British Journal of Rheumatology 1988;27:431-435
A COMPARISON OF AUTOANTIBODIES AND COMMON DNA
ANTIBODY IDIOTYPES IN SLE PATIENTS AND THEIR SPOUSES
BY D. A. ISENBERG, W. WILLIAMS, S. LE PAGE, G. SWANA*, R. FELDMANt,
I. ADDISON, R. BAKIMERJ AND Y. SHOENFELD*
'Immunology Department, St. Thomas' Hospital; ^Departments of Rheumatology Research and Medicine,
University College and The Middlesex Hospital Medical School; tDepartment of Medicine 'D' & Unit for
Autoimmune Disease Research, Soroka Medical Centre, Ben Curion University, Beer Sheeva, Israel
KEY WORDS:
Genetic influence. Familial influence, Autoimmunity, Anti-DNA antibodies, Anti-K30
antibodies.
lupus erythematosus (SLE) is an
autoimmune disease characterized by a wide
variety of clinical features and a broad spectrum
of autoantibodies [1]. The aetiopathogenesis of
SLE remains uncertain though it is clearly
multifactorial with genetic, hormonal, dietary
and other environmental influences [2-5]. Most
studies of aetiopathogenesis have focused on
genetic and hormonal abnormalities in patients
themselves. If environmental influences such as
viruses are important, it is reasonable to ask
whether those who share the same environment
might also develop similar sets of autoantibodies: in effect to examine the other side
of the coin from the healthy lupus relatives who
have been found to have antibodies to single
stranded DNA [6], cardiolipin [7] and poly
(ADP-ribose) [8] amongst others.
In this study we have analysed the sera from
20 matched pairs of SLE patients and their
spouses. Antibodies to DNA, poly(ADPribose), cardiolipin, the extractable nuclear
antigens (Sm, RNP, Ro, La) and rheumatoid
factor were all determined. In addition, using
polyclonal rabbit anti-idiotypic antibodies, the
sera were examined for the presence of three
SYSTEMIC
Submitted 7 January; revised version accepted 28
March 1988.
Address for correspondence: Dr. David Isenberg, The
Bloomsbury Rheumatology Unit. The Department of
Rheumatology Research, University College and The
Middlesex Hospital Medical School, Arthur Stanley
House, Tottenham Street, London W1P9PG.
common idiotypes first identified on anti-DNA
antibodies.
METHODS
The 20 SLE patients studied each met four
or more of the American Rheumatism
Association's revised criteria for the
classification of the disease [9]. Nineteen of the
patients were female and one was male. The
duration of the disease ranged from 6 months to
15 years, mean 4.8 years. The major disease
manifestations in these patients (cumulative
historical rather than necessarily current) were:
renal disease n=4; disease of the central
nervous system n=2; serositis n=6, joint and
skin manifestations only «=7; thrombocytopenia n = \. The criteria used to determine the
involvement of these different systems are
described elsewhere [10]. In each case the
spouse studied was in good health, had no personal history of autoimmune disease and had
lived with the patient prior to the onset of their
lupus (mean=8 years, SD=5.5, range 1-18
years).
The presence of antinuclear antibodies was
tested using serial dilutions of serum on a
human epithelial cell line (HEp-2) and antidouble stranded (ds) DNA antibodies by using
the Amersham kit. Values greater than 25 U/ml
were regarded as positive (as per manufacturer's recommendation). Anti-poly (ADPribose) antibodies were estimated by an ELISA
method [8]. Anti-cardiolipin antibodies were
assayed as follows.
