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(The FASEB Journal. 1999;13:1415-1422.)
© 1999 FASEB

Abnormal DNA methylation and deoxycytosine-deoxyguanine content in nucleosomes from lymphocytes undergoing apoptosis

SYLVIE HUCK*, ERIC DEVEAUD§, ABDELKADER NAMANE{dagger} and MONCEF ZOUALI*1

* Département d'Immunologie;
§ Unité d'Immunochimie Analytique;
{dagger} Laboratoire de Chimie Struturale des Macromolécules; Institut Pasteur, 75015 Paris, France

1Correspondence: Moncef ZOUALI, Département d'Immunologie, Institut Pasteur, 28, rue du Dr Roux, 75015 Paris, France.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Systemic lupus erythematosus (SLE) is characterized by an accelerated apoptosis of peripheral lymphocytes and an impairment of the clearance of apoptotic cells. Since changes in DNA methylation and in deoxycytosine and deoxyguanine (GC) content have been shown to enhance the potential of DNA to activate murine and human B lymphocytes, we tested the capacity of lymphocytes undergoing apoptosis (under conditions that mimic the deletion of self-reactive cells after antigen receptor engagement) to generate nucleosomes with a particular base composition. Using two cell culture systems and four apoptosis triggers, we found an increase of deoxymethylcytosine in fragmented chromosomal DNA of apoptotic B and T lymphocytes. However, this increase was not associated with modulation of DNA (cytosine-5) methyltransferase, the enzyme that methylates eukaryotic DNA, which suggests that the changes in DNA methylation patterns are not linked to the process of de novo DNA methylation during cell death. In addition, we could not detect a unique methylation pattern in highly repetitive Alu sequences present in the human genome of SLE subjects, as compared with controls. However, the abnormal DNA methylation of apoptotic nucleosomes was associated with an unusual pattern of nuclease-resistant, GC-rich regions in these DNA fragments. We propose that the combination of an accelerated apoptosis with a defect in the clearance of apoptotic cells results in release of increased amounts of nucleosomes with abnormally methylated, GC-rich DNA and provides an autologous stimulation that could bypass tolerance to self in systemic autoimmune diseases. These findings support the concept that the structure and dynamics of nucleosomes are critical in determining their immunogenicity in SLE.—Huck, S., Deveaud, E., Namane, A., Zouali, M. Abnormal DNA methylation and deoxycytosine-deoxyguanine content in nucleosomes from lymphocytes undergoing apoptosis.


Key Words: autoimmunity • SLE • GC% • C2-ceramide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SYSTEMIC LUPUS ERYTHEMATOSUS (SLE)2 is characterized by the production of antibodies to native DNA that cause tissue damage. Although the origin of this autoantibody hypersecretion remains unclear, three lines of evidence indicate that endogenously released DNA may be a potent trigger. First, apoptosis of circulating lymphocytes from SLE patients was found to be accelerated in vitro, and this acceleration was correlated with disease activity (1) . This abnormal cell survival was specific for SLE, and the rate of apoptosis of lymphocytes from rheumatoid arthritis patients was indistinguishable from that of control subjects (1) . More recently, it was recognized that in SLE, the clearance of apoptotic cells is impaired and this abnormality underlies the process of autoantibody production (2) . Third, the circulating DNA trapped in plasma immune complexes (IC) from SLE patients exhibits abnormal characteristics, with a high variability in size ranging from 20 bp to 17 Kb (3) , an increase in deoxyguanine (G) and deoxycytosine (C) percent (3 , 4 ), an abnormal methyldeoxycytosine (mC) content (5 , 6 ), and a relative immunogenicity in rabbits (3) . Although cross-hybridization with bacterial DNA was noted (7) , the prevailing view is that plasma lupus DNA is essentially released from apoptotic cells. Notably, Alu repetitive regions, which account for 3–6% of total genomic DNA (8) , also exhibit an abnormal mC content.

