Published December 1, 1985

8/14/2019 Published December 1, 1985

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G E N E T I C S U S C E P T I B I L I T Y T O M U R I N E C O L L A G E N I IA U T O I M M U N E A R T H R I T I S

P r o p o s e d R e l a t io n s h i p t o t h e I g G 2 A u t o a n t i b o d y S u b cl as s R e s p o n s e ,C o m p l e m e n t C 5 , M a j o r H i s to c o m p a t i b i l i t y C o m p l e x ( M H C ) a n d

N o n - M H C L o c iB Y W I L L I A M C . W A T S O N A N n A L E X A N D E R S . T O W N E S

From the Veterans Administration Medical Center and D epartment o f Medicine, U niversity ofTennessee Center for the Health Sciences, Memphis, Tennessee 3810 4

C o l l a g en I I a u t o i m m u n e a r th r i ti s ( C I I A I A ) , ~ a n e x p e r i m e n t a l m o d e l o fr h e u m a t o i d a r t h r it i s , i s i n d u c e d i n r a ts o r m i c e b y i m m u n i z a t i o n w i t h h e t e r o l o -g o u s C I I ( 1 , 2) . T h e r e i s c o n s i d e r a b l e e v i d e n c e t o s u p p o r t t h e c o n c e p t t h a t t h ei n i ti a t io n o f d i s e as e is re l a t e d t o t h e p r o d u c t i o n o f a c r o s s r e a c t i v e I g G a u t o a n t i -b o d y t o C I I , w h i c h d e p o s i t s i n a r t i c u l a r c a r t i l a g e a n d a c t i v a t e s t h e c o m p l e m e n tc a s c a d e . A f t e r i m m u n i z a t i o n w i t h h e t e r o l o g o u s C I I , a r t h r i t i c r a t s p r o d u c e a nI g G a n t i b o d y w h i c h is m o r e c r o s s r e a c ti v e w i th r a t C I I t h a n t h e I g G p r o d u c e d b yn o n a r t h r i t i c r a t s (3 ). A f f i n i t y - p u r i f i e d r a t a n t i - r a t C I I I g G w i ll p a s s i v el y t r a n s f e ra r t h r i t i s t o n o r m a l r a t s ( 4 ) . C o m p l e m e n t d e p l e t i o n i n h i b i t s t h e d e v e l o p m e n t o fa r t h r i t i s a f t e r a c t i v e i m m u n i z a t i o n w i t h C I I ( 3 ) o r p a s s i v e t r a n s f e r w i t h C I Ia n t i b o d y ( 5) . I n a d d i t i o n , w e r e c e n t l y h a v e s h o w n t h a t i n c o n t r a s t t o C 5 n o r m a lB l 0 . D 2 / n e w l in e m i c e, C 5 - d e f i ci e n t B l 0 . D 2 / o l d l in e m i c e a r e r e s is t a nt t o ar -t h r it i s b y p a s s i v e t r a n s f e r w i t h C I I a n t i b o d y , a n d t h a t , w h e n b o u n d t o c a r t i la g ei n v i t r o , e i t h e r r a t o r m o u s e C I I a n t i b o d y w i l l a c t i v a t e C 5 t o C 5 a ( 6 ) .

I n t h e m u r i n e m o d e l o f C I I A I A , W o o l e y e t al . ( 7 , 8 ) s u g g e s t e d t h a t s u s c e p t i-b i li ty to C I I A I A is li n k e d t o t h e I - A q h a p l o t y p e o f th e H - 2 l o c u s o f t h e M H C ,w h i c h i n t u r n i s l i n k e d t o h i g h a n t i b o d y r e s p o n s i v e n e s s t o C I I . H o w e v e r , t h e s ei n v e s t i g a t o r s n o t e d t h a t s o m e s t r a i n s p r o d u c e s u b s t a n t i a l a n t i b o d y r e s p o n s e s , b u tf ai l t o d e v e l o p a rt h r i ti s . B a s e d u p o n s t u d i e s r e p o r t e d h e r e i n , w e p r o p o s e t h a t t h es p e c if i c r e s p o n s e s o f t h e c o m p l e m e n t - f i x i n g I g G 2 s u b c la s s es , as w e l l a s t h ea v a i la b i li t y o f c o m p l e m e n t C 5 , r e p r e s e n t i m p o r t a n t d e t e r m i n a n t s o f s u s c e p t ib i l it yt o C I I A I A . I n a d d i t i o n , t h e s e s t u d i e s i m p l i c a t e g e n e s o u t s i d e t h e M H C i nA p r e l i m i n a ry a c c o u n t o f p a r t o f th i s w o r k w a s p re s e n t e d t o t h e A S B C / A A I F A S E B J u n e , 1 9 8 4m e e t i n g i n S t . L o u is , M i s s o u r i [ W a t s o n , W . C . , a n d A . S . T o w n e s . 1 9 8 4 . G e n e t i c r e s t r i c t i o n o f t h eI g G 2 a a u t o a n t i b o d y s u b c l a s s r e s p o n s e t o c o l l a g e n I I ( C I I ) : a n h y p o t h e s i s f o r r e s i s t a n c e t o C l la u t o i m m u n e a r t h r i t i s i n C 57 B 1 m i c e . Fed. Proc. 4 3 : 1 9 9 2 ( A b s t r . 3 3 4 3 ) ] . T h i s w o r k w a s s u p p o r t e db y p r o g r a m d i r e c t e d r e s e a r c h f u n d s f r o m t h e V e t e r a n s A d m i n i s t ra t i o n . A d d r e s s c o r r e s p o n d e n c e t o :W . C . W a t s o n , R h e u m a t o l o g y R e s e a r c h L a b o r a t o r y , V A M e d i c a l C e n t e r ( 1 51 ) , 1 0 3 0 J e f f e r s o n A v e . ,M e m p h i s , T N 3 8 1 0 4 .Abbreviations used in this paper: C I I A I A , c o l l a g e n I I a u t o i m m u n e a r t h r i t i s ; C F A , c o m p l e t eF r e u n d ' s a d j u v a n t ; E L I S A , e n z y m e - l i n k e d i m m u n o s o r b e n t a ss a y; I F A , i n c o m p l e t e F r e u n d ' s a d j u v a n t ;M H C , m a j o r h i s t o co m p a t i b i li t y c om p l e x .1 8 7 8 J . E x e . M E I ). T h e R o c k e f e l le r U n i v e r si t y P r e s s 0 0 2 2 - 1 0 0 7 / 8 5 / 1 2 / 1 8 7 8 / 1 4 $ 1 . 0 0

V o l u m e 1 6 2 D e c e m b e r 1 9 8 5 1 8 7 8 - 1 8 9 1

Published December 1, 1985


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WATSON AND TOWNES 1879determining susceptibi l i ty and provide addit ional support for an important rolefor auto anti body to CI I in the initiation of arthritis in this animal model.

