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The Origin Sequence Structure and Consequences of Developing Anti-DNA Antibodies.

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ARTHRITIS & RHEUMATISM Volume 37
Number 2, February 1994, pp 169-180
0 1994, American College of Rheumatology
169
~~~
VIEWPOINT
THE ORIGIN, SEQUENCE, STRUCTURE, AND
CONSEQUENCES OF DEVELOPING
ANTI-DNA ANTIBODIES
A Human Perspective
DAVID A. ISENBERG, MICHAEL R. EHRENSTEIN, CELIA LONGHURST, and
JATINDERPAL K. KALSI
Although the etiology of systemic lupus erythematosus (SLE) remains enigmatic, it is likely that
antibodies to DNA are an integral component in many,
though not all, cases. The caveat is necessary, because
several reports (for review, see ref. 1) have indicated
that 30% or more of patients who meet the widely
accepted classification criteria (2) devised by the
American College of Rheumatology (formerly, the
American Rheumatism Association) do not have detectable anti-DNA antibodies. Although useful, the
classification criteria are broadly based and allow the
diagnosis of SLE in patients with diverse clinical and
serologic features. Most patients without anti-DNA
antibodies do have antinuclear antibodies of other
specificities (e.g., anti-Sm, anti-Ro, anti-La). It is
possible, though we doubt it, that more sensitive
assays would detect anti-DNA antibodies in all SLE
patients. Thus on balance, lupus should really be
considered a generic term, encompassing several individual conditions.
The presence of anti-DNA antibodies in the
serum of SLE patients has long been considered both
a marker of, and pathologic factor in, renal disease
(3,4). Indeed, in our group of nearly 200 SLE patients,
we have yet to see a patient with severe renal involveFrom The Division of Rheumatology, Department of Medicine, University College London.
Supported by The Arthritis and Rheumatism Council, the
Oliver Bird Fund, and the Gerd Cohn bequest.
David A. Isenberg, MD; Michael R. Ehrenstein, MRCP;
Celia Longhurst, BSc; Jatinderpal K. Kalsi, PhD.
Address reprint requests to David Isenberg, MD, Department of Rheumatology, Bloomsbury Rheumatology Unit, Arthur
Stanley House, 50 Tottenham Street, London WlP 9PG, England.
Submitted for publication March 9, 1993; accepted in revised form July 7, 1993.
ment who has not had elevated DNA antibody levels.
There are some reports of serum anti-double-stranded
DNA (anti-dsDNA) antibody levels correlating with
the severity of renal disease in SLE (for review, see
ref. 5). However, many of these reports are flawed,
having generally taken little account of the patients’
concomitant therapy, or having provided insufficient
histologic data for meaningful comparison. Reassuringly, a recent report by Okamura and colleagues (6)
on 40 untreated patients with lupus nephritis, has
shown a strong correlation between IgG anti-dsDNA
levels and renal histology that was not evident for IgM
anti-dsDNA or for anti-single-stranded DNA (antissDNA) antibodies of either isotype.
It is evident that the development of antibodies
per se, even anti-DNA antibodies, does not automatically lead to autoimmune disease. Healthy individuals, notably, the elderly and especially relatives of
patients with autoimmune disease, may develop a
range of autoantibodies without apparently coming to
any harm (for review, see ref. 7). This observation
must be critically considered in the case of anti-DNA
antibodies. While it is true that antibodies to singlestranded DNA have been detected in 20-30% of the
healthy relatives of SLE patients (for review, see ref.
8), antibodies to double-stranded, or native, DNA
virtually never occur in these relatives. Among 147
healthy relatives of 48 SLE patients, we found only 1
who had detectable anti-dsDNA antibodies (on Crithidia
luciliae substrate) (9). Thus, antibodies to dsDNA do
seem to be particularly associated with SLE.
The question of a direct role of anti-dsDNA
antibodies in the pathogenesis of murine lupus has
been reexamined recently. Vlahakos and colleagues
(10) have described the ability of murine monoclonal
170
anti-dsDNA antibodies (derived from MRLApr and
SNF 1 lupus-prone mouse strains) injected into
healthy-strain mice to form immune deposits at distinct glomerular and vascular sites. Following the
work of Shmiedeke et al (ll), who showed that histones can bind with high affinity to the glomerular
basement membrane (GMB), Brinkman and colleagues (12) reported that anti-DNA antibodies bind to
heparan sulfate proteoglycan, a constituent of the
GMB, via histones and DNA. In recent studies incorporating isolated GBM loops and renal perfusion studies in Wistar rats, that group of investigators (13) has
shown that anti-DNA antibodies bind to the glomerulus via complexes of histones and DNA and, to a lesser
extent, via DNA alone.
In another study, Vlahakos et a1 (14) provided
evidence that murine monoclonal antibodies were
able, in vivo, to penetrate cells and interfere sufficiently with their normal function as to contribute to
pathologic abnormalities. However, only 5 of a large
panel of over 30 monoclonal antibodies were able to do
this, which suggests that whatever the experimental
mechanism (e.g., Fc receptor-dependent process, reactivity with DNA on the cell surface), direct DNA
antibody penetration is unlikely to be a significant
pathologic process in SLE patients. The question of
antibody penetration into living cells now goes back
some 15 years (15), since the original suggestion that
anti-RNP antibodies might possess this ability. However, with rare exception (16), no other groups have
described this phenomenon. Given its great potential
interest, this strongly suggests to us that either the
techniques required to demonstrate antibody penetration are technically difficult to reproduce or its likely
importance is viewed with skepticism by many immunologists.