These observations suggest that the combination of an accelerated apoptosis with a defect in the clearance of apoptotic cells results in release of increased amounts of nuclear antigens into the extracellular milieu, thereby providing the source of driving an autoimmune response and combining with autoantibodies to form immune complexes (IC) (2 , 9 10 11 12 13 14 15 ). This view is supported by the demonstration that changes in DNA methylation and GC content enhance the potential of DNA fragments to activate murine and human B lymphocytes and could play a role in the pathogeny of systemic autoimmune diseases by augmenting the immunogenicity of nucleosomes released from apoptotic cells (13 , 14 , 16 17 18 ). However, little is known about the base composition of the nucleosomes released during cell death. To gain insight into the molecular characteristics of apoptotic nucleosomes, we used an in vitro model system to determine whether apoptosis of lymphocytes is associated with an alteration of DNA methylation, GC content, and/or expression of DNA (cytosine-5) methyltransferase (MTase), the enzyme that mediates methylation of deoxycytosines. To determine whether specific elements of the genome of SLE subjects exhibit a unique pattern of methylation-sensitive restriction sites, we also probed the methylation state of Alu repetitive DNA sequences in lupus peripheral lymphocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cells and culture conditions
A murine B lymphoma cell line, WEHI-231 (American Type Culture Collection, Rockville, Md.), was obtained from Dr. P. Sarthou (Institut Pasteur, Paris). It was cultured at 37°C in a 5% CO2 humidified incubator and maintained in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM 2-mercapto-ethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc., Rockville, Md.). Cells were counted and their viability was assessed by trypan blue exclusion using a hemocytometer.

BALB/c mice at 5 to 8 wk of age were killed, thymi were removed, and thymocyte single-cell suspensions were isolated. Human lymphocytes were obtained from peripheral mononuclear cells by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradient centrifugation and elimination of monocytes by adherence on plastic flasks for 1 h at 37°C, as described previously (19) . DNA fragmentation was analyzed by agarose gel electrophoresis.

Induction of apoptosis
WEHI-231 cells were cultured at a density of 0.5–1 x 106 cells/ml and apoptosis was induced by treatment with goat anti-IgM antibody (Pierce, Rockford, Ill.) at 1 µg/ml for 20 h or with N-acetyl-D-sphingosine (C2-ceramide) (Sigma, St. Louis, Mo.) at 10 µM for 20 h. For murine thymocytes, apoptosis was induced by treating 2 x 106 cells/ml with 5 mM of dexamethasone (Calbiochem-Behring Corp. La Jolla, Calif.) for 16 h or by dilution to a density of 5 x 105 cells/ml and culture for 16 h. Genomic DNA was isolated from peripheral mononuclear cells of SLE patients and control subjects as described previously (19) .

Characterization of apoptotic DNA
Briefly, cells were washed twice with phosphate-buffered saline (PBS) and lysed by addition of a hypotonic Nonidet P-40 lysis buffer (1% Nonidet P-40 in 20 mM EDTA, 50 mM Tris-HCl, pH 7.5) (20) . The extract was digested with RNase A for 2 h at 56°C and then with proteinase K at 45°C for at least 6 h. This method allows separation of apoptotic low molecular weight DNA fragments (referred to as LMW DNA) from high molecular weight genomic DNA (referred to as HMW DNA), which can be visualized on a 1% agarose gel containing 0.5% µg/ml ethidium bromide. Before hydrolysis, a further cleaning of DNA was performed by a phenol-chloroform extraction, followed by three successive ethanol precipitations in 2 M ammonium acetate.

High-performance liquid chromatography (HPLC)
Genomic DNAs were extracted with phenol-chloroform, washed, and ethanol-precipitated three times before cleavage. Samples (30–200 µg) were then chemically hydrolyzed with perchloric acid, neutralized with potassium hydroxide, centrifuged, and filtered as described in detail elsewhere (6) . Calibration standards (50 µg of the free nucleosides cytosine, guanine, thymine, adenine, and methyl-cytosine) were supplied by Sigma and were run in parallel. A volume of 5 µl, corresponding to 5–10 µg of hydrolyzed DNA or 2 µg of free nucleoside calibration standard, was injected into an HPLC analysis system equipped with a diode array detector (Hewlett-Packard 1090M). Nucleosides were separated by chromatography on a Nucleosil 5 µM C18 column (4.6 x150 mm, Macherey-Nagel), using a 20 mn linear gradient of acetonitrile (0 to 25%), and the eluted material was monitored at 260 nm wavelength. The retention times and the absorption spectra of each sample, as compared with calibration standards, served as a basis to identify the peaks. In the experimental conditions used, the retention times for cytosine, uracil, methyl-cytosine guanine, thymine, and adenine were 2.9, 3.6, 5.8, 6.2, 6.9, and 8.7 mn, respectively. Data are expressed as percent mC calculated according to the formula: percent mC = {mC/(mC+C)} x100.