Ma te r i a l s and MethodsMice . 7-8-wk-old mice were purchased from The Jackson Laboratory, Bar Harbor,ME. Breeding between C57BL/6 (B6) and DBA/1 (D1) mice and backcrosses to D1[(B6D1)F~ D1] was done at the Memphis VAMC. Complement, specifically C5, isthought to be requir ed for the initiation of CII AIA (6), and female mice are known tobe relatively deficient in C5, as well as C6 and C7 (9). To contro l for this sex-relateddiffer ence in com plement and its possible effect on susceptibility to CI I AIA , all studiesused male mice, with the exception o f the B6D1 D1 backcross study. Only fivebackcrosses were available and two of these were female. Since the study was performedprimarily to examine the mode of inheritance of IgG2a autoantibody responsiveness,rather than the susceptibility to arthritis o f B6D1 D1 backcrosses, these two femaleswere included.Collagen. Chick CII was extracted from sternal cartilage and purified according to

published methods (10). Mouse CII was a generous gift from Dr. Michael A. Cremer,who prepared it according to the same procedures used in preparing chick CII. Thepurity of both preparations was confirme d by electrophoresis (10).Immunization. Mice were injected subcutaneously in the tail with chick CII emulsified1:1 with complete Freund's adjuvant (CFA). The CFA was prepared by grinding myco-bacteria in incomplete Freund's adjuvant (IFA) (Difco Laboratories, Detroit, MI). Themycobacteria were heat-killed M. tuberculosis strains C, DT, a nd PN (Ministry of Agricul-ture, Fisheries and Food, Central Veterinary Laboratory, Weybridge, Surrey, England).Each mouse received 0.1 ml chick CII/CFA containing 100/~g CII and 10, 100, 250, or500 #g mycobacteria as indicated in the text.Arthritis. Mice were observed for 4- 5 mo for the presence or absence of arthritis. Ananimal was jud ged to be arthr itic if one or more join t regions (digits, wrist, ankle) werered and swollen.Purification o f Chick CII Antibo dy. The following proce dure was used to obtain purifiedD 1 and B6 antibody for studies o f crossreactivity and af finity and as a reference f orestimating IgG autoantibody subclass serum levels in individual mice as explained below.Chick CII-immune mice were bled from the retroorbital sinus at 7-10-d intervalsbeginning ~30 d after immunization and ending on about day 70. Serum aliquots werestored at -70C. Pooled serum was incubated with chick CII-Sepharose overnight at4C. The immunosorbent was prepared according to standard procedures using CNBr-activated Sepharose 4B (Sigma Chemical Co., St. Louis, MO). Unbound protein wasremoved by washing the immunosorbent wtih 0.1 M Tris, 0.15 M sodium chloride, pH7.4, on a sintered glass funnel until the filtrate absorbance at 280 nm achieved constantbackground levels (


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1 8 8 0 M U R I N E C O L L A G E N I I A U T O I M M U N E A R T H R I T I Sf o u n d t o b e < 1 0 % u n d e r t h e c o n d i t i o n s o f th i s s u b c la s s E L I S A o n t h e b a s i s o f th e i rr e l a ti v e r e a c ti v i ti e s w i t h p u r i f i e d m o u s e m y e l o m a I g G s u b cl a ss p r o t e i n s ( L i t t o n B i o n e t i c sI n c . , K e n s i n g t o n , M D ) . A f t e r w a s h i n g , t h e p l a te s w e r e i n c u b a t e d f o r a n o t h e r 2 h a t 4 Cw i t h p e r o x i d a s e - c o n j u g a t e d g o a t a n t i - r a b b i t I g G , F c - s p e c if i c s e r a ( C a p p e l l L a b o r a t o r i e s )d i l u t e d 1 : 7 0 0 0 i n E L I S A b u f f e r , w a s h e d a g a in , a n d t h e n i n c u b a t e d w i t h p e r o x i d a s es u b s t r a t e f o r 6 0 r a in a t r o o m t e m p e r a t u r e . T h e s u b s t r a t e c o n s is t e d o f 2 .2 1 m M 0 -p h e n y l e n e d i a m i n e , 2 6 . 5 9 m M c it ri c a c id , 5 1 . 4 0 m M s o d i u m p h o s p h a t e , 0 . 0 1 2 % h y d r o g e np e r o x i d e , a n d 0 . 1 % T w e e n - 2 0 . A v o l u m e o f 0 . 1 5 m l / w e l l w a s u s e d i n a ll i n c u b a t io n s .A b s o r b a n c e s at 4 5 0 n m w e r e m e a s u r e d w i t h a M i c ro e li sa A u t o R e a d e r M R 5 8 0 ( D y n a t e c hL a b o r a t o r i e s , I n c . ).T h e f o l lo w i n g p r o c e d u r e w a s u s e d t o c o n v e r t a b s o r b a n c e s t o /~ g / m l v a l u es f o r e a ch I g Gs u b cl a ss . V a r i o u s d i l u t i o n s o f M i le s L a b o r a t o r i e s m o u s e r e f e r e n c e s e r u m w e r e a s s a y e d o nt h e g o a t a n t i - m o u s e I g G F ( a b ' ) 2 - c o a t e d p l at e s. T h i s r e f e r e n c e s e r u m i s s u p p l i e d w i t h g g /m l v a l u es f o r e a c h I g G s u b c la s s. T h e a b s o r b a n c e v a l u e s (y ) a n d t h e c o r r e s p o n d i n g l o g # g /m l v a l u es (x ) w e r e e n t e r e d i n t o a T I - 5 5 c a l c u l a t o r ( T e x a s I n s t r u m e n t s , I n c . , D a ll as , T X ) ,p r e p r o g r a m m e d t o c o m p u t e s l o p e (m ) a n d y - i n t e r c e p t (b ) v a l u es i n t h e s t r a ig h t l i n ee q u a t i o n : y = ( m ) ( lo g x) + b . T h e c a l c u l a t o r u s e d a l in e a r r e g r e s s io n p r o g r a m t o s o lv e f o rm a n d b ; c o r r e l a t i o n c o e f f ic i e n ts ( r v a lu e s ) f o r t h e r e l a ti o n s h i p b e t w e e n E L I S A a b s o r b a n c ea n d t z g /m l v a lu e s o f I g G 1 , 2 a , 2 b , o r 3 w e r e n o t < 0 . 9 9 0 . T h e o b s e r v e d y a b s o r b a n c ev a l u e s fo r e a c h I g G s u b c l a ss i n t h e a f f in i t y - p u r i fi e d c h i c k C I I a n t i b o d y p r e p a r a t i o n s t h e nw e r e e n t e r e d i n t o t h e c a l c u l a t o r t o c o m p u t e t h e c o r r e s p o n d i n g # g / m l v a l u e s b y s o l v i n gf o r l og x i n t h i s e qua t i o n .lgG Autoantibody Subclass ELISA. A r t h r i t i c m i c e w e r e b l e d w i t h i n 4 8 h a f t e r t h e o n s e to f d is e a se a n d n o n a r t h r i t i c m i c e w e r e b l e d a t t h e p e a k o f t h e s e r u m r e s p o n s e , i . e. , d a y4 0 - 4 5 . T h e s e t w o t im e s w e r e s i m i la r , si n c e t h e m e a n o n s e t d a y ( + S E ) f o r a r t h r i ti c D 1m i c e ( T a b l e I ) w a s 4 3 + 2 , n = 2 5 . A n e x c e p t i o n t o t h e a b o v e p r o t o c o l i s n o t e d f o r t h es t u d y d e s c r ib e d i n T a b l e I V . S e r u m s a m p le s w e r e s t o r e d a t - 7 0 C b e f o r e a ss a y in g f o rI g G a u t o a n t i b o d y a s f ol lo w s . T h e s e r u m s a m p le s w e r e d i lu t e d i n E L I S A b u f f e r a n di n c u b a t e d o v e r n i g h t a t 4 C o n m i c r o t i t e r p la t e s c o a t e d w i t h 2 ~z g/m l m o u s e C I I i n 0 . 1 4 5M p o t a s s iu m p h o s p h a t e b u f f e r , p H 7 . 6 . A b s o r b a n c e v a l u e s f o r e a c h I g G s u b cl a ss w e r ed e v e l o p e d w i t h th e r a b b i t a n t i - m o u s e s u b c la s s s e r a, p e r o x i d a s e - c o n j u g a t e d g o a t a n ti -r a b b i t I g G F c - s p e c i f i c s e r u m , a n d p e r o x i d a s e s u b s t r a t e a s d e s c r i b e d a b o v e . K n o w nc o n c e n t r a t i o n s o f a n t i - c h i c k C I I I g G 1 , 2 a , 2 b , a n d 3 in a ff i n i ty - p u r i f i ed c h i c k C I I a n t i b o d yp r e p a r a t i o n s w e r e a s s a y e d s i m u l t a n e o u s l y . T h e s e c o n c e n t r a t i o n s w e r e e s t a b l i s h e d b y I g Gs u b c la s s E L I S A o n g o a t a n t i - m o u s e F ( a b ' ) 2 - c o a t e d m i c r o t i t e r p l a t e s a s d e s c r i b e d a b o v e .T h e r e s u l t a n t p a i r s o f v a lu e s (a b s o r b a n c e a n d # g / m l I g G a n t i b o d y s u b c la s s) w e r e s u b j e c t e dt o l i ne a r r e g r e s s i on a na l y s i s t o c om pu t e m a nd b va l ue s f o r e a c h s ubc l a s s ; c o r r e l a t i onc o e f f i c ie n t s ( r v a l ue s ) f o r t h e r e la t i o n s h i p b e t w e e n E L I S A a b s o r b a n c e a n d # g / m l I g G 1 ,2 a , 2 b, o r 3 w e r e n o t < 0 . 9 8 0 . T h e o b s e r v e d y a b s o r b a n c e v a l ue s fo r e ac h I g G a u t o a n t i b o d ys u b c la s s i n in d i v i d u a l s e r u m s a m p l e s t h e n w e r e e n t e r e d i n t o t h e c a l c u l a t o r to c o m p u t e t h ec o r r e s p o n d i n g / ~ g / m l v a l u e s .Competitive Inhibition ELISA. A n t i - c h i c k C I I I g G 2 a w a s p u r i f ie d f r o m a n a l iq u o t o fD 1 a n t i - c h i c k C I I I g G w i th p r o t e i n A - S e p h a r o s e C L - 4 B ( S i g m a C h e m i c a l C o .) , a c c o r d in gt o t h e g e n e r a l p r o c e d u r e d e s c r i b e d b y E y et al . (1 2 ) . T h e f o l l o w i n g m o d i f i c at i o n s w e r em a d e . A f t e r e lu t i n g I g M a n d I g G 1 w i t h p H 6 b u f f er , t h e b u f f e r p H w a s c h a n g e d t o 3 . 5 ,t o e lu t e I g G 2 a p l u s I g G 2 b p l u s I g G 3 . T h e p H o f t h is m i x t u r e w a s r e a d j u s t e d t o 5 . 5 , t h em i x t u r e l o a d e d b a c k o n t o t h e c o l u m n , a n d I g G 2 a w a s e l u t ed b y w a s h i n g t h e c o l u m n w i t hp H 5 . 5 b u f f e r . T h e I g G 2 a e l u a te w a s i m m e d i a t e l y n e u t r a li z e d w i t h 1 M T r i s , p H 8 ,c o n c e n t r a t e d i n a n A m i c o n c e l l, a n d d i a l y z e d i n t o P B S . A n a l y s is o f t h e e l u a t e b y I g Gs u b c la s s E L I S A r e v e a l e d 9 7 % I g G 2 a . H o r s e r a d i s h p e r o x i d a s e ( t y p e I V ; S i g m a C h e m i c a lC o . ) t h e n w a s c o n j u g a t e d t o t h e I g G 2 a a n t i b o d y w i t h g l u t a r a l d e h y d e , a c c o r d i n g t o th em e t h o d d e s c r i b e d b y A v r a m e a s e t a l. (1 3 ) . V a r i o u s c o n c e n t r a t i o n s o f p o o l e d D 1 o r B 6c h i ck C I I a n t i b o d y w e r e i n c u b a t e d w i th t h e c o u p l e d a n t i b o d y i n m o u s e C I I - c o a t e dm i c r o t i t e r w e l ls f o r 1 8 h a t 4 C u s i n g a v o l u m e o f 0 .1 m l / w e l l; c o n c e n t r a t i o n s w e r ee s t a b li s h e d b y I g G s u b c la s s E L I S A u s i n g p l a te s c o a t e d w i t h g o a t a n t i - m o u s e I g G F ( a b ') 2 ,a s e x p l a i n e d i n t h e p r e v i o u s s e ct i o n . T h e p l a te s t h e n w e r e w a s h e d a n d p e r o x i d a s e s u b s t r a te