In other studies of murine lupus, IgM antissDNA antibody-derived transgenes were expressed
in healthy-strain mice (17). It was shown that although
many B cells in these animals were able to express
anti-DNA immunoglobulin on their surface, the cells
were anergic-perhaps as a result of having been
exposed to antigen. The healthy-strain mice were
evidently able to control these potentially diseaseassociated antibodies. In partial agreement with this
report, Tsao and colleagues (18) described another
transgenic model utilizing H and L chain genes from a
hybridoma secreting an IgG monoclonal anti-DNA
antibody. Many of the B lymphocytes in this transgenic model expressed endogenous IgM, and some
expressed low levels of transgene-derived IgG, on cell
membranes. As in the initial model, the transgenic
ISENBERG ET AL
animals did not develop major manifestations of lupus.
However, these animals were found to have elevated
levels of IgG anti-DNA antibodies in their serum and a
minor degree of nephritis was noted. Interestingly,
immunizing these animals with DNA increased the
anti-DNA levels.
Intriguing as these recent results are, they cannot explain the origins of human lupus in general or the
development of anti-dsDNA antibodies in particular.
Three main concepts have emerged, though much of
the data used to support them are based on experiments performed in genetically predisposed animal
models. These models demonstrate various components of the human disease but not its totality. The first
concept proposes that systemic autoimmune disease is
the result of polyclonal B cell activation (19), of which
the anti-dsDNA antibody response is merely part. The
second suggests that autoreactive clones are the result
of antigen-driven specific stimulation (20). The third
envisages a 2-stage development, incorporating elements of both polyclonal activation and an antigendriven response (21). Based on evidence available
from human studies, we believe the third view is most
likely to be correct.
There is some debate in the literature about the
degree of polyclonal B cell activation in patients with
SLE. As Gharavi and colleagues (22) have emphasized, of -2,000 mammalian intracellular proteins
detectable by current methods, relatively few are
recognized by serum from SLE patients. While SLE
patients collectively may appear to have a broad range
of antinuclear antibodies, individual patients appear to
express a restricted range of antibody specificities and
lack, for example, antibodies to the nuclear transfer
RNA synthetases which are present in patients with
myositis. Furthermore, analyses of both human and
murine monoclonal antibodies have suggested that
some antibodies have a degree of cross-reactivity .
Thus some, although probably a minority, of the
anti-DNA antibodies bind cardiolipin (23) and others
have rheumatoid factor reactivity (24). More recently,
it has been shown that human monoclonal antibodies
may bind Ro and DNA (25). Thus, autoantibodies may
be truly polyreactive. In other words, the same antibody giving a positive signal in one assay may also
give a positive signal in another.
Alternatively, these data suggest that there may
be a restricted degree of polyclonal activation. However, it has been demonstrated (26), using an enzymelinked immunospot (ELIspot) assay method (which
enables the enumeration of both IgG and IgM antibodysecreting cells) that the mean numbers of peripheral
VIEWPOINT: ANTI-DNA ANTIBODIES
blood cells spontaneously secreting antibodies to such
common environmental antigens as influenza hemagglutinin, adenovirus hexon, and mannan from Cundidu
albicans, as well as to ssDNA and dsDNA, were
increased in SLE patients compared with healthy
controls. Even this evidence must not be taken as
confirmation of random polyclonal activation, since
there was no similar increased response to antigens
with which these patients were unlikely to have come
into contact, such as egg antigen from Schistosomu
mansoni (27). Thus, the polyclonal activation observed in human SLE might best be described as a
limited and “educated” response. It may, however,
simply reflect a stochastic representation of the individual’s previous antigenic experience. In other
words, all clones may be activated but it is easier to
detect those that were expanded prior to the polyclonal activation “event.”
In the past few years, particular aspects of
anti-dsDNA antibodies have become the focus of
considerable attention. In the following sections, we
review critically the likely stimulus of the production
of anti-DNA antibodies in SLE, the mechanisms that
are likely to be responsible for the long-term production of anti-DNA antibodies, the information that is
provided by sequence analysis of human anti-DNA
antibodies, and what is known about the epitopes that
these antibodies actually recognize. As far as possible,
we will concentrate on what is known about human
anti-DNA antibodies.
What is likely to be the stimulus of the
production of anti-DNA antibodies in SLE?
Mammalian DNA is not immunogenic in a
variety of animal species tested (for review, see ref.
28). However, as will be discussed later, sequence
analysis of anti-DNA antibodies shows features of
antigenic selection. A number of hypotheses with a
variable amount of supporting evidence have arisen to
explain this. In contrast to the lack of immunogenicity
of mammalian DNA, bacterial DNA can induce a good
antibody response in mice (29). Serum from patients
with lupus has also been shown to bind a widely
shared bacterial DNA epitope (30). However, the
binding profile of these antibodies was directed mainly
against ssDNA.
