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Presence of nucleosome-restricted antibodies in patients with systemic lupus erythematosus.

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ARTHRITIS & RHEUMATISM
Val. 38, No. 10, October 1995, pp 1485-1491
0 1995, American College of Rheumatology
1485
PRESENCE OF NUCLEOSOME-RESTRICTED ANTIBODIES IN
PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS
HENRI CHABRE, ZAHIR AMOURA, JEAN-CHARLES PIETTE, PIERRE GODEAU,
JEAN-FRANCOIS BACH, and SOPHIE KOUTOUZOV
Objective. To assess whether nucleosomerestricted antibodies, i.e., antibodies that react with the
whole nucleosome particle but not with its individual
components (double-stranded DNA [dsDNA] and histones), are present in the sera of patients with systemic
lupus erythematosus (SLE).
Methods. Antibodies were detected by enzymelinked immunosorbent assay using purified nucleosomes, dsDNA, or histones. These tests were applied to
the sera of 40 patients with SLE. Protein G-purified
IgGs of representative sera were sequentially adsorbed
on dsDNA- and histone-conjugated solid-phase supports
and further assayed for their nucleosome, dsDNA, and
histone reactivities.
Results. Of the 40 sera tested, 16 displayed
anti-dsDNA and/or antihistone antibody activity, which
was always associated with significant antinucleosome
reactivity. In addition, 3 sera showed antinucleosome
activity that was not associated with concomitant antidsDNA or antihistone activity. The presence of true
nucleosome-restricted antibodies was demonstrated, after solid-phase adsorption, in representative SLE sera
that showed anti-dsDNA or antihistone antibody activity, and also in sera that did not show these activities.
Conclusion. Our results provide evidence for the
presence of nucleosome-restricted antibodies in patients
with lupus. These nucleosome-restricted antibodies,
along with anti-dsDNA and antihistone antibodies, appear to belong to a broad set of antinuclear antibodies,
the antinucleosome family.
Henri Chabre, PhD, Zahir Amoura, MD, Jean-FranGois
Bach, MD, PhD, Sophie Koutouzov, PhD: Hbpital Necker, Pans,
France; Jean-Charles Piette, MD, Pierre Godeau, MD: HBpital de la
PitiC-SalpCtriere, Paris, France.
Address reprint requests to Zahir Amoura, MD, INSERM
U25, HGpital Necker, 161 Rue de Stvres, 75015 Pans, France.
Submitted for publication November 29, 1994; accepted in
revised form April 25, 1995.
The nature of the autoantigen that initiates the
production of anti-double-stranded DNA (antidsDNA) and antihistone antibodies in systemic lupus
erythematosus (SLE) has remained obscure. Nearly
10 years ago, Hardin (1) noted that patients with SLE
responded primarily to external features of intact
nucleosomal segments of chromatin, since the antibodies produced were directed to both histones and
dsDNA. The notion that the nucleosome-the fundamental repeating unit of chromatin-could be a major
autoantigen in lupus was later emphasized by Tan, in a
report on the “state of the art” of autoantibodies in
disease (2). Burlingame and colleagues (3) were the
first to provide evidence, in murine models of lupus,
that the production of antichromatin antibodies implicated a T-dependent immunization with self antigen.
In accordance with that observation, our group
showed that the onset of the autoimmune response in
lupus-prone MRL mice was characterized by the early
emergence of a serum antinucleosome activity that
was not associated with concomitant anti-dsDNA
and antihistone activities, and was due to bona fide
nucleosome-restricted antibodies (4). Later, as the
autoimmune response progressed, autoantibodies reacting against the individual components of the nucleosome, i.e., dsDNA and histones, appeared.
Taken together, these findings raise the strong
possibility that the nucleosome (or chromatin) is a
potential candidate immunogen in murine SLE. The
elegant demonstration by Mohan et a1 (5) that the
nucleosome is one of the major autoantigens reacting
with pathogenic autoantibody inducing-T cells in
SNFl lupus-prone mice lends support to the above
hypothesis.
A central role for chromatin in autoimmune
responses to histones and dsDNA in human lupus has
recently been suggested (6). Since circulating nucleosome oligomers have been demonstrated in the plasma
of SLE patients (7), this strongly suggests that the
1486
CHABRE ET AL
nucleosome itself is a potent immunogen in human
SLE. To assess this hypothesis, we investigated
whether nucleosome-restricted antibodies are present
in patients with overt clinical lupus.
