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Monoclonal lupus autoantibodies.

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MONOCLONAL LUPUS AUTOANTIBODIES
JOYCE RAUCH, EILEEN LAFER, CHESTER ANDRZEJEWSKI, 6. DAVID STOLIAR, and ROBERT S. SCHWARTZ
The autoimmune syndrome o f s y s t e m i c l u p u s erythema t o s u s (SLE) encompasses a d i v e r s i t y o f c l i n i c a l and
immunological a b n o r t n a l i t i e s , some o f w h i c h r e m a i n as
b a f f l i n g t o d a y as t h e y were t w e n t y y e a r s ago.
The
d i s o r d e r should, we b e l i e v e , be r e g a r d e d as a syndrome
because i t may e i t h e r a r i s e spontaneously, e r u p t a f t e r
exposure t o u l t r a v i o l e t l i g h t , o r d e v e l o p d u r i n g t r e a t ment w i t h a v a r i e t y o f drugs.
I t can, moreover, v a r y
i n c l i n i c a l s e v e r i t y from m i l d a r t h r a l g i a t o r a p i d l y
progressive glomerulonephritis.
A f e a t u r e commn t o
a l l forms o f t h e l u p u s syndrome i s t h e presence o f
a n t i b o d i e s t o one o r more c o n s t i t u e n t s o f t h e c e l l
n u c l e u s , i n c l u d i n g DNA ( e i t h e r s i n g l e strarlded DNA
sDNA , d o u b l e s t r a n d e d DNA dsDNA o r b o t h ) , n u c l e o p r o t e i n s , and h i s t o n e s .
In addition, other antibodies
may a r i s e :
autoimmune h e m o l y t i c anemia, thrombocytopenia, l u p u s a n t i c o a g u l a n t s ,
and b i o l o g i c a l l y f a l s e
p o s i t i v e t e s t s f o r s y p h i l i s a r e common problems.
The o r i g i n s o f l u p u s a u t o a n t i b o d i e s a r e unknown.
Whether n u c l e i c a c i d s themselves p r o v i d e t h e immunizing
s t i m u l u s remains e n i g m a t i c , e s p e c i a l l y because dsDNA i s
a v e r y weak immunogen -- even i n e x p e r i m e n t a l a n i m a l s
t h a t a r e g e n e t i c a l l y programmed t o produce anti-DNA
D e f i c i e n c i e s o f T lymantibodies spontaneously (1).
phocyte function, notably those o f suppressor c e l l s ,
have been i m p l i c a t e d i n SLE ( 2 ) . b u t t h e y l a c k t h e
s p e c i f i c i t y t h a t corresponds t o t h e s p e c i f i c i t y o f
l u p u s a u t o a n t i b o d i e s and t h e i r p r e d i l e c t i o n f o r n u c l e a r
constituents.
Moreover, t h e presence o f i d e n t i c a l
a b n o r m a l i t i e s i n some c l i n i c a l l y normal, s e r o n e g a t i v e
r e l a t i v e s o f p a t i e n t s w i t h SLE ( 3 ) a r g u e s t h a t i m p a i r e d
f u n c t i o n o f s u p p r e s s o r lymphocytes does n o t , by i t s e l f ,
i n s t i g a t e the production o f lupus autoantibodies.
Many o f t h e p u z z l i n g a s p e c t s o f SLE would be
c l a r i f i e d by d e t a i l e d a n a l y s e s o f t h e immunochemistry
of l u p u s a u t o a n t i b o d i e s .
Such work, however, has been
hampered by t h e f o r m i d a b l e t e c h n i c a l problem o f separ a t i n g i n d i v i d u a l a n t l b o d i e s f r o m t h e complex m i x t u r e
of a n t i b o d i e s i n l u p u s serum. The i s o l a t i o n o f i n d i v i d u a l a n t i b o d i e s i s e s s e n t i a l t o d i s t i n g u i s h t r u e speci f i c i t y from t h e cross r e a c t i o n s o f mixtures o f a n t i bodies.
T h a t problem has been s o l v e d by hybridoma
Fran the Departments o f Medicine and Biochemistry and
P h a m c o l o g y Tufts U n i v e r s i t y School o f Medicine. Boston.
Massachusetts
Supported by NIH Grant AM 27232
-
and RhcmtiSm, Vd. 25, No. 7 (July 1982)
technology.