Given other evidence that anti-DNA antibodies
cross-react with epitopes on infectious agents (e.g.,
Klebsiella) and that anti-DNA antibodies may be very
similar in structure to antibacterial antibodies (for
171
review, see ref. 31), it is tenable that anti-dsDNA
might arise from stimulation with foreign, rather than
self, DNA. Antibodies derived in this way probably in
the main represent natural antibodies, which provide
the first line of defense against infectious agents. They
are known to have a wide range of specificities,
including self, and are not representative of the antibodies associated with active lupus as delineated
above. Monoclonal IgG anti-DNA antibodies have
been shown to have a much more limited range of
specificities (32).
In an intriguing recent development, Krishnan
and Marion (33) examined the potential immunogenicity of a complex of native DNA and a synthetic DNA
binding protein. This peptide contained 27 amino acids
from an internal domain of a 52-amino acid carboxylfusion protein of ubiquitin in Trypanosoma cruzi. A
relatively small number (6 in all) of monoclonal antiDNA antibody derivatives of BALB/c mice immunized with the peptide-native DNA complexes were
found to share DNA specificities and V region structures with anti-DNA monoclonals derived from lupusprone (New Zealand black X New Zealand white)F,
(NZBNZW) mice. It remains to be demonstrated
however, that the antibodies produced from the mice
given the peptidenative DNA complex are actually
pathogenic.
Another possible explanation for the presence
of these antibodies is that DNA linked to histones,
abundantly found in nucleosomes, may be the stimulating antigen. We have compared a panel of 5 IgG and
5 IgM anti-DNA antibodies produced from the same
SLE patient and have found that although the IgG
antibodies were not cross-reactive with cardiolipin,
the two IgG anti-dsDNA antibodies did bind to histones which the IgM antibodies did not (34). Moreover, the binding to histones was enhanced by the
presence of DNA, detectable in the supernatant of the
hybridomas, which occupied the binding site of the
anti-dsDNA antibodies but not those of the antissDNA antibodies.
Nucleosomes have been found to interact with
murine spleen cells, which leads to the release of
interleukin-6 (35). Bell and colleagues (36) have demonstrated that cells in culture release nucleosomes that
stimulate proliferation and immunoglobulin synthesis
of normal mouse lymphocytes. This could enhance
polyclonal activation and autoantibody formation. Experiments with NZB/NZW mice showed that the polyclonal B cell activation precedes by several weeks,
and reliably predicts, an antibody response skewed
toward DNA and the development of nephritis (37). A
172
ISENBERG ET AL
Table 1. Characteristics of human IgM monoclonal anti-DNA antibodies*
~~
Antibody
2 1I28
8E10
III2R
C6B2
TH3
1812
1117
POP
B19.7
III3R
Kim 4.6
BEG-2
IC4
RT79
11-1
L16
ML1
A10
A43 1
A947
~
~
Origin
SLE PBL
Leprosy PBL
SLE SPL
Sickle cell anemia
SPL
Leprosy PBL
SLE PBL
SLE PBL
Leukemia and
peripheral
neuropathy PBL
Fetal BML
SLE SPL
Healthy child
tonsil Ls
Fetal liver Ls
Myeloma BML
SLE SPL
SLE SPL
Fetal liver Ls
Fetal spleen Ls
Healthy adult PBL
Healthy adult PBL
Healthy adult PBL
DNA
antigen
VH
Germline
counterpart
1
1
1
2
HA2
HA2
51P1
VCE- I
DXP' 1
ss
2
3
3
3
VCE-1
VH26
VH26
VH26
ss (+RF)
ds
ss
3
3
3
VH26
56P1
v,1,911 I
DXP' 1
DXP'I
4
4
4
V4.71-2
VH4.7 1-4
VH4.21
VH251
VH6
VH6
VH6
VH6
?VH7
ss
ss
ss
ss > ds
ss
ss
+ ds
ss
ss
+ ds
ds
ss > ds
ds
ss
ss
ss
ss
ss (+RF)
5
6
6
6
6
7
D
JH
L
DXP' 1
4
4
K
5
K
3
?
DXP' 1
DXP' 1
DXP'I
DLR4
4
-
5
5
K
4
-
?
4
4
6
A
5
4
3
4
3
4
A
?
?
?
?
DXP'I
?
Q52
~ 5 2
~ 5 2
?Q52
?
K
K
K
A
K
K
K
K
A
K
4
A
A
Germline
Ref.
hk137
-
47
47
48
49
-
-
47
47
47
50
DILPl
-
51
48
52
-
53
48
54
48
hk137
vk328
-
55
55
55
55
56
* SLE = systemic lupus erythematosus; PBL = peripheral blood lymphocytes; ss = single-stranded; SPL = splenic lymphocytes; ds
double-stranded; BML = bone marrow lymphocytes; RF = rheumatoid factor; Ls = lymphocytes.
recent report described the presence of these nucleosomes in much higher concentrations in the plasma of
SLE patients compared with normal controls (38). The
DNA present in the circulation of SLE patients consists of 200 basepairs and their multiples, and we have
detected this size DNA bound to the anti-dsDNA
antibodies in the supernatant of the anti-DNAproducing hybridomas mentioned above (Ehrenstein
M, Longhurst C, Isenberg D: unpublished observations). Cell apoptosis releases DNA of this size; cell
necrosis does not (33).