PATIENTS AND METHODS
Patients and sera. The sera of 40 patients (34 women
and 6 men; age range 18-65 years, mean 32 years) from the
Service of Internal Medicine, HBpital de la Pitik-Salpetri&e,
who fulfilled at least 4 of the American College of Rheumatology revised criteria for SLE (8), were studied. Serum was
decomplemented by heating at 56°C for 30 minutes and was
stored at -20°C until used. According to the Systemic Lupus
Erythematosus Disease Activity Index (9), 28 of the patients
had inactive lupus and 12 had active lupus. Clinical symptoms at the time of blood sampling in the 12 patients with
active disease were as follows: malar rash in 9, alopecia in 6,
oral ulcers in 1, arthritis in 9, serositis in 3, renal involvement in 7, central nervous system involvement in 3, and
vasculitis in 2. The control group was composed of 66
healthy blood donors from the HBpital Necker Blood Bank
(49 women and 17 men; age range 19-65 years, mean 34
years).
Antibody purification procedure. Purification of serum antibodies was performed according to the following
procedure: first, serum IgGs were purified on a protein
G-Sepharose fast-flow column (Pharmacia, Uppsala, Sweden). These IgGs were further depleted of anti-dsDNA
antibodies by means of adsorption on DNA-cellulose columns (10). Briefly, dsDNA-cellulose (Sigma, St. Louis, MO)
was equilibrated overnight in 10 mM Tris, 1 mM EDTA (TE)
and 150 m M NaC1, pH 7.4 (TE buffer) at 4”C, washed
extensively with TE and 2M NaCl, pH 8.0, and further
equilibrated in TE buffer. Purified IgGs from each SLE
serum were passed through individual columns, and the
effluent was further depleted of antihistone antibodies by
adsorption on histone columns made of a mixture of total
histones (Sigma) covalently linked to p-nitrophenyl chloroformate-activated trisacryl beads (IBF, Villeneuve la
Garenne, France) (10). The effluent from each histone column was taken as the source of purified adsorbed IgGs.
The presence of possible contaminating nucleosomal
histones in the recovered IgG preparations (i.e., complexed
antibodies) was checked by Laemmli-type sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE;
15%) followed by Coomassie blue staining. The presence of
histones was further checked by immunoblotting using complexed anti-dsDNA monoclonal antibodies (MAb) from
PME 77 hybridoma supernatants, which display crossreactive antihistone activity (10,ll).
Tests for antigen reactivity of sera and purified IgGs.
Autoantigen preparation. Nucleosomes were prepared as
previously described (4). Briefly, nucleosomes were isolated
by digesting chromatin from mouse erythroleukemia L1210
cell nuclei with Staphylococcus aureus nuclease. After stripping of H1 and nonhistone proteins with 0.55M NaCl at O’C,
samples of the stripped chromatin were applied to sucrose
gradients (5-20%) prepared in TE, 0.55MNaCI, and 0.2 mM
~
phenylmethlysulfonyl fluoride. After overnight centrifugation at 200,00Og, nucleosome fractions were collected at an
optical density (OD) of 260 nm and subjected to PAGE.
Fractions corresponding to purified mononucleosomes were
extensively dialyzed against TE, concentrated, and stored
on ice for no longer than 2 weeks. Histone gel analysis (12)
showed that the mononucleosome preparation consisted of
equal amounts of the 4 core histones.
Lambda phage dsDNA, mixtures of total histones,
and the individual H2A, H2B, H3, and H4 histones were
obtained from Boehringer Mannheim (Mannheim, Germany).
Determination of antigen concentration by enzymelinked immunosorbent assays (ELISAs).Antigen concentrations were determined by ELISAs, essentially according to
the method described by Burlingame and Rubin (13). Using
the microbicinchoninic acid protein assay (Pierce, Rockford,
IL), we first confirmed that equivalent amounts of histones
were bound to the ELISA plates in both the histone and the
nucleosome ELISAs (data not shown). In order to compare
the relative amount of dsDNA bound to the plates in the
dsDNA and the nucleosome ELISAs, 2 affinity-purified
MAb specific for dsDNA, PME 77 and H241 (kindly provided by Dr. B. Stollar, Tufts University, Boston, MA), and
2 SLE affinity-purified polyclonal anti-dsDNA antibodies
were used. Reactivity profiles shown in Figure 1 indicated
that anti-dsDNA and antinucleosome activities were superimposable with each of the anti-dsDNA MAb assayed, and
very similar to the 2 polyclonal anti-dsDNA antibodies. We
therefore assumed that the amount of dsDNA bound to the
plates and “available” for antibody binding was comparable
in the 2 ELISA systems.
