psoriasis. J. Mol. Med. 84: 175–182.
24. Jones, N. H., M. L. Clabby, D. P. Dialynas, H. J. Huang, L. A. Herzenberg, and
J. L. Strominger. 1986. Isolation of complementary DNA clones encoding the
human lymphocyte glycoprotein T1/Leu-1. Nature 323: 346–349.
25. Reiss, D., Y. Zhang, and D. L. Mager. 2007. Widely variable endogenous ret-
roviral methylation levels in human placenta. Nucleic Acids Res. 35: 4743–4754.
26. Hillion, S., S. Garaud, V. Devauchelle, A. Bordron, C. Berthou, P. Youinou, and
C. Jamin. 2007. IL-6 is responsible for aberrant BCR-mediated regulation of
RAG expression in SLE. Immunology 122: 371–380.
27. Robertson, K. D., K. Keyomarsi, F. A. Gonzales, M. Velicescu, and P. A. Jones.
2000. Differential mRNA expression of the human DNA methyltransferases
(DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and
tumor cells. Nucleic Acids Res. 28: 2108–2113.
28. Brown, S. E., M. F. Fraga, I. C. Weaver, M. Berdasco, and M. Szyf. 2007.
Variations in DNA methylation patterns during the cell cycle of HeLa cells.
Epigenetics 2: 54–65.
29. Pullmann, R., Jr., E. Bonilla, P. E. Phillips, F. A. Middleton, and A. Perl. 2008.
Haplotypes of the HRES-1 endogenous retrovirus are associated with develop-
ment and disease manifestations of systemic lupus erythematosus. Arthritis
Rheum. 58: 532–540.
30. Zhou, Y., and Q. Lu. 2008. DNA methylation in T cells from idiopathic lupus and
drug-induced lupus patients. Autoimmun. Rev. 7: 376–383.
31. Vigorito, E., K. L. Perks, C. Abreu-Goodger, S. Bunting, Z. Xiang, S. Kohlhaas,
P. P. Das, E. A. Miska, A. Rodriguez, A. Bradley, et al. 2007. microRNA-155
regulates the generation of Ig class-switched plasma cells. Immunity 27:
32. Wang, H., J. Feng, C. F. Qi, Z. Li, H. C. Morse 3rd, and S. H. Clarke. 2007.
Transitional B cells lose their ability to receptor edit but retain their potential for
positive and negative selection. J. Immunol. 179: 7544–7552.
33. Pang, Y., Y. Norihisa, D. Benjamin, R. R. Kantor, and H. A. Young. 1992. IFN-?
gene expression in human B-cell lines: induction by IL-2, PKC, and possible
effect of hypomethylation on gene regulation. Blood 80: 724–732.
34. Huber, L. C., J. Stanczyk, A. Ju ¨ngel, and S. Gay. 2007. Epigenetics in inflam-
matory rheumatic diseases. Arthritis Rheum. 56: 3523–3531.
35. Balada, E., J. Ordi-Ros, S. Serrano-Acedo, L. Martinez-Lostao, M. Rosa-Leyva,
and M. Vilardell-Tarre ´s. 2008. Transcript levels of DNA methyltransferases
DNMT1, DNMT3A and DNMT3B in CD4? T cells from patients with systemic
lupus erythematosus. Immunology 124: 339–347.
36. Ogasawara, H., M. Okada, H. Kaneko, T. Hishikawa, I. Sekigawa, and
H. Hashimoto. 2003. Possible role of DNA hypomethylation in the induction of
SLE: relationship to the transcription of HERV. Clin. Exp. Rheumatol. 21:
37. Balada,E., J.Ordi-Ros, S.Serrano-Acedo,
M. Vilardell-Tarre ´s. 2007. Transcript overexpression of the MBD2 and MBD4
genes in CD4? T cells from SLE patients. J. Leukocyte Biol. 81: 1609–1616.
38. Sawalha, A. H., M. Jeffries, R. Webb, Q. Lu, G. Gorelik, D. Ray, J. Osban,
N. Knowlton, K. Johnson, and B. Richardson. 2008. Defective T-cell ERK sig-
naling induces IFN-regulated gene expression and overexpression of methyla-
tion-sensitive genes similar to lupus patients. Genes Immun. 9: 368–378.
39. Qiang, Y. W., M. Kitagawa, M. Higashi, G. Ishii, C. Morimoto, and K. Harigaya.
2000. Activation of MAPK through alpha5/?1 integrin is required for cell cycle
progression of B progenitor cell line, Reh, on human marrow stromal cells. Exp.
Hematol. 28: 1147–1157.
40. Gysin, S., S. H. Lee, N. M. Dean, and M. McMahon. 2005. Pharmacologic in-
hibition of RAF–?MEK–?ERK signaling elicits pancreatic cancer cell cycle
arrest through induced expression of p27Kip1. Cancer Res. 65: 4870–4880.
41. Richardson, B., D. Ray, and R. Yung. 2004. Murine models of lupus induced by
hypomethylated T cells. Methods Mol. Med. 102: 285–294.
42. Mi, X. B., and F. Q. Zeng. 2008. Hypomethylation of IL-4 and -6 promoters in
T cells from SLE patients. Acta Pharmacol. Sin. 29: 105–112.
5632IL-6 CONTROLS DNA METHYLATION AND CD5 IN LUPUS B CELLS