In a preliminary experiment, we have shown
that the nucleosomes contained in the supernatant of
the above-mentioned hybridomas were not immunogenic in BALB/c mice, but that the nucleosome complexed with the IgG anti-dsDNA antibody produced
from the hybridoma led to the production of antidsDNA antibodies in the mice (39). The antibody
alone was not immunogenic. The lack of immunogenicity of nucleosomes alone has been observed by
other groups (Sylviane Muller, Institute of Molecular
Biology, Strasbourg, France: personal communication), but one study showed that DNA-protein complexes found in sera of SLE patients could provoke the
development of anti-DNA antibodies in normal mice
(40) after a single injection. However, it was not clear
whether there was any antibody in their preparation,
=
that is, in the form of immune complexes. The immunization of the animals was unusual; in particular, no
adjuvant was used, it was not clear how many booster
injections were given, nor was the route of immunization apparent. Moreover, no control mice were included. A comprehensive study exploring whether
DNA complexed with a variety of proteins might be
antigenic has not, to our knowledge, been reported.
It has also been suggested that activated phagocytic cells can release highly reactive oxygen species
(ROS) which can penetrate cell membranes, interact
with nuclear DNA, and cause the release of altered
DNA, which in turn, could stimulate anti-DNA antibodies. This possibility was explored by Gordon et a1
(41), who compared 41 SLE patients with 37 disease
controls and 20 healthy controls. The SLE group had
statistically significantly greater anti-ROS DNA binding than the control groups. The ELISA used was
more sensitive than conventional radioimmunoassay
for anti-DNA antibodies.
What mechanisms are likely to be responsible
for the long-term production of anti-DNA
antibodies?
Once initiated, the anti-DNA response is unlikely to be limited by the lack of antigen, since DNA
173
VIEWPOINT: ANTI-DNA ANTIBODIES
Table 2.
Characteristics of human IgG monoclonal anti-DNA antibodies*
Antibody
Origin
H2F
32.B9
33.Hll
33.F12
35.21
19.E7
1-2a
SD6
T14
2A4
33.c9
KS3
D5
SLE PBL
SLE PBL
SLE PBL
SLE PBL
SLE PBL
SLE PBL
SLE SPL
Healthy adult PBL
SLE PBL
Myeloma BML
SLE PBL
SLE SPL
SLE PBL
IgG
subclass
DNA
antigen
?
2
ds
ss + ds
ss + (ds)
ss + ds
ds > ss
ds > ss
ds
ds
ss + ds
ds
ss + ds
?
1
ss > ds
3
1
1
2
1
?
?
3
?
dS
VH
3
3
3
3
3
3
3
3
4
4
4
4
4
Germline
counterpart
VH26
VH26
VH35.21
V,3-8
HI1
56P1
56P1
?
VH4.21
V4.71-2
V4.4B
?
Vu4.21
D
JH
L
Germline
?
4
6
4
3
4
6
4
KIV
AVIII
NI
KIIIb
A
KIII
Vk4
AVIII
?
A11
KIIIb
DLW
?
DNI
DMV2
DHFL16
?
?
DXP’1
DM2
?
?
?
4
6
3
?
5
K1
KI
KI
A11
KIIIa
?
kv325
?
vg
DILpl
A11
kv325
DILpl
hk102
A11
K
Ref.
48
57
51
51
51
51
48
58
59
60
60
59
54
* See Table 1 for definitions of abbreviations.
antigen (with or without histones) is present in normal
individuals and is increased in patients with SLE.
Thus, the negative signals that are seen in immune
responses to infectious agents brought about by the
elimination of the foreign antigens would not occur in
the anti-DNA response. It seems more likely that
abnormal immunoregulation leads to the persistence of
the immune response toward DNA.
It seems certain that T cell drive must also be
participating in the persistent production of anti-DNA
antibodies. Linker-Israeli and colleagues (42) have
proposed that in lupus patients, CD8+ T cells directly
stimulate B cells to produce antibodies, rather than
suppressing CD4+ T cell stimulation. Thus, disordered T cell-B cell interaction leads to uncontrolled
production of antibodies.
Evidence from mouse models shows that persistence of anti-DNA reactivity may be due to faulty
apoptosis of B cells. Strasser et a1 (43) have used a
transgenic model in which a single dysregulated gene,
bcl-2, capable of enhancing B cell life span when
expressed in B cells, provoked hypergammaglobulinemia, high titers of anti-DNA antibodies, and widespread systemic autoimmunity in nonautoimmune
mice. This implies that the rapid turnover of B cells in
the normal mouse prevents the development of autoimmunity. In the first of what is likely to be a flood of
reports, Graninger (44) has described the overexpression of bcl-2 messenger RNA in unstimulated peripheral blood lymphocytes from 19 of 24 SLE patients
tested. The overexpression of the bcl-2 gene was still
more pronounced in patients with active SLE. It
would be interesting to determine whether this increased expression was in B cells or T cells. Studies in
our own laboratory of more than 70 SLE patients,
however, have not confirmed this bcl-2 overexpression (Rose, Isenberg, Latchman: unpublished
data).