431
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SUMMARY
In order to determine whether environmental influence per se might influence autoantibody production, sera
from the spouses of 20 SLE patients wereexamined. No antibodies to cardiolipin, poly (ADP-ribose), or ENA
were detected and none had detectable rheumatoid factor. One weakly positive ANA reaction was noted, one
had anti-DNA antibodies (by RIA and ELISA) and in two sera the common DNA antibody ldiotype 16/6 was
found. The idiotype was not, however, present on either anti-DNA or anti-K30 antibodies. Although longterm analyses arc required, it is evident that sharing the same environment with patients who commonly
express a wide range of autoantibodies and common idiotypes-rarely leads to their expression in nonautoimmune subjects
432
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXVII NO. 6
Serum samples from two spouses found to
have raised 16/6 ID levels were diluted 1:100
(for the anti-DNA ELISA) and 1:40 (for the
anti-K30 test) and an equal volume of inhibitor
added to give a final dilution of 1:200 or 1:80.
The inhibitors used were single-stranded DNA
at concentrations of 5 /J-g/ml, 2.5 Mg/ml and 1
/Mg/ml; and K30 antigen at 10 Mg/ml, 5 fig/m\
and 1.25 /xg/ml. PBS was used as a control. The
mixtures were incubated for 30 min at 37°C and
then overnight at 4°C, and were tested in standard ELISAs [14, 15] for IgM and IgG
antibodies to single stranded DNA and K30.
RESULTS
A full comparison of the autoantibody and
idiotype profiles in the SLE patients and their
respective spouses is shown in Table I. The
results in Table I are expressed in +/— form for
simplicity, but the tests of the SLE spouses
were overwhelmingly negative (full details of
the results are available from the authors). One
spouse had a positive ANA reaction (1:40 only)
but two husbands had raised 16/6 idiotype
levels, in one case (spouse of CW) this was
accompanied by a raised anti-dsDNA antibody
level (Amersham kit, 36 U/ml). This serum was
also positive for anti-single stranded and
dsDNA antibodies by ELISA when tested
(using a method described recently) [15],
though negative by Chthidia. No clear temporal
relationship between either disease duration or
length of time spent with the patient before
disease onset was evident when comparing
those few spouses with detectable antibodies/
idiotypes and the majority without. For
example, the spouse of DB (ANA positive) had
lived with his spouse for 2 years before her
lupus began, whereas the spouses of JA and
CW (16/6 ID positive) had spent 7 and 15 years
respectively.
The sera from the spouses of lupus patients
CW and JA, found to be 16/6 ID positive, were
also found to contain IgG antibodies binding
K30 (66% and 84% respectively — upper limits
of normal = 50% of a known high positive). As
the JA spouse's serum also bound DNA we
tried to determine, by absorption experiments,
whether the 16/6 ID was present on anti-DNA
or anti-K30 antibodies. The results of these
experiments are shown in Table II. It is evident
that whilst absorption with DNA and K30
effectively removed most of the antigen binding
activity, the 16/6 levels were not affected. Thus
the idiotype must be present on antibodies with
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Dynatech Immulon II plates were coated
with 50 /xl/well of cardiolipin (Sigma, 100 ^ig/
ml). The cardiolipin was diluted in absolute
alcohol and allowed to evaporate overnight at
4°C. The wells were blocked for 2 hours with
100 fx\ 10% fetal calf serum (FCS) in phosphate
buffered saline (PBS). The plates were washed
three times with PBS and 50 fi\ of test sera were
added in duplicate, diluted 1:50 in 10% FCS.
After a 2-hour incubation and further washing
(three times with PBS) alkaline phosphatase
linked goat anti-human immunoglobulin
(Sigma, 1:1000 in 10% FCS) was added for a 2hour incubation. The plates were again washed
three times with PBS. 50 /xl of substrate was
added to each well and incubated for 40 min at
37°C. The plates were read on a Dynatech 580
ELISA reader at 405 nm. On each plate six
normal sera were included and a known high
positive control in quadruplicate. All the results
were expressed as a percentage of a high
positive run in quadruplicate on each plate. The
upper limit of normal (> mean ±2 SD of 30
healthy controls) for IgG anticardiolipin was
35% and for IgM 25%.
Rheumatoid factor was measured by
standard Latex test and positive dilutions of
1:40 or greater were regarded as positive.
Counter immunoelectrophoresis was used to
determine the presence or absence of
antibodies to Sm, RNP, Ro and La.