The method we adapted f r o m t h e o r i g i n a l
t e c h n i q u e o f K o h l e r and M i l s t e i n ( 4 ) i n v o l v e s t h e
v i t r o f u s i o n o f an a u t o a n t i b o d y - p r o d u c i n g B c e l l w i t h
~ i m m m o r t a l " plasmacytoma c e l l ( 5 ) , and p r o v i d e s an
u n l i m i t e d s o u r c e o f monoclonal l u p u s a u t o a n t i b o d i e s .
Such a n t i b o d i e s a r e i d e a l r e a g e n t s because t h e y can
define rigorously not only ligand binding specificit i e s , b u t g e n e t i c markers, and even p r i m a r y s t r u c t u r e s
o f autoantibodies.
Moreover, i f a n t i b o d i e s a r e f o o t p r i n t s o f a n t i g e n s , t h e n i t i s p o s s i b l e t h a t monoclonal
a u t o a n t i b o d i e s c o u l d d e f i n e immunogens can i n c i t e a u t o immunization,
A u t o a n t i b o d y p r o d u c i n g hybridomas prepared f r o m
s p l e e n c e l l s o f NZB, (NZBxNZW)F
and MRL/l m i c e i n c l u d e t h o s e a g a i n s t e r y t h r o c y t e s t i - 8 ) . thymocytes ( 9 ) ,
r i b o s o m a l RNA (10). DNA (11-15). and t h e Sm a n t i g e n
( 1 6 ) . We have c o n c e n t r a t e d on two a s p e c t s o f MRL/l derived,
h y b r idoma produced anti-DNA a u t o a n t i b o d i e s :
t h e i r l i g a n d b i n d i n g p r o p e r t i e s and t h e i r i d i o t y p e s .
We have a n a l y z e d a n t i b o d i e s t h a t were produced by o v e r
t h i r t y c l o n e d hybridomas and we have p r e p a r e d a l i b r a r y
of a n t i - i d i o t y p i c a n t i b o d i e s a g a i n s t monoclonal antiDNA a u t o a n t i b o d i e s .
By means o f c o m p e t i t i v e radioimnunoassays, we
f o u n d t h a t each monoclonal a u t o a n t i b o d y had a u n i q u e
a n t i g e n b i n d i n g p r o f i l e , and i n a l l cases t h e r e was
binding t o several d i f f e r e n t nucleic a c i d antigens
(11).
The m u l t i p l i c i t y o f t h e b i n d i n g r e a c t l o n s we
observed i s e x p l a i n a b l e b y t h e presence o f c r o s s r e a c t i n g e p i t o p e s among t h e v a r i o u s l i g a n d s . One i m p l i c a t i o n o f those r e s u l t s i s t h a t heterogeneity o f autoa n t i b o d i e s i n SLE s e r a may r e f l e c t n o t o n l y d i v e r s i t y
among c l o n e s o f a u t o a n t i b b d y p r o d u c i n g c e l l s b u t a l s o
serological cross reactions o f individual autoantibodies.
Some hybridoma a u t o a n t i b o d i es have s p e c i f i c i t y f o r
n u c l e i c a c i d bases.
Our hybridoma H43, f o r example,
produces an a u t o a n t i b o d y w i t h a p r e f e r e n c e f o r guanosine residues.
Others, by c o n t r a s t , r e a c t w i t h a
s t r u c t u r e common t o a wide range o f n a t u r a l and synt h e t i c polynucleotides. That s t r u c t u r e i s probably t h e
s u g a r phosphate backbone, w h i c h c o n s i s t s o f phosphate
groups i n p h o s p h o d i e s t e r l i n k a g e s e p a r a t e d by t h r e e
c a r b o n atoms o f a d j a c e n t sugar m o l e c u l e s . O t h e r b i o l o g i c a l m o l e c u l e s a l s o c o n t a i n such phosphate groups; a
t y p i c a l example i s p h o s p h o l i p i d .
T h i s resemblance
between p h o s p h o l i p i d s and p o l y n u c l e o t i d e s l e d us t o
t e s t t h e a b i l i t y o f p h o s p h o l i p i d s t o r e a c t w i t h mono-
MONOCLONAL LUPUS ANTIBODIES
c l o n a l anti-DNA a n t i b o d i e s ( 1 2 ) . Some, b u t n o t a l l , o f
t h e a u t o a n t i b o d i e s bound s t r o n g l y t o s e v e r a l phosphol i p i d s , and i n a few cases t h e p h o s p h o l i p i d s were
s t r o n g e r c o m p e t i t o r s i n r a d i o i m m n o a s s a y s t h a n DNA
itself.