Watanabe-Kukunaga and colleagues (45) have
shown that mice carrying the lpr mutation have defects
in the Fas antigen gene. This protein, which is encoded by this gene, is important in cell apoptosis.
Thus, autoreactive T cells are not deleted as a consequence. An abnormality of B cells in lpr mice has also
been found by Nemazee and coworkers (46), who have
postulated that faulty tolerance induction also lies at
the level of the B cell as well as the T cell in this model
of lupus.
One apparent contradiction seems to be emerging. On the one hand, investigators have found increased amounts of nucleosomes, a product of apoptosis, in SLE; on the other hand, evidence of the
prolongation of the life of immune cells and the prevention of apoptosis has emerged. This dichotomy
could be resolved either by postulating that the cells
undergoing apoptosis are not lymphocytes or that
there is a much-increased production of lymphocytes
and that their destruction is mediated by immunoregulatory cells in their attempt to suppress the autoantibody response.
What information and messages can we glean
from sequence analysis of human anti-DNA
antibodies?
In Tables 1 and 2 and in Figure 1, the established characteristics of the human monoclonal anti-
Germline Gene1 lsotype
Monoclonal
VH26
1812
!xP
819.7
H2F
32.89
VH26
1812
R3p
819.7
H2F
32.89
56P1
Ill-3R
19. €7
1- 2a
56P1
I l l-3R
I S . E7
1- 2a
F R3
CDR3 / J H
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
IQM _ _ _ _ _ _ _ _ A _ _ S _ _ _ _ _ _ _ _ _ _ _ - _ _ - _ _ _ _ _
GRMWERWFGESPPFDYWGQGTLVTVSS
IgG _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
EINCYGGFHYYNYGMDVWGQGTTVTLS
I@ _ L _ _ _ _ _ S A _ T S _ _ _ _ S _ _ _ _ _ _ _ _ _ _ _ _ _ _ S P G K V E K W E L P F D Y W G Q G T L V T V S S
S
JH4
JH6
J H4
VH4. 21
RT79
D5
T14
VH4. 21
RT79
D5
T14
IgM
lgG
lgG
FR3
RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
C D R 3 IJ H
________________________________
VRRSGRVVVPAAPRNRDAFDI WGQGTMVTVSS
_ _ I M _ _ _ _ _ _ _ _ _ - _ N _ T _ _ _ _ _ _ _ _ _ _ F - _ _ _ _ _ _ _ _ _ _ _ _ _ - _ _ _ _ R _ A - _ _ - _ _ _ _ - _ _ -
GAPNFGNYYKARRGWFDPWGQGTLVTVSS
GWPYYYGAGSYYKRGYFDYWGQGI RVTVSS
JH3
J H5
JH4
VH4 71- 2
BEG 2
2A4
VH4 71- 2
BEG 2
2A4
F R3
R V T l S V D T S K N Q F SL K L S S V T AADT AVY Y CAR
C D R 3 IJ H
IgM
________________________________
I&
_____I__________________________
QSNWFDPWGQGTLVTVSS JH5
D S I MGEl A R G P R A K G Q G Y G M D V W
~~
GQGTTVTVSS
JH6
6-1G1
L16
MLl
A10
A431
6-1G1
L16
ML1
A1 0
A431
Figure 1. Comparison of the germline gene sequences and representative amino acids constituting human IgM and IgG monoclonal anti-DNA
antibodies. Key points to note when studying these sequences are as follows: 1) The distribution of replacement mutations within the
complementarity determining regions (CDRs) is evidence of an antigen-drivenresponse. 2) The basic amino acids that are present in the CDRs
have been shown to be important in DNA binding. Winkler et a1 (57) found a balanced distribution of basic amino acids throughout the CDRs;
however, RT79 and D5, which use the germline gene V 4 . 2 1 , have a concentration of arginine residues within the CDR3 region. 3) The length
of the CDR3 regions; anti-DNA antibodies tend to have D-D fusions, resulting in extended CDR3 regions. FR = framework region.
174
VIEWPOINT: ANTI-DNA ANTIBODIES
DNA antibodies of which we are aware are compared
(47-6 1).
Although many hybridoma-derived IgM antibodies to ssDNA or dsDNA have been described
(Table l), it has been difficult to immortalize IgG
anti-DNA-secreting B cells from SLE patients. A
limited number of IgG anti-DNA antibody sequences
have been published from which to perform a valid
sequence/structure analysis (Table 2). As more information becomes available, it should be possible to
determine with greater accuracy whether individuals
with SLE have a set of immunoglobulin V genes that
are different from those who do not, and if there are
differences between anti-DNA antibodies derived
from different sources. However, it is already clear
from the data shown in Table 1 that IgM DNA antibodies may utilize elements of any of the 6 wellestablished V, families and that the choice of light
chain, K or A, is not critical. In contrast, it appears that
IgG anti-DNA antibodies have thus far only been
found to be encoded by members of the V,3 or vH4
families.