The 16/6 and 134 DNA antibody idiotypes
were first identified on human hybridoma
derived monoclonal antibodies [11]. The
lymphocytes used in these fusions were taken
from unrelated patients with SLE. In contrast
the PR4 idiotype was first identified on a human
hybridoma derived antibody from a patient
with leprosy [12]. The 16/6 and 134 idiotypes
were detected by previously described ELISAs
in which the test serum was initially diluted on
the plate and the anti-idiotypic antibody added
[8, 13]. The PR4 idiotype was measured by an
ELISA coating the anti-idiotypic antibody onto
the plate, then incubating with the test sera and
then adding an alkaline phosphatase linked
anti-human Ig. A normal range was established
(> mean ± 2 SD) using 32 healthy controls.
Full details of the assay are described elsewhere
[14].
In order to try and determine a fuller antigenbinding profile of two sera from lupus spouses
found to be 16/6 idiotype (ID) positive, their
binding to the Klebsiella antigen K30 (anti-K30)
was measured as previously described [14].
ISENBERG ETAL.: AUTOANTIBODIES AND DNA ANTIBODY IDIOTYPES 433
different antigen binding capacities in these
sera.
DISCUSSION
Whilst it may be tempting to use these data as
a means of 'discrediting' the influence of local
environmental factors on autoantibody
production, and possibly aetiopathogenesis as a
whole, this is premature. The patients had a
broad cross-section of lupus manifestations and
TABLE I
RESULTS OF LUPUS PATIENTS AND THEIR SPOUSES BINDING TO THE AUTOANTIGENS TESTED, AND THE COMMON
ANTIBODY IDIOTYPES DETECTED
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25"
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Patienty
spouse
ANA
RF
JA(F)
1:256 1:1056
Spouse
MM(F)
1:320 1:160
Spouse
JS(F)
1:2560 1:1280
Spouse
MC(F)
1:280
Spouse
FL(F)
1:32
Spouse
PC(F)
1:10240 Spouse
BMS (F) 1:1280
Spouse
JT(F)
1:40
Spouse
HN(F)
1:1280
Spouse
1:640
NV(F)
Spouse
GH(M)
1:160
Spouse
DB(F)
1:320
Spouse
1:40
MJ(F)
1:160
Spouse
MS(F)
1:2560 1:256
Spouse
CW(F)
1:160
Spouse
CBh (F) 1:1280
Spouse
AT(F)
1:320
Spouse
CBr(F)
1:640
Spouse
BK(F)
1:80
Spouse
MW(F)
1:640
Spouse
-
AntiAnti-DNA Anti-poly cardiopilin
antibodies (ADP-nbose) antibodies
ENA
Ro
La
-
+
Idiotypes
Sm RNP
-
+
16/6
134
PR4
-
+
-
+
+
+
ND
-
+
+
+
i
+
-
+
+
+
-
+
-
+
+
Results are generally given as + or - as most of the spouses tested were negative in every assay. Absolute values are
available from the authors. Anti-DNA results refer only to the Amersham kit method looking for antibodies to double
stranded DNA. F=Female, M=Male, ND = Not done.
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1
2
DNA
434
BRITISH JOURNAL OF RHEUMATOLOGY VOL. XXVII NO. 6
TABLE II
THE LEVELS OF IGG AND IGM SINGLE STRANDED DNA ANTIBODIES, IGM ANTI-K30 ANTIBODIES AND 16/6 ID PRE- AND
POST-ABSORPTION WITH DNA AND K30
IgG anti-DNA
antibodies
Pre
701
424
(40%)
Pre
246
Post
144
(51%)
IgM anti-K30
antibodies
16/6 ID level
Pre
Post
Pre
313
27
(93%)
45
(90%)
663
453
544
Post DNA
absorption
617
(-)
Post K30
absorption
526
(21%)
535
(2%)
The JA spouse's serum did not bind DNA and neither JA nor CW spouse's serum had IgG anti-K30 antibodies The
post-absorption figures (percentage inhibition is given in parentheses) represent the maximum inhibition found, with 5
/xg/ml ssDNA and 10 jig/ml K30 respectively. The figures given are the OD x 10'.
disease activity (data not shown) but serial
studies of both patients and their household
contacts will be required to clarify the
environmental influences. To date, conflicting
data on the balance of environmental and
genetic influences have been reported.
