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Evidence for detection of low molecular weight DNA В Эanti-DNA complexes in systemic lupus erythematosus.

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EVIDENCE FOR DETECTION OF
LOW MOLECULAR WEIGHT DNA-ANTI-DNA COMPLEXES
IN SYSTEMIC LUPUS ERYTHEMATOSUS
VINCENT AGNELLO and TADAYUKI MITAMURA
T h r e e immune complex assays, t h e monoclonal Rheum a t o i d F a c t o r i n h i b t i o n (mRF), t h e C l q s o l i d phase
(ClqSP) and t h e C l q f l u i d phase b i n d i n g assay (ClqBA)
were compared w i t h n a t i v e DNA (nDNA) a n t i b o d y t i t e r s
and serum h e m o l y t i c complement (CH50) l e v e l s i n s e r i a l
analyses o f p a t i e n t s w i t h systemic lupus erythenatosus
No C o r r e l a t i o n was e v i d e n t among immune complex
(SLE).
assays.
A p o s i t i v e c o r r e l a t i o n was observed between
t h e ClqSP and nDNA a n t i b o d y assays and a n e g a t i v e c o r r e l a t i o n was observed between t h e ClqSP a n d CH50 assays.
E v i d e n c e was p r e s e n t e d t h a t low m o l e c u l a r w e i g h t
DNA-anti-DNA complexes were d e t e c t e d by t h e ClqSP.
The
complexes d e t e c t e d b y ClqSP w e r e 8 s t o 19s i n s i z e and
some were s e n s i t i v e i n p a r t t o d e o x y r i b o n u c l e a s e t r e a t ment.
I n t h e p a s t t e n y e a r s a wide v a r i e t y o f immune
complex assays have been d e v e l o p e d and s e v e r a l have
been used t o s t u d y p a t i e n t s w i t h SLE.
Recently there
has been c o n s i d e r a b l e c o n t r o v e r s y c o n c e r n i n g t h e c l i n i c a l u t i l i t y o f t h e s e assays and t h e i r r e l a t i o n s h i p t o
t h e mare e s t a b l i s h e d s e r o l o g i c p a r a m e t e r s o f d i s e a s e
a c t i v i t y i n p a t i e n t s w i t h SLE, nDNA a n t i b o d i e s and
complement l e v e l s ( 1 - 3 ) .
C l i n i c a l Immunology Laboratory, Department o f Medicine
Tufts-New England Medical Center, Boston, Mass
02111
This study was supported by National I n s t i t u t e o f
Health Grant 5 R01 16984, an A r t h r i t i s Foundation C l i n i c a l
Research Center Grant and The Lupus Erythematosus Foundation
o f Boston
Arthritis and Rheumatisni. Vol. 25, No. 7 (.Id? 1982)
MATERIALS AND METHODS
The diagnosis and e v a l u a t i o n o f SLE were based on t h e
American Rheumatism Association p r e l i m i n a r y c r i t e r i a ( 4 ) and
previously reported c r i t e r i a f o r designating episodes o f
c l i n i c a l a c t i v i t y ( 5 ) . Total CH50 and nDNA antibody l e v e l s
were q u a n t i t a t e d as previously described (5). The mRF ( 6 )
and the ClqBA ( 7 ) were performed as previously described.
The ClqSP ( 8 ) was adapted t o p o l y v i n y l m i c r o t i t e r p l a t e s
(Cooks Laboratory, D i v i s i o n o f Dynatech Labs., Inc., Alexand r i a , VA).
'251 l a b e l l e d Staph p r o t e i n A was used i n place
D i l u t i o n s o f a standard
o f L751 anti-human immunoglobulin.
heat aggregated IgG preparation were determined. The m i n i mal concentration o f aggregate detected i n the mRF was
1 ug/ml, i n the ClqSP was 2 ug/ml and i n the ClqBA was
8 L,g/ml.
I s o k i n e t i c sucrose d e n s i t y g r a d i e n t u l t r a c e n t r i Co-efficient o f
fugation was performed as described ( 9 ) .
c o r r e l a t i o n was determined using Hewlett-Packard C a l c u l a t o r
9810A S t a t i s t i c a l Pack.
S i g n i f i c a n c e o f c o r r e l a t i o n coe f f i c i e n t s was determined using s t u d e n t ' s t - t e s t .