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W A T S ON A N D T O W N E S 1 8 8 1w a s a d d e d . A b s o rb a n c e s w e re m e a s u re d w i th th e Mic ro e li s a A u to R e a d e r . R e la t iv ea n t ib o d y a f f in i ty w a s d e f in e d a s th e c o n c e n t ra t io n o f u n c o u p le d D 1 o r B 6 a n t ib o d yr e q u i r e d t o r e d u c e t h e E L I S A a b s o r b a n c e b y 5 0 % .

R e s u l t sAna lys is of the IgG Au toant ibody Subclass Response in the CI I AIA -suscep t ible D1Strain. T h e r e l a ti o n s h ip b e t w e e n s u sc e pt i bi l it y to C I I A I A a n d t h e m a g n i t u d e

o f th e t o t a l I g G a u t o a n t i b o d y r e s p o n s e i n t h e D 1 s t r a i n i s s h o w n i n T a b l e I . W i t hv a r i a b l y i n c r e a s i n g a m o u n t s o f m y c o b a c t e r i a u s e d i n t h e a d j u v a n t , t h e s e r u ml ev e l o f I g G a u t o a n t i b o d y i n c r ea s e d f r o m a n a v e r a g e o f 1 4 g g / m l , w i t h o u tm y c o b a c t e r i a , t o a m a x i m u m o f 7 89 # g / m l w i t h 2 5 0 g g m y c o b a c t e r i a p e r m o u s e .A r t h r i t i s f a i l e d t o d e v e l o p i n m i c e i m m u n i z e d w i t h o u t m y c o b a c t e r i a in w h i c hs e r u m l ev e ls v a r i e d b e t w e e n 8 a n d 2 9 # g / m l . I n m i c e i m m u n i z e d w i t h m y c o b a c -t e r ia , a r t h r i t i s w a s f o u n d t o b e a s s o c i at e d w i t h s e r u m l e ve l s o f 8 1 - 1 , 9 4 3 / ~ g / m l .A n a l y s is o f t h e I g G s u b c l as s r e s p o n s e r e v e a l e d t h a t m o s t o f t h is I g G a u t o a n t i b o d yw a s I g G 2 a , a v e r a g i n g 7 3 % o f th e t o ta l I g G a u t o a n t i b o d y ( r an g e , 4 5 - 9 7 % ; n =2 5 ). I t is i n t e r e s t i n g t o n o t e t h a t a h i g h p e r c e n t o f I g G 2 a a u t o a n t i b o d y r e s p o n -s i v en e s s a p p e a r s t o b e a n i n h e r e n t t r a i t o f th e D 1 s t r ai n , s in c e I g G 2 a a u t o a n t i b o d ya l so w a s t h e p r e d o m i n a n t I g G s u b cl a ss i n t h e n o n a r t h r i t i c D 1 m i c e i m m u n i z e dw i t h o r w i t h o u t m y c o b a c t e r i a , a v e r a g i n g 5 5 % ( r a n g e , 4 0 - 7 1 % ; n = 9) . I n t e r m so f th e m a g n i t u d e o f t h e I g G 2 a a u t o a n t i b o d y r e s p on s e , s u s ce p ti b il it y a p p e a r e d t ob e a s so c i a t e d w i th a r e s p o n s e o f ~ 5 0 # g / m l o r m o r e , s i n ce t h e l o w e s t s e r u mI g G 2 a a u t o a n t i b o d y l e ve l a s s o c i a te d w i t h d i se a s e w as 6 2 g g / m l a n d t h e h i g h e s tl e v e l a s s o c i a t e d w i t h t h e a b s e n c e o f d i s e a s e w a s 4 4 u g / m l ( F i g . 1 ) .