Lymphocytoxic antibodies in 57% of SLE
family members and in 68% of the close
household contacts have been reported [16].
The corresponding figures for anti-RNA
antibodies were 21 and 0% respectively [17].
No major difference in respect of DNA
antibodies was found. An increased incidence
of immunoglobulin and properdin at the
dermal-epidermal junction in normal skin of
first degree household contact relatives and
spouses
supports
the importance of
environmental factors [18]. However, amongst
the relatives with raised ANA titres no
distinction between household and nonhousehold contacts was found, implying that
genetic factors are more important. Future
studies will need to examine the patients and
several
household contacts at initial
presentation with follow-up assessments of the
contacts as well as the patients.
It has been shown by absorption experiments
in other reports that the 16/6 ID is present on
anti-DNA antibodies in SLE [19] and anti-K30
antibodies in individuals with Klebsiella
infections [14]. We could not identify the 16/6
ID on either of these antibodies in the two sera
from lupus spouses who had high 16/6 ID levels.
The antigen binding specificity of the antibodies
bearing the 16/6 ID in these two individuals
remains unknown.
The detection of two spouses with raised 16/6
levels was of interest although the number
studied was small. We reported previously that
4-5% of the normal population are 16/6
idiotype positive. The sharing of idiotypes is
often thought to represent sharing of common
germ-like genes, though the antigen binding
specificities of antibodies with the same
idiotype are not necessarily identical.
ACKNOWLEDGEMENT
We gratefully acknowledge support from the
Arthritis and Rheumatism Research Council and the
Daniel Falkncr Charitable Trust.
REFERENCES
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ISENBERG ETAL.: AUTOANTIBOD1ES AND DNA ANTIBODY IDIOTYPES 435
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NOTICE
2ND FRENCH CONGRESS OF RHEUMATOLOGY
Xllth Symposium of the French Society of Rheumatology
Dates: 14,15 March 1989.
Venue: Centre International de Conferences de la Cite des Sciences et de l'lndustrie de la Villette
(La Villette International Conference Centre of the City of Science and Industry).
Symposium will comprise plenary sessions on selected subjects and parallel sessions with
introductory lectures by invited speakers, oral presentations of papers and poster exhibits.
Registration fees: members of the French Society of Rheumatology, 400 francs; non-members of
the French Society of Rheumatology, 600 francs; residents, students preparing specialization,
rheumatologists with under 2 years' practice (documented proof requested), 200 francs.
Further information: Secretariat de la Societe Franchise de Rhumatologie, Service de
Rhumatologie, Hopital Henri Mondor, 51, Av. du Mai de Lattre de Tassigny, 94010 Creteil,
France. Tel: (16 1) 49 81 21 11, extension 12709.
Downloaded from http://rheumatology.oxfordjournals.org/ at Pennsylvania State University on May 12, 2016
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High incidence of anticardiolipin antibodies in
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Dudeney C, Shoenfeld Y, Rauch J, et al. A study
of anti-poly (ADP-ribose) antibodies and an
anti-DNA antibody idiotype and other immunological abnormalities in lupus family members.
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revised criteria for the classification of systemic
lupus erythematosus.
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Parry HF, Snaith ML. Useful laboratory
measurements in the management of systemic
lupus erythematosus. Q J Med 1982;52:125-38.
Shoenfeld Y, Rauch J, Massicotte H, et al. Polyspecificity of monoclonal lupus autoantibodies
produced by human-human hybridomas. N
EnglJ Med 1983;308:414-20.
Williams W, Zumla A, Behrens R, et al. Studies
of a common idiotype PR4 in autoimmune
rheumatic disease. Arthritis Rheum (in press).