ELISAs. The antinucleosome, anti-dsDNA, and antihistone (total or individual) ELISAs were set and processed
as described previously (4). Briefly, antigen-coated plates
were washed with phosphate buffered saline-0.1% Tween
(PBS-Tween), pH 7.4, and postcoated for 2 hours with 0.1
ml of PBS-10% fetal calf serum, pH 7.4. After washing, sera
diluted in PBS-Tween (1: 100) were added and the reaction
was carried out for 2 hours. Bound antibodies were detected
with peroxidase-conjugated goat anti-human IgG (heavy and
light chain-specific) antisera (Biosys, Compiegne, France).
Binding was measured by adding ABTS substrate (Southern
Biotechnology, Birmingham, AL), and OD at 405 nm was
read with an automated spectrophotometer (Dynatech,
Alexandria, VA).
To determine IgG concentrations, the same ELISA
method was used, except that plates were coated with goat
anti-human IgG. The ODs generated by sera were compared
with a standard curve generated using known serial concentrations of protein G-purified IgGs.
Uncoated plates were used to determine background
activity, which was subtracted from values obtained with
antigen-coated plates. To minimize day-to-day variations in
the ELISAs, sera were thawed only once for antigen reactivity determinations. Appropriate positive and negative
control sera were always added to the plates on the day of
the assay, and interassay variations of these controls never
exceeded 1%.
Expression of results. In order to compare serum
reactivity against the 3 antigens, the results were expressed
as positive or negative for each ELISA. The threshold value
1487
NUCLEOSOME-RESTRICTED ANTIBODIES IN SLE
*Oo0
1M*
1500]
Mab W4’
PME 77
800-
3
d
0
SLE patient #2
600-
400-
200-
, , , , , , -10,
OT
1
Antibody concentration (nglml)
,
;i.
100
1000
low0
Figure 1. Comparative activity of the anti-double-stranded DNA (anti-dsDNA) and antinucleosome enzyme-linked immunosorbent assays. Affinity-purified monoclonal anti-dsDNA
antibodies (MAb) PME 77 and H241 and polyclonal anti-dsDNA antibodies from 2 systemic
lupus erythematosus (SLE) patients were used to compare the detection of DNA within the 2
substrates: A phage dsDNA (0)
and purified mononucleosomes (0).Peroxidase-conjugated
anti-mouse or anti-human IgGs (Fc specific) were used as detecting agents. O.D. = optical
density.
for positivity was defined as 3 SD above the mean in the 66
sera (diluted 1:lOO) from healthy blood donors. The OD
cutoff values for each ELISA were as follows: antinucleosome 0.101, anti-dsDNA 0.105, and antihistone 0.090. All
values in the healthy blood donor group fell within these
ranges. SLE sera (diluted 1: 100) with OD values greater than
the cutoff value were considered positive.
Statistical analysis. The correlation between antinucleosome reactivity and clinical symptoms was determined
by chi-square analysis, with Yates’ correction when necessary.
RESULTS
Antinucleosome, anti-dsDNA, and antihistone
reactivities of SLE patient sera. Of the 40 SLE patient
sera studied, 19 (47.5%), 15 (37.5%), and 13 (32.5%)
were found to be positive in the antinucleosome,
anti-dsDNA, and antihistone ELISAs, respectively.
Of the 19 sera reacting with nucleosomes, 16 (84%)
exhibited concomitant anti-dsDNA and/or antihistone
activities. The other 3 sera (16%) were shown to react
with nucleosomes without reacting with dsDNA and
histones. Of note, all sera that had anti-dsDNA or
antihistone antibody activity also exhibited reactivity
to nucleosomes.
Correlation between clinical symptoms and antigen reactivities. Antibody reactivity was found to
correlate with disease activity. In the group with
active SLE, 12 of 12 (loo%), 11 of 12 (92%), and 10 of
12 (83%) of the sera displayed antinucleosorne, antidsDNA, and antihistone reactivity, respectively, as
opposed to 7 of 28 (25%), 4 of 28 (14%), and 3 of 28
(1 1%) of the sera in the inactive SLE group (P < O.OOO1
for all 3 comparisons). No statistically significant correlation was found between serum antinucleosome activity and the presence of any specific clinical symptoms.
Characterization of nucleosome-restricted antibodies. Four representative patients, 2 with active
SLE and 2 with inactive SLE, whose sera exhibited
reactivity to nucleosomes at a dilution of 1:lOO in the
CHABRE ET AL
1488
600 -
-
Ma
Pa
Ad
800 -I
800 7
500 1
-
400 -
600
400
-
400 -
200
-
200
4
300 -
200 -
-
100
0
0
(I
0-
Ba
300
200
100
-
0-
0
SLE serum antibody activity
6
800-
800 -I
0
600:
600
400
200
-
0-
400
200
500
-
-
400 -
-
300
-
200
-
100
-
-
0’
screening ELISA were selected for further study.