Western blot analysis
After cell culture, WEHI-231 cells were collected by centrifugation and washed in PBS. The cell pellet (106 cells) was resuspended in 50 µl of cold lysis buffer containing 1% Triton-X100 and transferred to a 1.5-ml microcentrifuge tube. Cell lysate supernatant was subjected to SDS-PAGE through a 7.5% reducing gel, and the gels were trans-blotted onto polyvinylidene difluoride (PVDF) membranes (Amersham, Little Chalfont, U.K.) using a Bio-Rad Trans-Blot apparatus at 250 mA for 1 h. Blots were processed according to the manufacturer's recommendations and probed with a rabbit anti-MTase antibody (1/2 000) provided by Dr. M. Szyf (McGill University, Montreal) in PBS buffer containing 5% of dry milk and 0.5% Tween-20. After washing in PBS buffer containing 0.5% Tween-20, binding was revealed using a horseradish peroxidase-labeled anti-rabbit conjugate 1/5 000 diluted.

Polymerase chain reaction
The PV-PR fragment, corresponding to Alu repetitive sequences, was obtained by molecular amplification using the 18-mer PV1 antisense primer (5'-GGC CGG GCG CGG TGG CTC-3', designed from nucleotide positions 1–18 in the PV sequence) and the 18-mer PV2 sense primer (5'-CTC CGC CTC CCG GGT TCA-3', designed from nucleotide positions 217–200 in the PV sequence) (21) . Polymerase chain reaction amplification of 50 ng of DNA template was achieved with TaqI DNA polymerase for 30 cycles at 72°C for 30 s, 68°C for 30 s, and 94°C for 1 mn.

Methylation analysis of Alu repeats
Genomic DNAs were digested with the methylation-insensitive enzyme MspI, which recognizes and cuts at both CCGG and CmCGG restriction sites, or with the methylation-sensitive enzyme HpaII, which cuts only unmethylated CCGG restriction sites. Samples (5 µg of digested DNAs) were loaded, size-fractionated on a 2% agarose gel, and blotted onto Hybond N+ filters (Amersham) by vacuum transfer. Subsequently, filters were hybridized to an Alu probe consisting of a 210 bp fragment derived from Alu `PV-PR' (21) and cross-hybridizing with the Precise and PV Alu subfamilies (22) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of apoptosis in WEHI-231 B cells
To determine whether apoptosis in B lymphocytes is associated with mC and GC% changes, we chose as a model system the well-defined WEHI-231 cell line expressing the sIgMhigh, sIgDlow phenotype. This lymphoma cell line undergoes growth and apoptosis after cross-linking of sIgM, which mimics the deletion of self-reactive B cells in the bone marrow after antigen receptor engagement (23 , 24 ). To induce DNA fragmentation characteristic of the known morphological changes of apoptotic cells, we incubated the cells with medium or anti-IgM antibody. Since ceramide, the second messenger for the sphingomyelin pathway, is also able to mediate downstream death signaling through the cell surface receptor for TNF, the CD95 receptor, and after gamma irradiation (25) , we also used the synthetic ceramide analog N-acetyl-D-sphingosine (C2-ceramide) to deliver a potent signal for apoptosis in the WEHI-231 cell line. From cells incubated with anti-IgM antibody or C2-ceramide, we were able to separate fragmented LMW DNA forming the ladder that defines apoptotic cells from HMW DNA (Fig. 1 ). Fifteen to 30% of the DNA extracted from WEHI-231 cells undergoing apoptosis in culture was fragmented.