RESULTS
The c o m p a r i s o n of t h e t h r e e immune complex assays
w i t h nDNA a n t i b o d y t i t e r s and CH50 i n one s e r i a l s t u d y
o f a 1 4 - y e a r - o l d p a t i e n t w i t h SLE i s shown i n F i g u r e 1.
F i v e f l a r e s o f d i s e a s e a c t i v i t y were observed.
The
ClqSP showed t h e b e s t c o r r e l a t i o n o f t h e immune complex
assays.
The d a t a s u g g e s t a p o s i t i v e c o r r e l a t i o n between t h e . C l q S P and nDNA a n t i b o d y t i t e r s .
The c o r r e l a t i o n s o f t h e d i f f e r e n t assays a r e summarized i n
T a b l e 1. The o n l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n was
f o u n d between t h e ClqSP and nDNA a n t i b o d y t i t e r s ; a
n e g a t i v e c o r r e l a t i o n was f o u n d between ClqSP and CH50.
Second serum ( 7 / 1 5 / 8 0 ) w i t h h i g h ClqSP and nDNA
t i t e r s was s t u d i e d by s u c r o s e d e n s i t y g r a d i e n t u l t r a c e n t r i f u g a t i o n ( F i g u r e 2 ) . The peak ClqSP a c t i v i t y was
8s and was d e c r e a s e d a p p r o x i m a t e l y 30% b y d e o x y r i b o -
LOW MOLECULAR WEIGHT DNLANTI-DNA
0
.
789
1
(OOr
1
0
I
I
25
50
I
n
do
'O
XGR4drmfWLUM
Figure 2. 5-20% lsokinetic sucrose density gradient o f SP
serum 7/15/80 (see Figure 1). 0-0 shows protein d i s t r i bution i n the gradient 0 - 4 shows ClqSP results. The dlst r i b u t l o n o f I96 and I @ i n gradient was also determined
(only p a k s shown).
Peak Ig6 and ClqSP t i t e r occurred a t
the same point i n the gradient.
m
m
fsw
Figure 1. Serial study o f patient SP showing results o f
three innrne complex assays, mFR. ClqSP and ClqBA, nDM
a n t i k d y assay and 0150 assay. Mom1 l l n i t s a n shown by
the dashed line. Mom1 l i m i t f o r nDNA antibody was 2.5
vg/nl.
Clinical flares are Indicated by the arrows. The
nmber o f vertical lines i n the a r m I s proportional t o the
severity o f the flare.
nuclease treatment.
I n a similar study o f 10/30/80,
increased ClqBA i s shown i n Figure 3. This peak,
however, was r e l a t i v e l y low molecular weight, 14s and
was not sensitive t o deoxyribonuclease.
Correlation o f the ClqSP assays with nDNA antibody
and CH50 could be shown with most studies o f SLE sera
The kidney o f patient HP manifested heavy
(Table 2).
subendothelial g l m r u l a r deposits o f DNA containing
cqnplexes which ware detected i n association with
increased t i t e r s o f DNA antibody and decreased CH50
(10) (Figure 4). A sucrose density gradient ultracent r i f u g a t i o n o f HP serum (9/15/75) demonstrated l o w
molecular weight complexes by ClqSP (Figure 2).
No significant correlation was found between ClqSP
and nDNA antibody f o r patients BD and NC (Table 2). A
previous study (5) indicated that complement f i x i n g
nDNA antibody correlated with hypocomplementemia and
disease a c t i v i t y whereas non-complement f i x i n g nDNA
antibodies d i d not show a similar correlation. ND had
non-complement f i x i n g nDNA antibody, frequently i n high
t i t e r and the ClqSP was negative. There i s no correlat i o n between nDNA antibody and ClqSP. BD had i n t e r mittent elevated t i t e r s o f complement f i x i n g nDNA
antibody.
Correlation coefficients restricted t o
segments o f the s e r i a l study where complement f i x i n g
nDNA antibody were present, the correlation c o e f f i c i e n t
for ClqSP/ nDNA was increased t o 0.88, p < 0.02.
Table 1. SP Serlal study: correlation coefficients f o r innune complex assays, hemolytic
complement and nDNA antibody (duration of study: February 1979 t o May 1981)
Tests
Compared
ClqBA ClqBA
ClqSP mRFRI
No Specimen 53
r
.15
.3
P value
34
0
-
ClqV
nRFRI
40
0
-
ClqffA
0150
53
.19
.2
Clqp
0150
MFRl
0150
%b
ClpSP
nDNAAb
56
51
.31
.02
39
-.13
.4
42
29
.a3
.17
.3
-.46
C.001
<.001
AFRI
nDW
AGNELLO AND MITAMURA
790
presence o f h i g h t i t e r s o f nDNA a n t i b o d i e s suggests t h e
p o s s i b i l i t y t h a t s o l u b l e DNA-anti-DNA complexes a r e
p r e s e n t i n a n t i b o d y excess i n t h e sera o f p a t i e n t s w i t h
a c t i v e SLE.