Comparison of lgG A utoant ibod y Subclass Responses in Suscept ible D 1 and Resis tantB6 S t ra ins . A s s h o w n i n F i g. 1 , I g G 1 , 2 b , a n d 3 a u t o a n t i b o d y r e s p o n s e s o fn o n a r t h r i t i c B 6 m i c e g e n e r a l l y o v e r l a p p e d t h o s e o f a r t h r i t i c D 1 m i c e . In m a r k e dc o n t r a s t , t h e r e w a s a c l e a r s e p a r a t i o n b e t w e e n I g G 2 a a u t o a n t i b o d y r e s p o n s e s:

T A B L E IA Comparison of Arthritic and Nonarthritic IgG Autoantibody Serum Levels in D1 Mice

Anti-mouse Cl l IgGArthri- Mean percent (range)Grou p (n) tis Total

1 2 a 2 b 3~g/ml

DI , (7) - 14 (8-29) 28 (17-43 ) 55 (40-71) 14 (7-24) 3 (2-6)D12 (8) + 364 (81-836) 16 (7-33) 69 (57-84) 12 (5-25) 3 (1-9)DI2 (1) - 69 7 55 29 9DI~ (10) + 420 (171-858 ) 14 (3-27) 67 (45-87) 9 (1-32) 10 (2-36)DI4 (7) + 789 (260-1943) 12 (1-31) 83 (63-97) 3 (1-5) 2 (1-4)DI4 (1) - 81 4 54 33 9Mice were imm unized w ith 100 ug chick CII and either 0 (D ll), 10 (DI2), 100 (DI3), or 250 (D14)ttg mycobacteria in 1FA . Total and percent IgG autoantibody subclass values were derived fromindividual ttg/ml autoantihody subclass values deter min ed by Ig G autoantibody subclass ELISA onmouse CI I-co ated microtiter plates as explained in Materials and M ethods.



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1 2 a 3A N T I - M O U S E C l l Ig G

F IG U RE 1 . C o m p a r i s o n o f Ig G a u t o a n t i b o d y s u b c la s s r e s p o n s e s o f t h e C I I A I A - s u s c e p t i b l eD 1 s t r a i n ( T a b l e 1 ) w i t h t h o s e o f t h e r e s i s t a n t B 6 s t ra i n . I m m u n i z a t i o n s c h e d u l e f o r D 1 m i c ei s d e s c r i b e d in t h e l e g e n d t o T a b l e 1. B 6 m i c e w e r e i m m u n i z e d w i t h 1 0 0 # g c h i c k C I I a n d 2 5 0g g m y c o b a c t e r i a i n I F A ( B 6 - 4) . S e r u m I g G a u t o a n t i b o d y s u b c l as s l e v e ls w e r e d e t e r m i n e d b yl g G a u t o a n t i b o d y s u b c la s s E L I S A o n m o u s e C I I - c o a t e d m i c r o t i t e r p la t e s as e x p l a i n e d inM a t e r i a l s a n d M e t h o d s . ( 0 ) A r t h r i t i c m i c e , ( O) n o n a r t h r i t i c m i c e .T A B L E I I

Compa rison of the Crossreactivity of Anti-Chick CII IgG Affinity Purified from the PooledSerum o f Arthrit ic D1 a nd Nonarthrit ic B6 MiceP e r c e n t c r o s s r e a c t i v i ty o f a n t i - c h i c k C I I l g GG r o u p 1 2 a 2 b 3

D 1 5 7 ( 5 0 - 6 4 ) 5 0 ( 4 8 - 5 1 ) 5 6 ( 5 5 - 5 7 ) 5 3 ( 5 0 - 5 5 )B 6 5 3 ( 4 6 - 6 3 ) 5 4 ( 4 8 - 6 0 ) 5 5 ( 4 7 - 6 4 ) 4 9 ( 4 4 - 5 5 )

A n t i b o d y w a s p u r i f i e d w i t h c h i c k C I I - S e p h a r o s e a n d a s s a y e d o n e i t h e r m o u s e C I I - o r c h i c k C I I -c o a t e d E L I S A p l a te s as d e s c ri b e d i n M a t e r i a ls a n d M e t h o d s . D a t a r e p r e s e n t a n t i - m o u s e C I I E L I S Av a l u e d i v id e d b y t h e a n t i - c h i c k C I I E L I S A v a l u e m u l t ip l i e d b y 1 0 0 ; v a lu e s r e p r e s e n t t h e m e a n ( r a n g eo f t r i p l i c a t e d e t e r m i n a t i o n s ) .

t h e m a x i m a l B 6 I g G 2 a a u t o a n t i b o d y r e s p o n s e w a s 1 4 # g / m l ( g r o u p m e a n , 8 g g /m l ) c o m p a r e d w i t h t h e l o w e s t I g G 2 a a u t o a n t i b o d y r e s p o n s e a s s o c i a t e d w i t ha r t h r i t i s i n D 1 m i c e , i .e . , 6 2 ~ g / m l , m e a s u r e d i n a n a r t h r i t i c D 1 m o u s e i m m u n i z e dw i t h 1 0 Iz g m y c o b a c t e r i a .

Crossreact iv i ty wi th Mouse CI I of Ant ibodies f ro m Susceptible D1 an d Resistant B6Strains. T a b l e I I e x a m i n e s w h e t h e r t h e r e l a ti v e d e f i c i e n c y o f th e l g G 2 a a u t o a n -t i b o d y s u b c l a s s r e s p o n s e o f t h e B 6 s t ra i n s h o w n i n F i g . 1 m i g h t r e p r e s e n t a