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  		Figure 1. Low molecular weight (LMW) fragmented DNA and high molecular weight (HMW) DNA in B and T lymphocytes undergoing apoptosis. A) Apoptosis in the murine IgM+ pre-B cell line WEHI-231. Cells were exposed to varying concentrations of anti-IgM (Anti-µ) antibody (0.5, 1, or 5 µg/ml) for 20 h. Trypan blue staining revealed that this treatment resulted in 15, 20, and 45% of dead cells, respectively. LMW DNA, corresponding to apoptotic fragmented DNA (S) and HMW DNA (P), were extracted by the Nonidet P-40 lysis method from the supernatant and the pellet of the cells, respectively. Loading represents the content of 106 cells. Restriction fragments of {lambda} phage DNA digested with HindIII were used as molecular weight markers (Kb). For C2-ceramide treatment (C2-C), cells were exposed to varying concentrations of N-acetyl-D-sphingosine (10, 20, or 40 µM) for 20 h. Loading represents the content of 0.5 x 106 cells. B) Apoptosis in murine thymocytes. Culture of ex vivo isolated cells in dilution conditions induced death in 12% of the cells, as assessed by trypan blue staining. DNA extraction was carried out as described above, and loading represents the content of 106 cells. Restriction fragments of {Phi}X174 phage DNA digested with HaeIII were used as molecular weight markers (Kb).

GC% and mC content in DNA of apoptotic WEHI-231 cells
To see whether in lymphocytes DNA fragmentation preferentially targets nucleosomes undergoing apoptosis with a particular GC content, we extracted HMW and LMW DNA from WEHI-231 cells cultured with medium, anti-IgM antibody, or C2-ceramide. DNA samples were hydrolyzed chemically into nucleosides and their GC content was quantified using an HPLC column calibrated with nucleoside standards. Both anti-IgM antibody and C2-ceramide treatment resulted in as much as a 50% increase in GC content of fragmented LMW DNA (Fig. 2 ). This high GC% of LMW DNA could reflect a lower sensitivity of GC-rich regions contained in fragmented DNA to nucleases responsible for apoptotic DNA cleavage compared with AT-rich regions of fragmentation-resistant DNA. To determine the DNA methylation pattern in lymphocytes undergoing apoptosis, we then quantified mC by HPLC in LMW and HMW DNA of WEHI-231 cells. These analyses revealed that the mC content of fragmented DNA was also augmented, although not as dramatically as the GC content (Fig. 3 ). This high proportion of mC in apoptotic DNA may also result from a higher resistance of methylated cytosines to nucleases.



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  		Figure 2. Percentages of deoxycytosines (C) and deoxyguanines (G) in DNAs from B and T lymphocytes undergoing apoptosis. The murine IgM+ pre-B cell line WEHI-231 was treated with either anti-IgM antibody (1 µg/ml) or C2-ceramide (10 µM) for 20 h or was untreated. For murine thymocytes, apoptosis was induced by treating 2 x 106 cells/ml with 5 mM of dexamethasone for 16 h or by dilution to a density of 5 x 105 cells/ml and culture for 16 h. LMW DNA, corresponding to apoptotic fragmented DNA and absent in untreated control cells (hatched bars), and HMW DNA (white bars) were extracted from apoptotic cells, hydrolyzed, and processed for HPLC analysis, as described in Materials and Methods. The results are from one representative experiment.



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  		Figure 3. 5-Methylcytosine (mC) content in DNAs from B and T lymphocytes undergoing apoptosis. Experimental conditions were as described in legend to Fig. 2 . Data are expressed as mC percent calculated according to the formula: percent mC = {mC/(mC+C)} x100. The results are from one representative experiment.

GC% and mC content in DNA of apoptotic thymocytes
Since T lymphocytes from SLE patients were found to exhibit an abnormal DNA methylation (5) , we asked whether the increase in mC content of fragmented DNA we found was unique to the B cell line used. To test this possibility, we undertook a similar set of experiments using murine thymocytes able to enter apoptosis after glucocorticoid exposure or growth factor withdrawal (26) . We found that addition of dexamethasone or dilution of cell cultures resulted in an increase in DNA fragmentation and cell death in a time-dependent manner. We also demonstrated that both treatments induced an increase in GC% and mC content of fragmented LMW DNA of T cells (Figs. 2 , 3) . Before apoptosis induction, the mC content was slightly higher in the WEHI-231 immortalized cell line (mean =15%) than in mouse thymocytes (mean =10%). Other investigators also found mC content differences, depending on the source of mouse lymphocytes tested (27) . These experiments show there is a global methylation of deoxycytosines and an increase in GC% in fragmented DNA of both B and T lymphocytes induced to die by apoptosis.