We ( 1 2 ) a l s o found t h a t t h e p h o s p h o l i p i d c a r d i o l i p i n i n h i b i t e d the antinuclear antibody reaction o f
some monoclonal anti-DNA a u t o a n t i b o d i e s :
t h a t one o f
them p r o l o n g e d t h e a c t i v a t e d p a r t i a l t h r o m b o p l a s t i n
t i m e t e s t i n a manner c h a r a c t e r i s t i c o f a l u p u s a n t i c o a g u l a n t (presumably b y b i n d i n g t o t h e p h o s p h o l i p i d
t h a t t h e t e s t r e q u i r e s ) ; and t h a t p h o s p h o l i p i d s , as
w e l l as p o l y n u c l e o t i d e s , s p e c i f i c a l l y i n h i b i t e d i d i o t y p e a n t i - i d i o t y p e r e a c t i o n s o f some anti-DNA a n t i bodies. Those r e s u l t s a g a i n emphasize t h a t some o f t h e
d i v e r s i t y of a u t o a n t i b o d y r e a c t i o n s i n SLE stems f r o m
t h e presence i n a v a r i e t y o f b i o l o g i c a l m o l e c u l e s o f a
relatively
simple,
recurrent antigenic structure.
Monoclonal a u t o a n t i b o d i e s a r e i d e a l l y s u i t e d t o
i d i o t y p i c a n a l y s i s because o f t h e ease o f p u r i f y i n g t h e
i d i o t y p e . Ue have produced a n t i - i d i o t y p e a n t i b o d i e s i n
r a b b i t s a g a i n s t f i v e anti-DNA hybridoma a n t i b o d i e s .
The a n t i - i d i o t y p e a n t i b o d i e s were d e f i n e d by t h e i r
a b i l i t y t o i n h i b i t b i n d i n g o f t h e homologous a u t o a n t i I d i o t y p e a n t i - i d i o t y p e i n t e r a c t i o n s were
body t o DNA.
a l s o a n t i g e n i n h i b i t a b l e , w i t h t h e same o r d e r o f a n t i gen i n h i b i t i o n as i n c o m p e t i t i v e l i g a n d b i n d i n g assays
(11.17).
Q u a n t i t a t i v e radioimmunoassays, e s t a b l i s h e d
f o r each i d i o t y p e a n t i - i d i o t y p e p a i r , were used b o t h t o
seek c r o s s r e a c t i v e i d i o t y p e s and t o measure serum
l e v e l s o f i d i o t y p e (17).
One system, H130/anti-H130,
i s o f c o n s i d e r a b l e i n t e r e s t because i t i d e n t i f i e s a
h i g h frequency, p r o b a b l y dominant, i d i o t y p i c f a m i l y i n
a l l MRL/1 mice.
Anti-H130,
p r e p a r e d i n r a b b i t s by
i m m u n i z a t i o n w i t h t h e m n o c l o n a l anti-DNA a n t i b o d y
H130, c r o s s r e a c t s w i t h a b o u t two t h i r d s o f o t h e r
h y b r i d m a anti-DNA a u t o a n t i b o d i e s and i t i s p r e s e n t i n
m i l l i g r a m amounts i n MRL/l serum.
About one t h i r d t o
one h a l f o f MRL/l serum anti-DNA a n t i b o d i e s have t h e
H130 i d i o t y p e , b u t c u r i o u s l y , a c o n s i d e r a b l e excess o f
serum immunoglobulins w i t h t h i s i d i o t y p e do n o t b i n d
DNA.
Thus, H130 i s r e p r e s e n t a t i v e o f a r e l a t i v e l y
l a r g e f a m i l y o f m o l e c u l e s , some o f w h i c h o v e r l a p w i t h a
s i m i l a r f a m i l y o f anti-DNA immunoglobulins.
The p r a c t i c a l ( t h e r a p e u t i c ) i m p l i c a t i o n s o f a dominant i d i o t y p e
among anti-DNA a n t i b o d i e s a r e u n d e r i n v e s t i g a t i o n .