Among the vH4 family members, VH4.21 has
excited much interest (for review, see ref. 61). It was
initially found to be utilized by cold agglutinins synthesized as monoclonal antibodies by B cell tumors
(62). Probing for the utilization of VH4.21 has been
greatly facilitated by the observation that this gene
encodes for an idiotope (defined as a structure on the
variable region of the heavy or light chain of an
immunoglobulin), designated 9G4, for which a rat
monoclonal antiidiotypic antibody is available (62).
Subsequently, it was shown (59) that T14, a monoclonal anti-DNA antibody, has 94.8% homology with
V,4.21. In our own study (54), we found that the
presence of several arginine and lysine residues in the
CD3 region (see Figure 1) was important for enabling
the antibodies RT79 (an IgM) and D5 (an IgG), both
derived from V,4.21, to bind DNA. A panel of other
V~4.21-deriVed antibodies which lacked these residues in CDR3 were cold agglutinins without DNA
binding ability (54). Arginine residues have also been
shown to play an important role in determining the
specificity of anti-DNA antibodies from MRLllpr and
NZBNZW mice (63).
The particular importance of arginines in CDR3
for DNA binding has recently been confirmed by
Radic et a1 (64). Using in vitro mutagenesis experiments, they showed that substitution of an arginine on
the CDR3 heavy chain by a glycine resulted in the loss
of DNA binding capacity of the parent antibody 3H9.
This change in reactivity may have been due to a
175
CDR3 conformation that interferes with DNA binding
at the combining site of VH3H9due to loss of positive
electrostatic potential in the combining cleft of the
glycine substitution variant.
The light chain of antibody T14 (59) has 98.6%
homology with a germline VJIIb member kv325. The
VH4 and VJIIb genes expressed by the monoclonal
T14 differ from their respective germline counterparts
by 14 and 5 nucleotides, respectively. A nonrandom
distribution of silent and replacement mutations was
found. Such a distribution of mutations within an
antibody V region is consistent with a process of Ig
receptor-dependent stimulation and selection of mutations (64,65). Only 2 replacements were found within
the framework regions. Four of 10 replacement mutations resulted in arginine or asparagine residues.
Six IgG monoclonals reacting with dsDNA
were described by Winkler et a1 (57). These were
derived from 3 SLE patients. These antibodies were
shown to be of high affinity, and limited polyreactivity
was observed. The V region genes used in these
monoclonals were somatically mutated. The pattern of
mutations was suggestive of an antigen-driven response in at least 4 of these antibodies. Five of the 6
antibodies use vH3 gene segments and 1 a vH4 gene.
Interestingly, 2 of the monoclonals used V, genes
which code for IgM natural antibodies. Of the 35
mutations in the CDR regions, 15 yielded arginine,
asparagine, or lysine residues. However, these mutations did not show the bias toward CDR3 described
above for V,4.21. There was in fact a balanced
distribution of these basic amino acids over the CDRl ,
CDR2, and CDR3 regions of the heavy and light
chains, suggesting a broader contribution of all CDR
regions for DNA reactivity.
Dersimonian et a1 (47) highlighted the importance of the VH26 member of the vH3 family. They
isolated 6 IgM anti-DNA antibodies, 5 derived from an
SLE patient and the sixth from a patient with leprosy.
Thus, 4 of the SLE monoclonals described, including
18/2 and 1/17, carried the 16/6 idiotype (a collection of
idiotopes), levels of which have been found to be
elevated in the sera of patients with active lupus (66).
The nucleotide sequences of these antibodies were
found to be identical and had 99% homology with the
germline gene segment VH26. The third SLE monoclonal, 21/28, was found to have 93% homology to the
vH1 germline gene segment HA2 and was idiotypically
related to 8E10, a monoclonal antibody derived from a
patient with leprosy. Monoclonals 21/28 and 8E10
shared 100% homology at the nucleotide level, thus
176
showing strong evidence of the conservation of this
germline gene.
Sequence analysis of an anti-DNA IgM monoclonal, POP, from a patient with chronic lymphatic
leukemia and peripheral neuropathy (50), has shown
96% homology with germline gene VH26 and 99%
homology to a VJIIa germline gene (67). This antibody has serine-phenylalanine at positions 31 and 32
instead of the serine-tyrosine found in an IgM antiDNA (ss and ds) C6B2, which was derived from a
6-month-old without autoimmune disease. This replacement could explain why POP monoclonal binds
to ssDNA only.
B19.7 (51), a 16/6 idiotype-positive IgM antissDNA of fetal origin, was compared to 18/2 because
its binding to ssDNA was inhibited by the 16/6 antiidiotype. The sequences, however, had no homology
in the CDR3 region and the antibodies used different
light chains, B19.7 using a A and 18/2 using a K chain.
The results showed that antibodies with identical VH
regions and expressing the same idiotype may have
different DNA binding sites. These differences prevented the inhibition of ssDNA binding by the 16/6
antiidiotype in B19.7.