DNA methyltransferase expression during apoptosis
In eukaryotic cells, methylation of deoxycytosines is mediated by the enzyme MTase, giving rise to methyldeoxycytosines. Since abnormal levels of MTase have been observed in patients with idiopathic SLE (28) , we asked whether expression of this enzyme is modulated during apoptosis induced in vitro. WEHI-231 cells were cultured with medium or with anti-IgM antibody and MTase expression was probed by Western blotting. Levels of MTase were evaluated by scanning the specific bands and determining their relative intensity. Under these experimental conditions, apoptosis of WEHI-231 cells was not associated with up-regulation of this enzyme (Fig. 4 ).



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  		Figure 4. Effect of apoptosis on expression of DNA (cytosine-5) methyltransferase (MTase) expression in WEHI-231 B-cell line. Apoptosis was induced in the murine IgM+ pre-B-cell line WEHI-231 by culture in the presence of anti-IgM antibody for 20 h. Lymphocyte lysates (5 x105 cells) were extracted by the Triton-X-100 method. Proteins were separated on a 7.5% acrylamide-SDS-gel under reducing conditions, transferred onto PVDF membranes, and immunoblotted with an antibody to MTase. Molecular mass is indicated by markers shown on the right. MTase is a protein of an apparent molecular mass of 170 kDa and is marked by an arrowhead. C, control cells. A1: Cells treated with anti-IgM antibody (0.5 µg/ml); A2: cells treated with anti-IgM antibody (1 µg/ml).

Methylation state of Alu repetitive sequences in human SLE
To determine whether specific elements of the human genome of SLE subjects exhibit a unique pattern of methylation-sensitive restriction sites, we investigated the methylation of highly repetitive sequences distributed throughout the genome (22) . The majority of such sequences belong to the Alu family, which accounts for 3–6% of total genomic DNA. Any particular DNA fragment of >= 5 000 bp has a high probability of containing Alu sequences, which may be subdivided into groups of related subfamily members based on nucleotide divergence and time of appearance in the human lineage. With 500-2000 members in the human genome, the most recently formed subfamily is termed the `Predicted Variant' (PV) subfamily. The next older group is called the `Precise' subfamily and encompasses as many as 105 members. Notably, the frequency of CpG dinucleotides in these Alu subfamilies is higher (up to ninefold) than in human DNA (29 , 30 ). In addition, four polymorphic CCGG sites are present in the PV consensus sequence and are highly methylated in human spleen DNA (31) , implying that they are frequently the targets of methylation. Since Alu CpG clusters account for one-third of all potential methylation sites in the genome (22) , we investigated the methylation status of young Alu repeats in SLE patients by Southern blot hybridization at two methyl-sensitive restriction sites. Using a 200 bp probe corresponding to the Precise and PV Alu subfamilies (22) , we found that the restriction enzyme HpaII, which cuts only when the deoxycytosine in the 5' position of the sequence CCGG is demethylated, has a recognition site that is very frequent in Alu consensus sequences. In DNA samples isolated from SLE patients and control subjects, no HpaII site demethylation was detectable (Fig. 5 ), indicating that their Alu sequences are methylated. We then used the HpaII isoschizomer MspI, which cuts the sequence CCGG regardless of deoxycytosine methylation. Digests of human DNAs with this enzyme showed three restriction fragments of 70, 130, and 200 bp, the 70 and 130 fragments resulting from a partial but reproducible digest of the 200 bp band. A hybridization smear (>200 bp) due to repetitive Alu sequences, was also present. However, there was no difference in the hybridization patterns between lupus samples and controls. This similarity was confirmed by scanning the three different bands and determining their relative intensity relative to the signal obtained with the sample digest (data not shown). Thus, the extent of Alu methylation in SLE lymphocytes was comparable to that of control samples.