We assume t h a t monoclonal a n t i b o d i e s a r e r e p r e s e n t a t i v e o f i n v i v o c l o n a l p o p u l a t i o n s , and t h a t t h e
s p e c i f i c i t i e s o f t h e monoclonal a n t i b o d i e s do n o t
depend on new c o m b i n a t i o n s o f c h a i n s from s p l e e n c e l l
and myeloma f u s i o n p a r t n e r s .
The l a t t e r assumption i s
v a l i d w i t h myeloma c e l l l i n e s t h a t do n o t s y n t h e s i z e o r
s e c r e t e I g c h a i n s , b u t when I g s e c r e t i n g myeloma c e l l s
were used, i t may be n e c e s s a r y t o p u r i f y t h e r e l e v a n t
s u b p o p u l a t i o n o f hybridoma p r o d u c t s .
E l i a t and Laskov
( 1 8 ) have d e s c r i b e d such a procedure, w h i c h i n v o l v e s
p r e p a r a t i o n o f Fab fragments and a f f i n i t y chromatography on a n t i g e n Sepharose columns t o remove a l l
c o m b i n a t i o n s w i t h myeloma c h a i n s . To t e s t t h e assumpt i o n t h a t hybridoma p r o d u c t s a r e r e p r e s e n t a t i v e of
v i v o autoantibody production, p a r a l l e l analyses o f
G l e x a n t i b o d y p o p u l a t i o n s i n autoimnune sera and
monoclonal a n t i b o d i e s . The i n f o r m a t i o n g a i n e d f r a n t h e
l a t t e r w i l l g u i d e a r a t i o n a l approach t o s t u d i e s o f t h e
serum p o p u l a t i o n s .
A l o n g s t a n d i n g q u e s t i o n i n autoimmunity, re-emphasized b y s t u d i e s o f monoclonal a u t o a n t i b o d i e s ,
concerns t h e n a t u r e o f t h e i m n u n i z i n g s t i m u l u s i n t h e s e
conditions.
The s e r o l o g i c a l r e a c t i v i t y o f a u t o a n t i b o d i e s w i t h a p a r t i c u l a r a n t i g e n c o u l d be a f o r t u i t o u s
745
r e s u l t because t h e s e l e c t e d r e a g e n t i s a c t u a l l y a c r o s s
r e a c t i n g antigen.
The f i n d i n g t h a t a s i n g l e h bridoma
p r o d u c t can r e a c t w i t h d e n a t u r e d DNA, p o l y {I), and
phospholipids r e i n f o r c e s t h i s ambiguity.
We m i g h t
c o n s i d e r such an i m m u n o g l o b u l i n t o be a n anti-DNA a n t i body, b u t we c a n n o t c o n c l u d e t h a t DNA s t i m u l a t e d i t s
production.
The d e m o n s t r a t i o n t h a t i m m u n i z a t i o n o f
experimental animals w i t h c a r d i o l i p i n leads t o t h e
p r o d u c t i o n o f a n t i b o d i e s t h a t b i n d t o DNA ( 1 9 ) f u r t h e r
i l l u s t r a t e s the caution required i n the interpretation
o f serological results.
The r e c e n t a v a i l a b i l i t y o f s u i t a b l e f u s i o n p a r t n e r s f o r human l y m p h o c y t e s (20,21) now p e r m i t s e x t e n s i o n o f s t u d i e s o f m u r i n e l u p u s t o t h e human d i s o r d e r .
We have embarked upon an a n a l y s i s o f monoclonal human
lupus autoantibodies,
and a f o r t h c o m i n g r e p o r t w i l l
describe our results.
The a p p l i c a t i o n o f hybridoma
t e c h n o l o g y t o a u t o a n t i b o d i e s has been an i m p o r t a n t
advance i n s t u d i e s o f autoimnune diseases. The method
p e r m i t s a n a l y s e s o f u n p a r a l l e l e d p r e c i s i o n . and i t
s h o u l d l e a d t o a new l e v e l o f u n d e r s t a n d i n g o f t h e s e
e n i g m a t i c d i s o r d e r s . The o u t s t a n d i n g a n a l y t i c a l advant a g e o f monoclonal a u t o a n t i b o d i e s i s t h e h i g h degree o f
s p e c i f i c i t y t h e y possess. Such s p e c i f i c i t y , moreover,
m i g h t have i m p l i c a t i o n s f o r t h e r a p y ; a p r o s p e c t a l r e a d y
demonstrated i n p r i n c i p a l w i t h e x p e r i m e n t a l a n i m a l s
(22,23).
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