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WATSON AND TOWNES 1 8 8 3s u b c l as s -s p e c if i c d e c r e a s e i n c r o s s r e a c t i v i t y w i t h m o u s e C I I . D e t a il s o f t h e m e t h o da r e d e s c r i b e d i n t h e l e g e n d t o T a b l e I I . A s s h o w n , t h e r e w e r e n o m a j o rd i f f e r e n c e s i n t h e r e l a t i v e c r o s s r e a c t i v it i e s o f B 6 a n t i - c h i c k C I I I g G 1 , 2 a , 2 b ,a n d 3 . T h e r e a ls o w a s n o m a j o r d i f f e r e n c e i n c r o ss r e a c ti v i ty w h e n c o m p a r i s o n sw e r e m a d e w i t h D 1 a n t i - c h i c k C I I I g G 1 , 2 a , 2 b , a n d 3 . T h e o v e r a l l m e a np e r c e n t c r o s s re a c t iv i t y o f D 1 o r B 6 a n t i - c h i c k C I I I g G ( % I g G 1 + % I g G 2 a +% I g G 2 b + % I g G 3 / 4 ) w a s 5 4 % ( r a n g e , 5 0 - 5 7 % ) a n d 5 3 % ( r a n g e , 4 9 - 5 5 % ) ,r e s p e c t i v e l y .Affinity of Autoantibody from Susceptible D1 and Resistant B6 Stra ins. T h er e l a t iv e a ff i n it ie s o f D 1 a n d B 6 a n t i b o d y f o r m o u s e C I I w e r e c o m p a r e d u s i n g ac o m p e t i t i v e i n h i b i t i o n E L I S A a s d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s . T h ec o m p a r i s o n r e v e a l e d t h a t 2 7 t ~g /m l o f e i t h e r D 1 o r B 6 c h i c k C I I a n t i b o d yi n h i b i t e d b y 5 0% t h e b i n d i n g o f p e r o x i d a s e - l a b e l e d D1 a n t i - c h i c k C I I I g G 2 a t om o u s e C I I - c o a t e d m i c r o t i t e r p l a te s . T h e r a n g e s f o r 5 0 % i n h i b i t io n i n d u p l i c a t ed e t e r m i n a t i o n s w e r e 2 6 - 2 7 u g / m l f o r D 1 a n t i b o d y a n d 2 4 - 2 9 ~ tg /m l f o r B 6a n t i b o d y .IgG Autoantibody Subclass Response in (B6D1)Fl Hybrids. T h e s u g g e s t e d a s s o -c i a ti o n b e t w e e n s u s c e p ti b il it y a n d t h e I g G 2 a a u t o a n t i b o d y r e s p o n s e w a s f u r t h e re x a m i n e d i n (B 6 D 1 ) F t h y b r i d s . A s s h o w n i n T a b l e I I I , 1 2 o f 1 2 B 6D 1 m i c e w e r eh i g h - p e r c e n t I g G 2 a a u t o a n t i b o d y r e s p o n d e r s ( r a n g e, 5 0 - 9 5 % ) . I n t h e f o u r w it ha r t h r it i s, t o t a l I g G a u t o a n t i b o d y l e v el s r a n g e d f r o m 6 5 t o 3 5 8 u g / m l . A s a re s u l to f h i g h - p e r c e n t I g G 2 a a u t o a n t i b o d y r e s p o n s iv e n e s s , m o s t o f th i s a u t o a n t i b o d yw a s I g G 2 a a n d s e r u m l ev e ls o f I g G 2 a a u t o a n t i b o d y w e r e w i t h in t h e r a n g e o fa r t h r i t i c D 1 l e v e l s . T a b l e I I I a l s o s h o w s t h a t , i n 4 o f 1 2 n o n a r t h r i t i c B 6 m i c e ,t o t a l I g G a u t o a n t i b o d y l e v e l s w e r e s i m i l a r t o a r t h r i t i c B 6 D 1 l e v e l s ( B 6 r a n g e ,7 0 - 1 8 2 / ~ g / m l ) . H o w e v e r , s e r u m I g G 2 a a u t o a n t i b o d y l e ve ls r e m a i n e d s u b st an -t i al l y b e l o w a r t h r i t i c B 6 D 1 l e v el s ( B 6 r a n g e , 6 - 1 4 ~ g / m l ) as a r e s u l t o f l o w -p e r c e n t I g G 2 a a u t o a n t i b o d y r e sp o n s i v e n e s s ( B 6 r a n g e , 6 - 1 6 % ) .Comparison of lgG Autoantibody Subclass Responses in Arthritic a nd NonarthriticB6 and BIO Mice . A s t u d y w a s d e s i g n e d t o c h a l l e n g e t h e r e s i s ta n c e o f t h e B 6s t ra i n b y i n c r e a s in g t h e q u a n t i t y o f m y c o b a c t e r i a - c o n t a i n i n g C F A f r o m 2 5 0 t ~ g/m o u s e to 5 0 0 u g / m o u s e , b o o s t i n g , a n d t r e a t i n g t h e m i c e w i th l o w -d o s e ( 1 0 m g /k g ) C y t o x a n 2 d b e f o r e t h e p r i m a r y a n d b o o s t e r i m m u n i z a t i o n s , i n a n a t t e m p tt o r e d u c e T s u p p r e s s o r c e l l a c t i v i ty ( 1 4 ). A s s h o w n i n T a b l e I V , t h e s e t r e a t m e n t sd i d n o t p r o d u c e a n y m a j o r c h a n g e s i n e i th e r t h e m a g n i t u d e o f t h e t o ta l I g Ga u t o a n t i b o d y r e s p o n s e o r t h e r e l a t i v e I g G s u b c la ss d i s tr i b u t io n o f t h e t o t a l I g Ga u t o a n t i b o d y r e s p o n s e i n m o s t o f t h e B 6 m i c e .H o w e v e r , a r t h r i ti s d i d d e v e l o p i n 2 o f 2 0 o f t h e s e m i c e , o n e t h a t r e c e i v e d t h eh i g h e r d o s e o f C F A a n d t h e o t h e r , t h e h i g h e r d o s e o f C F A p l u s lo w - d os eC y t o x a n . T o t a l I g G a u t o a n t i b o d y l ev e ls w e r e m a r k e d l y e l e v a t e d (T a b l e I V ) i nt h e s e a r th r i t i c m i c e , b u t m o s t o f t h e I g G a u t o a n t i b o d y w a s I g G 2 b . T a b l e I V a l s os h o w s t h e r e s u lt s o f a st u d y w i t h t h e B 1 0 s u b l in e i n w h i c h 1 o f 1 2 m i c e d e v e l o p e da r t h r it i s w i t h t h e l o w e r d o s e o f C F A ( 2 5 0 ~ g / m o u s e ) . A g a i n , t o t a l I g G a u t o a n -t i b o d y l ev e ls w e r e i n c r e a s e d , b u t I g G 2 b a n d n o t I g G 2 a w a s t h e p r e d o m i n a n tI g G a u t o a n t i b o d y s u bc la ss .Comparison of IgG2a Autoantibody Responses in Strains of Mice Differing in H-2and lg-1 Haplotypes and Susceptibility to CII AIA. T a b l e V p r e s e n t s t h e I g G 2 a



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1 8 8 4 MURINE COLLAGEN II AUTOIMMUNE ARTHR ITISTABLE III

Comparison of Arthritic and Nonarthritic IgG Autoantibody Responses in 'B6D1)F1 Hybridsand B6 MiceAnti-mous e CII IgGStrain ArthritisTotal IgG2a IgG2a

#g/ml % #g/mlB6D1 358 60 214 +B6D1 210 95 200 +B6D1 168 53 89 +B6D1 65 83 54 +B6D1 39 72 28B6D1 35 74 26B6D1 29 66 19B6D1 21 71 15B6D1 18 50 9B6D1 18 50 9B6D1 15 60 9B6D1 13 69 9B6 182 6 11B6 86 16 14B6 73 10 7B6 70 9 6

Mice were immunized with 100 ttg chick CII and 250 /~g mycobacteria m IFA. Serum IgGautoantibody subclass levels were determi ned by IgG autoantibody subclass ELISA on mouse CI I-coated microtiter plates as described in Materials and Methods. Total and percent IgG2a weredet erm ine d from individual #g/m l values of IgG1, 2a, 2b, and 3 (only those B6 mice with totalresponse s within the ra nge of B6D1 re sponses are shown here; see Table IV for the analysis of all 12B6 mice).

a u t o a n t i b o d y r e s p o n s e a s a si m p l e m a t h e m a t i c a l p r o d u c t o f t h e # g / m l v a l u e o ft o ta l I g G a u t o a n t i b o d y a n d t h e p e r c e n t I g G 2 a a u t o a n t ib o d y . H - 2 q / I g - l c D 1m i c e w e r e h ig h - to t a l a n d h i g h - p e r c e n t l g G 2 a r e s p o n d e r s , r e s u l t i n g i n I g G 2 aa u t o a n t i b o d y s e r u m l e ve l s o f 6 2 # g / m l o r m o r e i n t h e a r t h r it i c a n i m a l s. I n t h eo n e o f e i g h t D 1 m i c e t h a t f a i l e d t o d e v e l o p a r t h ri t is , t h e s e r u m I g G 2 a a u t o a n t i -b o d y l e ve l w a s 4 4 # g / m l .