The e f f e c t o f deoxyribonuclease on t h e
ClqSP b i n d i n g o f SP's serum i s c o n s i s t e n t w i t h t h i s
p o s s i b i l i t y . While these r e s u l t s a r e c o n t r a r y t o those
of I z u i e t a1 (ll), they a r e c o n s i s t e n t w i t h those o f
Bruneau and Benveniste (12).
The low s r a t e s o f the complexes detected by ClqSP
a r e s i m i l a r t o t h e f i n d i n g s from u l t r a c e n t r i f u g a t i o n
s t u d i e s where t h e m a j o r i t y o f DNA-anti-DNA complexes
detected were s m a l l e r than 21s (12). There i s i n d i r e c t
evidence t h a t t h e predominant DNA-anti-DNA complex
detected by t h e ClqSP may c o n s i s t o f a s i n g l e antibody
and a s i n g l e small DNA molecule:
a ) The peak ClqSP
r e a c t i v i t y was 8s which i s s m a l l e r than a dimer o f IgG;
b) t h e mRF which d e t e c t s dimers o f IgG d i d n o t d e t e c t
t h e ClqSP r e a c t i v e m a t e r i a l i s o l a t e d on g r a d i e n t u l t r a c e n t r i f u g a t i o n ; c ) monomers o f IgG a r e p o o r l y detected
by Clq. DNA has been shown t o d i r e c t l y r e a c t w i t h Clq
(13). Hence, small complexes o f IgG and DNA then would
b i n d more a v i d l y t h a n IgG alone.
The s t u d i e s on pat i e n t s BD and NC suggest t h a t h i g h a v i d i t y complement
f i x i n g a n t i b o d y may be r e q u i r e d f o r f o r m a t i o n o f such
complexes.
Several previous f i n d i n g s a r e c o n s i s t e n t w i t h t h e
above evidence.
The predominant form o f IgG antibody
b i n d i n g t o DNA has been shown t o be b i v a l e n t bonds t o
s i n g l e molecules w i t h l i t t l e c r o s s l i n k i n g (13).
The
minimal DNA fragment which binds t o nDNA antibody i s 20
t o 25 base p a i r s ; 35 base p a i r s a r e needed f o r b i v a l e n t
b i n d i n g (14).
DNA r e c e n t l y i s o l a t e d from sera o f
p a t i e n t s w i t h a c t i v e SLE was small, 30 t o 40 base p a i r s
(15).
I t remains t o be determined whether o r n o t small
DNA-anti-DNA complexes a r e r e l a t e d t o t h e hypocmplementemia and t h e DNA-anti-DNA canplexes t h a t have
been demonstrated i n t h e r e n a l d e p o s i t s i n lupus nephritis.
The s t u d i e s presented on p a t i e n t HP demons t r a t e d t h a t these small complexes were present a t t h e
time of hypocomplementemia and d e t e c t i o n o f DNA cont a i n i n g r e n a l imnune conlplexes.
I t i s p o s s i b l e , however, t h a t l a r g e r complexes a r e r e s p o n s i b l e f o r b o t h
t h e l e s i o n s and t h e hypocomplementemia and t h a t t h e
small complexes r e p r e s e n t residua. The r e c e n t s t u d i e s
on complement f i x a t i o n by DNA-anti-DNA complexes would
PeokIgG Concentroilon8s
I
X GRADENT VOLUME
F i g u r e 3. 5-20% i s o k i n e t i c sucrose d e n s i t y g r a d i e n t o f SP
serum 10/3/80 ( s e e F i g u r e 1). 0--0 shows p r o t e i n d i s t r i b u t i o n . 0 - 4 shows ClqEA r e s u l t s . The d i s t r i b u t i o n o f IgG
and IgM i n the g r a d i e n t was a l s o determined ( o n l y peaks
shown).
DISCUSSION
The usual s e r o l o g i c f i n d i n g o f hypocomplementemia
i n the presence o f h i g h nDNA a n t i b o d y l e v e l s i n a c t i v e
SLE has always presented a paradox s i n c e i n animal
models o f i m n e complex disease, disease a c t i v i t y was
associated w i t h hypocomplementemia and c i r c u l a t i n g
immune complexes formed i n a n t i s e n excess.