Among the other VH3-derived antibodies, Kim
4.6, an anti-ssDNA IgM monoclonal (from a normal
healthy adult) had 100% homology with the germline
V, gene segment VH9.111 and 98% homology with a
V, gene segment expressed in a patient with Burkitt’s
lymphoma (52). Kim 4.6 contains a region of 25
nucleotides which matches the germline segment
DXP’ 1. On alignment with other anti-DNA antibodies,
it was found that a conserved core of 16 bp, identical
with a region of DXP’ 1, occurred in many of them. A
4-amino acid stretch tyrosine-tyrosine-glycine-serine
is encoded with this 16-bp region and appears to be
conserved in human anti-DNA antibodies. The utilization of this 16-bp core in the CDR3 of anti-DNA
antibodies points to the importance of the CDR3
residues in defining DNA binding specificity.
Studies by Logtenberg et a1 (55) revealed that
antibodies encoded by the V,6 gene family display
binding patterns reminiscent of autoantibodies present
in the sera of SLE patients. Nucleotide analysis reveals that the CDR3 region is of conserved length. The
VH6-encoded autoantibodies were found to bind
ssDNA. It was believed that binding was not simply
charge-related, since these antibodies did not bind to
similar negatively charged molecules, such as RNA.
Comparison of the sequences of the VH6 genes expressed by the fetal lines L16 and ML1 to germline
gene 61G1 revealed 100% homology. This germline
ISENBERG ET AL
gene has been isolated from unrelated individuals and
demonstrates that VH6 is highly conserved in the
population.
Monoclonals A10 and A431, derived from normal adult lymphocytes, differed from the prototype
VH6germline by 6 and 3 nucleotides, respectively (56).
Eight of 9 nucleotide differences were concentrated in
the CDRl and CDR2 regions, and resulted in amino
acid replacements. This pattern of nucleotide substitutions suggests somatic mutation. Further studies of
these antibodies showed that A10 and A431 had undergone somatic mutation in the absence of class
switching.
Eight anti-dsDNA antibodies were described by
Mannheimer-Lory et al (48). These antibodies carry
the 31 idiotope which is K light chain associated. As
described above, it has been reported that many DNA
binding antibodies are enriched for arginine, asparagine, and glutamine residues (63-65,68-70). A random
sampling of V,1 chains showed an average of 2.8 of
these residues in CDR1. The V, chains of the 8
monoclonals described contain an average of 3.75 of
such residues within the CDRl region.
In summary, there appears to be no V, restriction within the IgM anti-DNA antibodies. All V,
families are represented. In contrast, the IgG antiDNA antibodies which have been described appear to
use only members of vH3 or V,4. This may only be a
reflection of the limited number of IgG monoclonals
available for study, The K and A light chain isotypes
are equally represented in these antibodies, and no
particular V, or V, member is predominant.
It is now established that both natural and
lupus-derived anti-DNA antibodies can be encoded by
germline genes without somatic mutation. However,
somatic mutation may well be the event which distinguishes natural antibodies from those which are pathogenic. Thus the V, sequence of BEG-2, an IgM
anti-ssDNA antibody of human fetal origin (53), was
found to be very similar to that of an IgG DNA-binding
antibody, 2A4 (60). The sequences differ by 15 bp,
specifying 8 amino acid changes. These mutational
events lead to enrichment of arginine, asparagine,
lysine, and tyrosine residues, which have been noted
to be important in DNA binding in mouse model
systems. It is clear, however, that some antibodies can
still bind DNA without enrichment of these residues.
The information gleaned from the available sequences
points to the existence of a variety of mechanisms that
can result in the “acquisition” of DNA binding potential.
VIEWPOINT: ANTI-DNA ANTIBODIES
What is known about the epitopes that antiDNA or anti-DNMhistones actually
recognize?
The nature of the epitopes that are bound by
anti-DNA antibodies is only partially elucidated. A
range of specificities is attributed to these immunoglobulins. Monoclonal antibodies and sera from SLE
patients and lupus-prone animal models show that
reactivity may be greater toward denatured forms of
DNA than to the fully helical form (71). Anti-DNA
antibodies may react directly with nucleosides and
nucleotides, with some antibodies exhibiting marked
preference for particular basesbase pairs. Thus, reactivity for poly(dA-dT), poly(dG-dC), poly A, poly
(dT), poly(d[BrU]) with and without ssDNA activity
has been reported (71-76), as well as for poIy(ADPribose), a polymer that is produced in response to
certain forms of DNA damage (77). Additional specificities include other forms, such as ssRNA, dsRNA,
triple-helical RNA, RNA.DNA hybrids, and of
course, dsDNA, which can occur in a supercoiled or
relaxed form, depending on the physicochemical conditions of the environment (30).
Although the exact nature of the epitopes
bound by anti-DNA antibodies is not known, DNA of
various lengths has been isolated from the sera of SLE
patients, especially those with vasculitic and central
nervous system involvement (78). Using plasmapheresis
fluids from SLE subjects, Krapf et a1 (79) demonstrated
the presence of nucleic acids, approximately 20kilobases long, which were found to be immunogenic in
rabbits. These animals were injected with the isolated
nucleic acid, and the resultant antiserum reacted not
only with the immunogen, but also with dsDNA. It has
been suggested that the higher-than-expected GC content of these isolated DNA fragments may contribute
to enhanced antigenicity since Z-DNA formation may
become favored (79,80).