T h e H - 2 d / I g - l c D 2 s t ra i n a ls o w a s a h i g h - p e r c e n t I g G 2 a r e s p o n d e r , b u t a lo w -t o t a l Ig G r e s p o n d e r . A s a r e s u lt , I g G 2 a a u t o a n t i b o d y s e r u m l e ve l s r e a c h e d am a x i m u m o f o n l y 2 2 # g / m l a n d a r t h r i t is w a s n o t o b s e r v e d . T o e v a l u a t e t h ep o s s ib i li t y o f a li nk b e t w e e n h i g h - p e r c e n t I g G 2 a r e s p o n si v e n e s s a n d t h e H - 2 dh a p l o t y p e , w e a n a l y z e d t h e r e s p o n s e o f t h e B 1 0 . D 2 / n s t ra i n , w h i c h h a s t h e H -2 d h a p i o t y p e o f t h e D 2 s t r ai n o n t h e B 1 0 ( I g - l b ) b a c k g r o u n d . U n l i k e t h e D 2s t r a i n , t h is H - 2 d c o n g e n i c s t ra i n w a s a l o w - p e r c e n t I g G 2 a r e s p o n d e r , s i m i l a r t ot h e B 1 0 s t r a in . B o t h o f t h e s e l a t t e r s t r a i n s a l so w e r e l o w - t o t a l Ig G r e s p o n d e r s ,r e s u l t i n g i n s e r u m I g G 2 a a u t o a n t i b o d y l e v e l s t h a t f a i l e d t o r e a c h a r t h r i t i c D 1l e v e ls . T h e 8 % v a l u e f o r i n c i d e n c e o f d i s e a s e in t h e B 1 0 s t r a i n s h o w n i n T a b l eV r e f e r s t o t h e 1 o f 1 2 m i c e t h a t d e v e l o p e d d i s e a s e a n d m a r k e d l y e l e v a t e d l e v el so f I g G 2 b a u t o a n t i b o d y ( T a b l e I V ).B 6 D 1 F l h y r i d s ( H - 2 b / q , I g - 1 b / c ) w e r e l o w - t o t a l I g G r e s p o n d e r s s i m i l a r t o



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W A T S O N A N D T O W N E S 1 8 8 5T A B L E I V

Comparison of Arthrit ic a nd N onarthrit ic lgG Autoantibody Responses in B6 and B IO MiceA n t i - m o u s e C I I I g G

G r o u p ( n ) A r t h r i t i s M e a n p e r c e n t ( r a n g e)p g / m l T o t a l1 2 a 2 b 3

B 6 * ( 1 2) - 5 7 ( 1 7 - 1 8 2 ) 2 0 ( 3 - 6 0 ) 2 0 ( 6 - 4 4 ) 4 5 ( 1 6 - 7 3 ) 1 6 ( 6 - 3 9 )B 6 2 ( 9) - 3 1 ( 1 9 - 6 5 ) 8 ( 5 - 1 1 ) 1 4 ( 1 1 - 2 1 ) 5 9 . ( 4 9 - 6 8 ) 1 8 ( 1 5 - 3 0 )B6~ (1) + 573 2 6 78 14B 6 ~ / C Y T ( 9) - 8 9 ( 2 9 - 1 6 6 ) 6 ( 2 - 1 0 ) 1 7 ( 7 - 2 5 ) 6 2 ( 4 8 - 8 5 ) 1 6 ( 6 - 3 0 )B6 2 / C Y T (1 ) + 5 5 5 4 4 8 8 4B 1 0 , ( 1 1) - 1 9 ( 7 - 4 3 ) 1 1 ( 5 - 2 3 ) 2 4 ( 1 6 - 3 0 ) 5 0 ( 2 9 - 6 5 ) 1 7 ( 1 3 - 3 2 )B10t (1) + 122 2 13 78 7Mi ce w ere i m mu n i zed w i t h 1 0 0 p g ch ick CII an d e i t h e r 2 5 0 (B1 0 1) o r 5 0 0 (B6~ ) p g my co b ac t e r i ai n I F A : B 6 m i c e a l s o w e r e b o o s t e d w i t h 1 0 0 # g c h i c k C I I i n I F A 8 0 d a f t e r t h e p r i m a r yi m m u n i z a t i o n . C y t o x a n ( C Y T ) ( 1 0 m g / k g ) w a s a d m i n i s t e r e d b y t h e i n t r a p e r i t o n e a l r o u t e 2 d b e f o r et h e p r i m a r y a n d b o o s t e r i m m u n i z a t i o n s ; B 6 m i c e w e r e b l e d 1 5 d a f t e r t h e b o o s t e r , 4 a n d 7 d b e f o r et h e o n s e t o f a r t h r i ti s i n t w o m i c e ; B 1 0 m i c e w e r e i m m u n i z e d o n c e a n d b l e d o n d a y 4 4 , 5 d a f t e rt h e o n se t o f a r t h r i t is i n o n e m o u se . To t a l an d p e rcen t Ig G au t o an t i b o d y su b c la s s v a l u es w e red e r i v e d f r o m i n d i v i d u a l # g / m l a u t o a n t i b o d y s u b c l a s s v a l u e s d e t e r m i n e d b y I g G a u t o a n t i b o d ys u bc l as s E L I S A o n m o u s e C I I - c o a t e d m i c r o t i t e r p la t es , a s e x p la i n e d i n M a t e r i al s a n d M e t h o d s.* N o n a r t h r i t i c B 6 m i c e im m u n i z e d o n c e w i t h 1 0 0 p g c h i c k C I I a n d 2 5 0 p g m y c o b a c t e r i a in I F A a n db l ed o n d ay 4 4 (F i g . 1 ) .

T A B L E VGenetic Variation in the Total IgG Autoantibody Response, the Percent IgG2a Autoantibody

Response, and Susceptibil i ty to C II AIAA n t i - m o u s e C I I l g G [ m e a n ( r a n g e ) ]S t ra i n (n ) H -2 Ig -1 A r t h r i t i sT o t a l I g G 2 a I g G 2 a

pg/ml % #g/ml %D 1 ( 8) q c 7 0 0 ( 8 1 - 1 9 4 3 ) 7 9 ( 5 4 - 9 7 ) 5 6 0 ( 4 4 - 1 4 4 8 ) 8 8D 2 (6 ) d c 2 0 (8 -4 7 ) 6 3 (4 7 -8 2 ) 1 2 (5 -2 2 ) 0B I 0 ( 1 2 ) b b 2 7 ( 7 - 1 2 2 ) 2 4 ( 1 3 - 3 8 ) 5 ( 2 - 1 6 ) 8B1 0 .D 2 / n (6 ) d b 6 (4 -1 0 ) 3 8 (2 0 -5 0 ) 2 (2 -3 ) 0B6 (1 2 ) b b 5 7 (1 7 - 1 8 2 ) 2 0 (6 -4 4 ) 8 (5 -1 4 ) 0B 6 D 1 ( 1 2 ) b / q b / c 8 2 ( 1 3 - 3 5 8 ) 6 7 ( 5 0 - 9 5 ) 5 7 ( 9 - 2 1 4 ) 3 3B 6 D 1 D 1 ( 5) - - - - 8 4 0 ( 2 4 2 - 1 2 3 1 ) 7 9 ( 7 0 - 8 5 ) 6 6 6 ( 1 8 7 - 9 7 0 ) 1 0 0S W R ( 8) q c 7 7 0 ( 1 2 1 - 2 2 6 7 ) 7 9 ( 6 7 - 9 0 ) 6 2 6 ( 1 0 8 - 1 9 4 5 ) 0