The prel i m i n a r y data presented i n t h i s study may p r o v i d e some
i n s i g h t t o t h i s paradox. The f i n d i n g t h a t d e t e c t i o n o f
i m n e complexes by t h e ClqSP c o r r e l a t e s w i t h t h e
Table 2. SLE p a t i e n t s : comparison o f c o r r e l a t i o n c o e f f i c i e n t s f o r immune complex assays,
h e m o l y t i c complement and nDNA a n t i b o d y
T e s t s compared
Period
studied
ClqEA
ClqSP
ClqEA
ClqSP
Patient
mRFRI
MFRI
SP
LR
HP
ED
NC
2/79-5/81
8/76-3/81
9/75-2/76
4/76-2/81
6/75-4/80
.I5
-13
.01
.10
0
.28
.23
0
.55*
t
-p <.05
-D c.001
0
ClqEA
CH50
0
.I9
.74'
-.45*
.36* -.31
D
.I3
-.04
.31
ClqSP
mRFRI
ClqEA
ClqSP
CH50
CH50
nDNAAb
nDNAAb
-.46'
-.60+
-.65'
-.46*
.35
.31*
-.42
-.09
.02
.17
-.I3
.06
-.I6
.28
-.39
.E3'
.59+
.81t
.29
-.OE
mRFRI
nDNAAb
.I7
.26
.13
-.01
-.11
LOW MOLECULAR WEIGHT DNA-ANTI-DNA
H.P35O
79I
c o n s i s t e n t w i t h p r e v i o u s r e p o r t s w h i c h f o u n d t h e monoc l o n a l r h e u m a t o i d f a c t o r r e a g e n t d e f i c i e n t i n SLE
s t u d i e s (13,19,20).
The d i f f e r e n c e s o b t a i n e d w i t h t h e
ClqSP and ClqBA h a v e a l s o been p r e v i o u s l y r e p o r t e d ( 3 )
and t h e f i n d i n g t h a t t h e ClqBA r e a c t a n t was n o t a f f e c t e d b y deox r i b o n u c l e a s e i s t h e same r e s u l t o b t a i n e d
b y o t h e r s (Ilf.Our f i n a l assessment o f t h e c l i n i c a l
u t i l i t y o f t h e d i f f e r e n t immune complex assays t e s t e d
must a w a i t c o m p l e t i o n o f t h e a n a l y s e s o f a l a r g e r
number o f s e r i a l s t u d i e s .
The p r e l i m i n a r y r e s u l t s
presented i n d i c a t e t h a t t h e C l q assays a r e s u p e r i o r t o
t h e mRF ( 3 ) ; t h e C l q s o l i d phase assay i s s u p e r i o r t o
t h e ClqBA f o r m o n i t o r i n g d i s e a s e a c t i v i t y i n SLE.
r
9
I5
ACKNOWLEDGEMENTS
We thank Ms. K. Beatty and Ms. C. MacLeod f o r t h e i r
e x c e l l e n t t e c h n i c a l assistance and Ms. J. Ginsberg f o r t h e
s t a t i s t i c a l computations.
REFERENCES
1.
1974
1975
1
1976
RENAL W P S Y
Figure 4. Serial study o f p a t i e n t HP showing marked drop i n
CH50 closely associated w i t h r i s i n g t l t e r s o f nONA antibody
and ClqSP. There was sane d e t e r i o r a t i o n o f renal function
a s indicated by r i s i n g p r o t e i n u r i a (not shown) and serum
c r e a t i n i n e levels.
Renal biopsy was performed a t date
fndicated by arrow. I m n o f l u o r e s c e n t studies showed heavy
deposits o f DNA, inmunoglobulin, Clq and C 3 i n the mesangium
and along the basement membrane. Electron microscopy showed
large subendothel i a l , subepi the1 i a l and intra-membranous and
mesangial deposits.
support t h i s p o s s i b i l i t y (16).
A minimum o f f o u r IgG
anti-nDNA a n t i b o d y m o l e c u l e s were r e q u i r e d f o r comp l ement f i x a t i o n .
S i n c e p a c k i n g o f a n t i b o d y on DNA a p p e a r s t o be
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t h e o r i g i n o f s m a l l non-complement f i x i n g complexes i s
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b y serum d e o x y r i b o n u c l e a s e .
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The
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