Their sera were serially diluted in order to determine
the linear portion of the curve comparing the OD
generated by antinucleosome ELISA and the log of
serum concentration. Anti-dsDNA and antihistone activities were determined at the dilution corresponding
to that of the first point on the linear portion of the
curve. As shown in Figure 2, the 2 representative sera
from patients with active disease, at dilutions of 1:200
and 1:800, respectively, exhibited antinucleosome activity and concomitant anti-dsDNA and/or antihistone
activity. Sera from 2 patients with inactive disease,
both diluted 1: 100, displayed antinucleosome, but not
anti-dsDNA or antihistone, activity.
IgGs of these 4 sera were purified on protein G
and then adsorbed on dsDNA- and histone-conjugated
columns in order to deplete the purified IgGs of
anti-dsDNA and antihistone antibodies, which could
account for antinucleosome reactivity. Recovery for 1
0-
300
-
200 -
100-
O A
representative serum (patient Pa in Figure 2) after IgG
purification and adsorption was as follows: from 0.650
ml of serum (13.5 mg/ml IgG), 8.3 mg of purified IgG in
6.5 ml was recovered after protein G purification (1.3
rng/ml; yield 94%). Effluents from successive adsorptions on DNA cellulose and trisacryl-histones contained 8.1 mg and 6.8 mg of purified IgGs (yields 98%
and 84%), respectively. These final IgG preparations
were adjusted to the initial serum IgG concentration,
and assayed for antibody reactivity. As shown in
Figure 2, the adsorptions abolished anti-dsDNA and
antihistone reactivities in the 2 sera from patients with
active SLE in which these activities had been demonstrated, indicating that this procedure truly depleted
serum of anti-dsDNA and antihistone antibodies.
Nonetheless, antinucleosome activity was still present,
and was found to be virtually unchanged compared with
that observed in the starting serum.
We next investigated whether the nucleosome
1489
NUCLEOSOME-RESTRICTED ANTIBODIES IN SLE
AD
MA BA
PA
-94
67
-
- 43
- 30
- 21
- 14
w4/
1
2
3
4
5
6
7
Figure 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified nucleosome-restricted antibodies. Lanes
4-7, 100 pg of purified and adsorbed IgGs from sera of the same 4 representative systemic lupus erythematosus patients
as in Figure 2. Note the presence of heavy and light chains and the absence of contaminant histone bands. A mixture of
histones (Boerhinger Mannheim, Mannheim, Germany) was used as control at 2.5 pg (lane l), 1 pg (lane 2), and 0.5 pg
(lane 3 ) . Staining was with Coomassie blue. Molecular weight markers are shown on the right.
reactivity found in the 2 sera from patients with
inactive SLE, with no anti-dsDNA or antihistone
antibody activity, was also due to antibodies reacting
exclusively with the whole nucleosome structure.
IgGs from these 2 sera were purified and adsorbed
using the chromatography procedures described
above. The recovered preparation was found to still
react strongly with nucleosomes (Figure 2), excluding the possibility that anti-dsDNA or antihistone
antibodies were participating in the nucleosome reactivity, which would have been undetectable in our
ELISAs.
To confirm that these nucleosome-restricted
activities were truly due to noncomplexed antibodies,
the purity of the IgGs after the sequential adsorptions
was assessed by SDS-PAGE. Gel staining (Figure 3)
confirmed the absence of contaminating histone bands
in each of the IgG preparations from individual SLE
patients, indicating that the IgGs were in a noncomplexed form. The purity of individual SLE IgG preparations was further confirmed by immunoblotting
(results not shown).
DISCUSSION
Recent evidence obtained in murine models of
SLE suggests that the nucleosome, a 160-basepair
DNA wrapped around a core histone octamer, is a
preferential target for lupus autoantibodies and a putative autoantigen triggering the production of antibodies against its components, dsDNA and histones (3,4).
In order to investigate whether the nucleosome might
play a key role in human lupus, we assessed, by means
of ELISAs, the serum antinucleosome antibody reactivity in 40 SLE patients, in comparison with their
anti-dsDNA and antihistone reactivities. Our data
suggest that the production of anti-dsDNA and antihistone antibodies is closely associated with that of
antinucleosome antibodies. Indeed, in the 40 SLE sera
tested, anti-dsDNA or antihistone activity was never
detected without antinucleosome antibody activity.
Conversely, 84% of the sera exhibiting antinucleosome antibody activity exhibited concomitant antidsDNA and/or antihistone activity.