M i c e w e r e i m m u n i z e d w i t h 1 0 0 p g c h i ck C I I a n d 2 5 0 p g m y c o b a c t e r i a in I F A a s d e s c r i b e d p r e v io u s l yfo r D I (Tab l e I ) , B1 0 (Tab l e IV ) , B6 (F i g . 1) , an d B6 D 1 (T ab l e l l I ) s t ra i n s ; s e ru m Ig G au t o an t i b o d ys u b cl a ss l e ve l s w e r e d e t e r m i n e d b y I g G a u t o a n t i b o d y s u b c l as s E L I S A o n m o u s e C l l - c o a t e d m i c r o t i t e rp l a te s a s d e s c r i b e d i n M a t e r i al s a n d M e t h o d s . T o t a l a n d p e r c e n t I g G 2 a w e r e d e t e r m i n e d f r o mi n d i v i d u a l # g / m l v a l u es o f Ig G 1 , 2 a , 2 b , an d 3 .

t h e B 6 p a r e n t s t r a in , b u t h i g h - p e r c e n t I g G 2 a r e s p o n d e r s s i m i l a r t o t h e D 1 p a r e n ts t r a in . I n t h o s e B 6 D 1 m i c e a t t h e u p p e r r a n g e o f t h e to t a l r e s p o n s e , I g G 2 aa u t o a n t i b o d y l e v e l s w e r e i n t h e r a n g e o f a r t h r i t i c D 1 l e v e ls a n d d i s e a s e w a s



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1 8 8 6 M U R I N E C O L L A G E N I I A U T O 1 M M U N E A R T H R I T I So b s e r v e d . I n t h e f i v e b a c k c r o s s e s t o D 1 , t o t a l I g G r e s p o n s e s w e r e l o w i n o n e a n dh i g h i n t h e o t h e r f o u r , w h i l e t h e p e r c e n t I g G 2 a r e s p o n s e w a s h i g h i n a ll fi v e. I nt h e m o u s e w i th l o w to t al I g G a u t o a n t i b o d y , h o w e v e r , t h e I g G 2 a a u t o a n t i b o d yleve l was i n t he r ang e o f a r t h r i t i c D1 l eve ls ; d i sea se was obse rve d i n t h is an ima la s it w a s i n th e o t h e r f o u r . B a s e d u p o n t h e r e s p o n s e s o f b o t h t h e B 6 D I a n d D 1b a c k c r o s s e s , h i g h - p e r c e n t I g G 2 a r e s p o n s i v e n e s s a p p e a r s t o b e i n h e r i t e d a s ad o m i n a n t t r a i t .F i n al ly , t h e I g G 2 a a u t o a n t i b o d y r e s p o n s e o f t h e re s i s t an t a n d C 5 - d e f i c i e n t (1 5 )H - 2 q / I g - l c S W R s t ra i n w a s c o m p a r e d w i t h t h a t o f t h e D 1 s t r ai n ( T a b l e V ) .A r t h r i t i s w a s a b s e n t i n a ll e i g h t S W R m i c e , d e s p i t e t h e i n d u c t i o n o f a n a u t o i m -m u n e r e s p o n s e t h a t w a s e q u i v a l e n t t o t h a t o f a r t h r i t i c D 1 m i c e in t e r m s o f t o t a lI g G a u t o a n t i b o d y a n d p e r c e n t I g G 2 a a u t o a n t i b o d y .

D i s c u s s i o nT h e r e s u lt s o f t hi s s tu d y d e m o n s t r a t e a n i m p o r t a n t r o le f o r t h e I g G a u t o a n t i -

b o d y s u b c la s s r e s p o n s e i n C I I A I A . I n t h e s u s c e p t i b l e D 1 s t r a in , t h e I g Ga u t o a n t i b o d y r e s p o n s e c o n s i s te d p r e d o m i n a n t l y o f t h e I g G 2 a s u b cl as s a n d , i n t h er e s i s t a n t B 6 s t r ai n , t h e I g G 2 a a u t o a n t i b o d y s u b c l a ss r e s p o n s e w a s re l a t iv e l yd e f i c i e n t , d e s p i t e s i m i l a r I g G l , 2 b , a n d 3 a u t o a n t i b o d y r e s p o n s e s . E s t i m a t e s o fB 6 a n t i b o d y c r o s s r e a c t i v i t y a n d a f f i n i t y f o r m o u s e C I I w e r e s i m i l a r t o t h o s e o fD 1 a n t i b o d y , i n d i c a t i n g t h a t t h e s e p a r a m e t e r s w e r e n o t r e l e v a n t t o r e s is t a n c e t oa r t h r i t i s i n t h i s s t r a i n . I n r e c e n t s t u d i e s , B 6 m i c e w e r e f o u n d t o r e a d i l y d e v e l o pa r t h r i t is a f t e r p a s s i v e t r a n s f e r o f a f f in i t y - p u r i f i e d r a t a n t i- C I I a n t i b o d y ( u n p u b -l i shed da t a ) , wh ich i s t ho ug h t t o cons i s t p r im ar i l y o f t he I gG 2a subc l a ss (5 , 18 ) .S t u d i e s b y F a r k a s e t a l. ( 1 6 ) a n d E y e t a l. ( 1 7 ) s u g g e s t t h a t r a t I g G 2 a a n d m o u s eI g G 2 a a r e s i m i l a r , s i n c e I g G 2 a f r o m e i t h e r s p e c i e s i s t h e m o s t p o t e n t a c t i v a t o ro f th e c la s si ca l c o m p l e m e n t c a s c a d e , w i th l g G 2 b b e i n g l es s p o t e n t b u t m o r ee f f e c t i v e t h a n I g G 1 . T h u s , t h is p as s i v e t r a n s f e r s t u d y t e n d s t o r u l e o u t t h ep o s s i b il i ty th a t r e s i s t a n c e i n t h e B 6 s t ra i n w a s d u e t o d e f e c t s in h u m o r a l e f f e c t o rs y s te m s , e .g . , c o m p l e m e n t c o m p o n e n t d e f i c ie n c ie s a n d / o r i n f l a m m a t o r y c e lld e f e c t s , a n d a l s o p r o v i d e s s u p p o r t f o r t h e l i n k b e t w e e n s u s c e p t i b il i ty a n d t h eI g G 2 a s u b c l a s s . I n ( B 6 D 1 ) F 1 h y b r i d s , a r t h r i t i s d e v e l o p e d o n l y i n t h o s e a n i m a l sw i t h I g G 2 a a u t o a n t i b o d y r e s p o n s e s th a t a p p r o x i m a t e d o r e x c e e d e d t h o s e i na r t h r i t i c D 1 a n im a l s , f u r t h e r s u p p o r t i n g t h e i m p o r t a n c e o f t h e I g G 2 a a u t o a n t i -b o d y r e s p o n s e i n d e t e r m i n i n g s u s c e p t i b i l i t y t o C I I A I A .A s a d d i ti o n a l e v i d e n c e f o r t h e p o t e n t i a l p a t h o g e n i c i t y o f t h e I g G 2 a s u b c l a ss ,w e r e c e n t l y f o u n d ( 6 ) t h a t p u r i f i e d D 1 I g G 2 a a n t i b o d y w i l l a c t i v a t e C 5 t o C 5 aw h e n b o u n d t o c a r t i l a g e i n v i t r o a n d t h a t C 5 - d e f i c i e n t m i c e a r e r e s i s t a n t t oa r t h r i t is b y p as s i v e t r a n s f e r o f C I I a n t i b o d y . T h u s , t h e f a i l u r e o f B 6 m i c e tod e v e l o p d i s ea s e , d e s p i t e th e p r o d u c t i o n o f to t a l I g G a u t o a n t i b o d y l e v e ls w i t h i nt h e r a n g e o f a r t h r i t ic D 1 m i c e , m i g h t b e e x p l a i n e d b y t h e r e l a ti v e d e c r e a s e i nt h i s p o t e n t c o m p l e m e n t - a c t i v a t i n g s u b c l a s s .A s i d e f r o m t h e g r e a t e r c o m p l e m e n t - a c t i v a ti n g p o t e n c y o f I g G 2 a , i t is a ls o o fi n t e re s t t h a t b o u n d i m m u n e c o m p l e x e s c o n si s ti n g o f I g G 2 a a p p e a r t o b e t h em o s t p o t e n t a c t i v at o r s o f F c r e c e p t o r - b e a r i n g m a c r o p h a g e s ( 19 ). T h i s f i n d i n gm i g h t b e r e l e v a n t t o a f u r t h e r p a t h o g e n i c r o l e fo r t h e l g G 2 a a u t o a n t i b o d y