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  		Figure 5. Methylation pattern of Alu sequences in human DNAs. DNA samples (5 µg) isolated from lupus (p) or control subjects (T) were digested with the methylation-sensitive restriction enzymes MspI (M) or HpaII (H), electrophoresed on a 2% agarose gel, and transferred to a Hybond N+ membrane. The probe used was a PV-PR fragment encompassing a consensus sequence of the Predicted Variant (PV) and the `Precise' subfamilies. Molecular mass is indicated in bp.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been known for some time that disease manifestations of human lupus are under environmental influences, as exemplified by the exacerbation of skin rashes (or even the systemic symptoms) after sun exposure and the disease aggravation after viral and bacterial infections (10 , 15 , 17 ). The induction of a drug-induced lupus syndrome by chronic treatment of patients with certain drugs, such as procainamide and hydralazine, is also well documented (32 , 33 ). For example, over 90% of patients undergoing treatment with procainamide for 1 to 2 years mount antinuclear antibodies and ~20% of them develop lupus-like symptoms. A common denominator of these lupus triggers is an abnormal pattern of DNA methylation and GC content. Procainamide, hydralazine, and UV light are inhibitors of DNA methylation, and prokaryotic DNA exhibits a low mC content and GC% (17) . That DNA from SLE patients contains unusual amounts of mC and GC provides further support for the conclusion that abnormal DNA composition of nucleosomes may play a pathogenic role in this disease (14) . Inasmuch as the capacity of DNAs to induce proliferation and Ig production by murine and human B cells correlates closely with their CpG methylation patterns (16 , 18 ), it has been inferred that the released nucleosomes could not only provide ligands for formation of immune complexes, but also drive the autoantibody response. We therefore asked whether lymphocyte death results in the release of potentially immunogenic nucleosomes.

This was approached by using two cell systems that are highly proliferative in culture and inducing growth arrest, followed by death using four triggers of apoptosis. The results presented here show an increase of mC in fragmented chromosomal DNA of apoptotic B and T cells. In other in vitro systems, a correlation was also noted in cultured cells between a change in DNA methylation and apoptosis (34 35 36) . Since DNA is methylated in mammals by the enzyme MTase at the carbon-5 position of deoxycytosine residues, generally in the context of a CpG dinucleotide pair (37) , we also probed MTase expression in cells undergoing apoptosis. However, we could not find a down- or up-regulation of this enzyme, suggesting that the changes in DNA methylation patterns are not linked to the process of de novo DNA methylation during cell death. Similarly, the low methylation observed in the DNA trapped in ICs of lupus patients (38) may not be due to an active DNA demethylation process occurring during apoptosis.

Previous studies showed that DNA methylation levels in the spleen of lupus-prone MRL/lpr mice increase significantly with age (27) and that treatment with a demethylating agent prolongs survival and inhibited glomerulonephritis (27 , 39 ), suggesting that DNA methylation levels may play a role in progression of the autoimmune syndrome. In humans, there is a hypermethylation of the HLA-DR{alpha} locus in SLE patients (40) . We therefore investigated the methylation status of highly repetitive sequences distributed throughout the human genome and encompassing up to 6% of total genomic DNA (29 , 31 ). Recent studies show that probing this DNA subset readily enables detection of abnormal DNA methylation in pathological conditions, as described for an autosomal recessive immunodeficiency syndrome called ICF (41) . Here, however, we found no specific pattern of methylation-sensitive restriction sites in the human genome of SLE subjects and the extent of Alu methylation in SLE lymphocytes was comparable to that of control samples.