Since dsDNA and histones are individual coun-
1490
terparts of the nucleosome, it is likely that anti-dsDNA
and antihistone antibodies can interact with, respectively, the dsDNA and the histones of the nucleosome,
and thus be part of the antinucleosome reactivity. To
further determine whether this antinucleosome antibody reactivity was due to anti-dsDNA and antihistone antibodies or rather due to antibodies directed
against the whole nucleosome, sera were sequentially
adsorbed on dsDNA and histones. Depletion of antidsDNA and antihistone antibodies from 2 representative SLE sera showed the persistence of antinucleosome reactivity, strongly suggesting the presence of
nucleosome-restricted antibodies in these sera. From
the data, we also concluded that, at least in these
patients, anti-dsDNA and antihistone antibodies accounted for a minor part of the serum antinucleosome
reactivity. Indeed, after matching for IgG content, the
antinucleosome activity remained virtually unchanged
after depletion of anti-dsDNA and antihistone antibodies from serum. These findings are in accordance with
a previous report by Burlingame et a1 (6), in which
antichromatin activity from 4 of 10 sera from patients
with active SLE was shown to remain unchanged after
adsorption on dsDNA. Actually, <25% of the antichromatin activity was due to anti-dsDNA antibodies,
and the authors found in addition that approximately
one-third of the SLE sera studied had high antichromatin with little anti-dsDNA or antihistone reactivity.
Thus, our data confirm those authors’ conclusions that
in SLE, antibodies to individual histones or to dsDNA
are minor components of the IgG antichromatin (antinucleosome) response.
Recent work by Kramers et a1 (1 1) showed that
immune complexes formed by nucleosomes and murine monoclonal nucleosome-restricted antibodies
exhibit antihistone and anti-dsDNA antibody activity. Thus, one may question whether part of the
serum anti-dsDNA and/or antihistone activity is due to
the presence of nucleosome-restricted antibodynucleosome immune complexes. In the present study,
although we did not purify serum IgGs under dissociating conditions, the lack of anti-dsDNA and antihistone antibody activities in the final nucleosomerestricted preparations strongly suggests that the
nucleosome-restricted activities observed were due to
noncomplexed antibodies. This is further supported by
the absence of nucleosomal histone antigens in IgG
electrophoretic and immunoblot profiles. Although the
possibility of the presence of nucleosome-antinucleosome complexes in serum cannot be completely excluded, we postulate that these immune complexes
CHABRE ET AL
were retained on the dsDNA and histone columns (ref.
11 and Chabre H et al: unpublished observations).
In the sera of 3 SLE patients, we detected an
antinucleosome reactivity which was not accompanied
by anti-dsDNA or antihistone antibody activity. The
fact that the antinucleosome activity persisted after
adsorption suggests the presence of true nucleosomerestricted antibodies in these sera. The reactivity
profiles of the overall group of SLE sera demonstrate
the presence of antibodies that are directed against
antigenic determinants generated by specific interactions between the nucleosome components. Our findings and those previously reported in the literature
(3,4,6,11) support the notion that nucleosomerestricted anti-dsDNA and antihistone antibodies belong to a broad family of antinucleosome antibodies.
Interestingly, early studies on the LE factors responsible for the so-called LE cell phenomenon showed
that mixing of LE factor-positive sera with core
mononucleosome completely inhibited positive morphologic reactivity in the LE test, while there was no
effect with free dsDNA or histones (14). It is tempting
to postulate that the LE cell phenomenon is related to
these nucleosome-restricted antibodies.
We demonstrated in a recent study that
nucleosome-restricted antibodies were present in the
kidneys of proteinuric lupus-prone mice (4), suggesting that these autoantibodies are involved in the pathogenesis of the disease. Interestingly, Kramers et a1 (1 1)
recently reported that murine MAb reacting with nucleosome but not with its individual components (i.e.,
nuclesome-restricted MAb) bind to the glomerular
basement membrane in vivo, but only when complexed with nucleosome antigens. Taken together,
these findings strongly suggest that glomerular deposition of nucleosome-restricted immune complexes
could be an important event in the pathogenesis of
lupus nephritis.
In our study, which included both patients
with active SLE and patients with inactive SLE, we
found that 16% of those who exhibited serum antinucleosome activity were negative for anti-dsDNA
and antihistone antibodies (compared with -30% in
Burlingame and colleagues’ study of patients with active disease [ 6 ] ) .Thus, it may be questioned whether,
by assessing only anti-dsDNA and antihistone activities in such patients, one is perhaps ignoring a potentially pathogenic antinuclear antibody population, i.e.,
the population of nucleosome-restricted antibodies.
NUCLEOSOME-RESTRICTED ANTIBODIES IN SLE
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