Given that anti-DNA antibodies may exhibit
polyreactivity, it is believed that at least some of the
epitopes bound by these autoantibodies must be
present in the sugar phosphate backbone common to
all polynucleotides. The backbone consists of phosphate groups in a phosphodiesterase linkage, separated by 3 carbon atoms of adjacent sugar molecules.
A similar arrangement is found in phospholipids,
which probably explains the cross-reactivity of antiDNA antibodies with cardiolipin (81). Exactly how the
binding might be mediated by these autoantibodies has
been examined for very few immunoglobulins. It has
been postulated that components of both the major
177
and minor grooves are involved in binding to dsDNA,
and for particular polynucleotides, antibodies vary in
the degree of binding to bases and the backbone.
Examining an IgG murine monoclonal antiDNA antibody raised from an MRL-lpr/lpr mouse
spleen, Stollar et a1 (71) reported a high selectivity for
oligonucleotides containing G and C residues, but not
for A and T basepairs. To explain the binding of their
antibody, it was suggested that this would happen if
the antibody binding site straddled one turn of helical
DNA, binding to components in both the major and
minor grooves of the molecule as well as to the sugar
phosphate backbone. Similarly, Cygler et a1 (76) described antibody HED10, a murine monoclonal that
not only bound strongly to poly(dT) and poly(d[BrU])
with recognition of 4 consecutive bases, but the interaction also involved phosphate groups. In another
study of an ssDNA-reactive antibody (BV04-Ol), however, phosphate groups were not thought to play a
significant role in the binding (75).
It is highly probable that anti-DNA binding is
charge related. Variable region gene analysis of monoclonal anti-DNA antibodies has indicated that highly
basic amino acids such as arginine and lysine may be
important in the binding to a complex polyanion such
as DNA. Such amino acids most likely form hydrogen
bonds with the phosphate groups on the nucleic acid.
Similar interactions may also occur at the glomerular
basement membrane, where anti-DNA antibodies are
found to bind directly to other negatively charged
polymers (82).
As discussed above, the initial stimulus for
anti-DNA production remains a matter of speculation.
Histones, in particular core proteins, are also regarded
as poor immunogens, and yet it is often observed that
in patients with anti-DNA antibodies, antihistone antibodies also occur (83,84). It has been found that as
DNA complexed to proteins becomes immunogenic,
so histones acquire enhanced antigenicity when linked
to nucleic acids. It has therefore been suggested that
the initiating antigen for the production of these autoantibodies may be the nucleosome (85). DNA is found
in chromatin in close association with histones. There
are 2 main classes of these small basic proteins, the
core, or nucleosomal, histones (H2A, H2B, H3, and
H4) and the more variable linker protein histone H1.
Arginine-rich H3 and H4 form a tetramer, which is
flanked by a dimer of lysine-rich H2A and H2B. Two
turns of DNA are then wound around these subunits,
and the strands of DNA are bound by H1 to other
so-formed units, producing a string of nucleosomes
which make up the chromatin (86).
178
While it is known that antibodies bind the complexes of DNA and histones, the epitopes to which they
bind are not well described. However, reactivity to
individual histones, particularly in drug-induced lupus
(84), has been well characterized. Antihistone immunoglobulins are well documented in this variant of
disease, and while reactivity to all members of the
histone family can occur, especially to H1, particular
specificities may prevail in lupus, depending on the
inducing drug. In symptomatic patients with procainamide-induced lupus, reactivity to H2A-H2B-DNA
complex is prominent (87). In hydralazine-induced
disease, Portanova et a1 (88) reported antibodies to H3
and H4 histones.
The epitopes which incite antibody production
have been examined using both Western blotting and
epitope mapping techniques (for review, see ref. 86).
In both idiopathic and drug-induced disease, epitopes
present mainly in the carboxyl region (amino acids
123-220) have been described for H1 (89,90). This is
also reported for H5, a variant of H1 which is frequently found in nonmammalian species (90). Epitope
mapping has also recently indicated some areas of
reactivity in the globular region. Similar studies of
H2B and H2A have indicated that the amino terminus
bears the appropriate epitopes. For the former, these
are ranged in the peptide 1-20, and for the latter,
peptides 1-1 1 and 119-129 are thought to be important.
For H3 the pertinent peptides appear to be
present in both the amino (peptides 1-26) and carboxyl
(peptides 13&135) regions. For H4, amino acids 1-29
are considered important. Since some of the antihistone antibodies may be directed against the DNA
binding sites of these proteins, it has been suggested
that such antibodies may be considered antiidiotypic
for anti-DNA antibodies (91), and perhaps this is one
way in which these may arise.
Conclusions
The prominence of antibodies to DNA, especially dsDNA, in most lupus patients continues to
puzzle and provoke. Given that the link between them
was made in 1957 (for review, see ref. 27), it is
frustrating that the questions posed in this review have
not yet been fully answered. However, it remains a
central dogma for most lupus researchers that understanding the origins and consequences of anti-dsDNA
formation is a vital component of comprehending and
controlling the disease itself. As we have discussed,
there are many intriguing developments taking place in
DNA antibody research, which both tantalize us and
ISENBERG ET AL
offer clues to our understanding of the enigmas that
surround these fascinating immunoglobulins.
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