Our second key observation is based on investigations of a potential relationship between apoptosis, DNA fragmentation, and GC%. Using a variety of stimuli, we found a significant increase in the nucleases-resistant, GC-rich regions in DNA fragments isolated from both B and T lymphocytes undergoing apoptosis in vitro. It is notable that in a related study, isolation and characterization of apoptotic nucleosomes provided evidence that they also exhibit an abnormal histone composition (42) . It is remarkable that studies of endogenous circulating DNA isolated from the peripheral blood lymphocytes of patients with SLE showed that it accumulated GC-rich DNA fragments (38 , 43 ), is complexed with histones, and exhibits structural characteristics similar to those of oligonucleosomes, the structural units of chromatin (44 , 45 ). Furthermore, when recombinant DNA cloned from the ICs isolated from SLE patients was tested for immune reactivity, human lupus polyclonal and monoclonal antibodies exhibited a higher reactivity with the GC-rich DNA fragments than with the AT-rich fragments (38) .

We would like to emphasize that the present studies were performed in murine cells and in two cell types, thymocytes and a B cell line. Other investigators have found differences in DNA methylation levels in mice depending on the source of the cells and the age of the mice (27) . Another group has found that the mitogenicity of DNA did not necessarily correlate with DNA methylation levels (18) , implying that the immunostimulatory properties of DNA from nonvertebrates is not simply a reflection of the lack of CpG methylation. It is therefore possible that, as suggested previously (18) , inhibitory sequences or structural motifs may influence the immunogenicity of bacterial DNA. It will be important to test the mitogenicity of the DNAs released from apoptotic human B and T cells.

Within the limitations of the experimental approach we and others have taken, these converging observations reveal that the DNA fragments released from apoptotic B and T cells exhibit abnormal mC content, GC%, and histone composition, suggesting that circulating DNA of patients with SLE is largely or entirely derived from the cell nucleus rather than from nongenomic integrated viruses. These molecular characteristics may represent the underlying mechanism responsible for the triggering potential of oligonucleosome complexes released from apoptotic cells. This view corroborates a body of work on nuclear structures derived from entirely different approaches and supports the concept that the structure and the dynamics of nucleosomes may be critical in determining their immunogenicity. First, it was shown that virus-induced apoptosis results in the coclustering of autoantigens and viral antigens exclusively in small surface blebs of apoptotic cells, providing a novel immune context for self-antigens to bypass immune tolerance (12) . Second, it has been demonstrated that a subset of autoantigens is specifically cleaved early during apoptosis and that one of the cleaved molecules is the catalytic subunit of the DNA-dependent protein kinase. Cleavage of these autoantigens may target the newly released molecules for an autoimmune response by revealing cryptic fragments (11) . Third, data suggest that proteins phosphorylated during apoptosis may be preferred targets for autoantibody production in SLE patients (46) . Fourth, studies of cell death in human lupus have shown that there is an accelerated lymphocyte apoptosis and an increased expression of the CD95 death receptor on T and B cells (1 , 47 48 49 ). This accelerated apoptosis could occur either as a result of an intrinsic abnormality of lymphocytes or as a result of the high number of cells undergoing rapid apoptosis. Since it was recently recognized that in lupus the autoantibody process is caused by impairment of the clearance of apoptotic cells (2) , we would like to propose that the accelerated apoptosis and the persistence of abnormally methylated and GC-rich nucleosomes released from apoptotic cells provide an autologous stimulation that could bypass self-tolerance in this systemic disease. The increased amounts of nucleosomes released, with their altered DNA methylation, GC composition (this report), and histone content (42) , may become immunogenic, able to drive an autoimmune response and to combine with autoantibodies to form immune complexes.


   ACKNOWLEDGMENTS
 
We thank Dr. M. Szyf (McGill University, Montreal) for the gift of the anti-MTase antibody, Dr. Enzo Recco for Southern blot analysis, and Dr. Philippe Kourilsky for his support. This work was supported by an institutional grant from the Institut Pasteur. S.H. and M.Z. are investigators of the Institut National de la Recherche et de la Santé Médicale.


   FOOTNOTES
 
2 Abbreviations: C, deoxycytosine; C2-ceramide; N-acetyl-D-sphingosine; G, deoxyguanine; mC, deoxymethylcytosine; HMW, high molecular weight; HPLC, high-performance liquid chromatography; IC, immune complex; LMW, low molecular weight; MTase, DNA (cytosine-5) methyltransferase; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; SLE, systemic lupus erythematosus.

Received for publication November 3, 1998. Revision received February 22, 1999.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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