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WATSON AND TOWNES 1887s u b cl as s i n p r o m o t i n g t h e a c t i v a ti o n o f m o n o n u c l e a r c e l l / m a c r o p h a g e - l i k e ce ll sf o u n d i n a r e a s o f c a rt i la g e e r o s i o n i n C I I A I A .I t sh o u l d b e n o t e d t h a t I g G 2 b a n d 1 a r e n o t d e v o i d o f e it h e r c o m p l e m e n t -( 1 7 ) o r m a c r o p h a g e ( 1 9 ) -a c t iv a t in g a c t iv i ty , b u t r a t h e r a r e le ss a c t iv e t h a n I g G 2 a .T h u s , i t w o u l d b e r e a s o n a b l e t o e x p e c t t h a t C I I A I A c o u l d b e i n d u c e d b y e i t h e rs ubc la s s , i f a s u f f i c i e n t qua n t i t y i s p r od uc e d a nd b i nds t o t h e a r t i c u l a r c a r t i la ge .T h e d e v e l o p m e n t o f a r t h r i ti s i n t h r e e o t h e r w i s e re s i st a n t B6 a n d B 1 0 m i c e( T a b l e I V ) a p p e a r s t o r e p r e s e n t e v i d e n c e f o r s u c h a p o s si b il it y in t h e c a se o f t h eI g G 2 b s u b c l a s s . T h e r e c e n t r e p o r t b y W o o l e y e t a l . p r o v i d e s a d d i t i o n a l s u p p o r tfor th i s poss ib i l ity (20) .T h e f i n d i n g t h a t s u s ce p t ib i li ty t o C I I A I A is l i n k e d t o t h e p r e d o m i n a n tr e s p o n s e o f a s i n g le I g G s u b cl as s, e . g ., I g G 2 a o r 2 b , h a s r e l e v a n c e t o i m m u n o -r e g u l a t o r y m e c h a n i s m s a s w e ll as t o p o t e n t i a l s t ra t e g ie s f o r m o d u l a t i n g d i s e a se si n v o l v in g a p a r t i c u l a r a u t o a n t i b o d y s u bc la s s. T h e r e g u l a t i o n o f I g G 2 a e x p r e s s io ni s l i nke d w i t h s pec i f ic T c e l l m e c ha n i s m s (21 ) . M o du l a t i o n o f the s e po t e n t i a ls u b cl as s r e g u l a t o r y m e c h a n i s m s i n t h e D 1 s t ra i n , f o r e x a m p l e , m i g h t p r o v i d e am o r e s e l ec t iv e i m m u n o s u p p r e s s i v e e f f e c t b y n o t i n t e r f e r i n g w i t h I g G 1 o r 3 ( o rI gE , A , e t c . ) a n t i body s ubc l a s s r e s pons e s t o o t he r a n t i ge ns , e . g . , ba c t e r i a l , v i r a l ,t u m o r , e t c .A n a l y si s o f t h e i m m u n o g e n e t i c s o f t h e I g G 2 a a u t o a n t i b o d y s u b cl as s re s p o n s ei n T a b l e V s u g g e s t s t h a t C I I A I A r e p r e s e n t s a m o d e l o f a u t o i m m u n i t y in w h i c hs u sc e p ti b il it y is d e p e n d e n t u p o n t h e i n t e r a c ti o n o f b o t h M H C a n d n o n - M H Cg e n e s . A c c o r d i n g t o t h is m o d e l , M H C g e n e s , s p e ci fi c al ly I- A g e n e s , r e g u l a t e t h em a g n i t u d e o f t h e t o ta l I g G a u t o a n t i b o d y r e s p o ns e , a n d n o n - M H C g e n e s, p e r h ap sI g h - C g e n e s , r e g u l a t e t h e r e l a t i v e fr a c t i o n o f I g G a u t o a n t i b o d y a s I g G 2 a . S u c ha m o d e l w a s in i ti a ll y s u g g e s t e d b y t h e d i ss o c ia t io n o f t h e m a g n i t u d e o f t h e t o t a lI g G a u t o a n t i b o d y r e s p o n se a n d t h e r e l a ti v e p e r c e n t o f I g G 2 a a u t o a n t i b o d yo b s e r v e d i n t h e D 1 - D 2 a n d B 6 - B 6D 1 c o m p a r i s o n s ( T a b l e V ). T h e g e n e r a lc o n c e p t t h a t H - 2 g e n e s a r e i n v o lv e d in r e g u l a t i n g t h e m a g n i t u d e o f h u m o r a lr e s pons e s i s w e l l e s t a b l i s he d ( 22 ) . I n t h e c a s e o f C I I A I A , W oo l e y e t a l . (7 ) a ndH u s e e t a l. ( 8) u s e d H - 2 r e c o m b i na n t s t r a i n s o f m i c e t o s ugge s t t ha t s u s c e p t i b il i tya nd t he h i gh ( t o t a l ) a n t i body r e s pons e t o C I I i n t he D 1 s t r a i n i s l i nke d t o t he I -A q h a p l o t y p e o f t h e H - 2 o r M H C .T h e D 2 - B 1 0 . D 2 / n c o m p a r i s o n i n T a b l e V p r o v i d e s t h e b a s i s f o r s u g g e s t i n gt h a t n o n - M H C g e n e s m i g h t b e i n v o l v e d in r e g u l a t i n g t h e r e l a t iv e p e r c e n t o f to t a lI g G a u t o a n t i b o d y a s Ig G 2 a . T h e H - 2 d / I g - l c D 2 s t ra in w a s a h i g h - p e rc e n t I g G 2 aa u t o a n t i b o d y r e s p o n d e r , w h i le th e H - 2 d / I g - l b B 1 0 . D 2 / n c o n g e n i c s tr a in w a s al o w - p e r c e n t I g G 2 a a u t o a n t i b o d y r e s p o n d e r . T h u s , h i g h - p e r c e n t I g G 2 a a u t o a n -t i b o d y r e s p o n s i v e n e s s w a s n o t l i n k e d t o t h e H - 2 h a p l o t y p e o f t h e D 2 s t r ai n . T h i st r a i t al so d o e s n o t a p p e a r t o b e l i n k e d t o t h e H - 2 h a p l o t y p e o f t h e D 1 s t r a in ,s i n ce I g G 2 b a n d n o t I g G 2 a w a s d e s c r i b e d as t h e p r e d o m i n a n t I g G a n t i b o d ys ubc la s s i n t he H - 2 c o nge n i c B 10 . Q s t r a in ( 20 ) . B o t h o f t he s e c om pa r i s onsp r o v i d e s t r o n g e v i d e n c e o f a li nk b e t w e e n t h e p e r c e n t I g G 2 a a u t o a n t i b o d ys ubc l a s s r e s pons e a nd a non - M H C l oc us ( o r l oc i ) .I d i o t y p e s a n d a l lo t y p e s a r e k n o w n t o b e l i n k e d to i m m u n o g l o b u l i n g e n e s . W es u g g e s t t h a t a n I g h - C g e n e ( s ) m i g h t b e i n v o l v e d i n d e t e r m i n i n g t h e r e l a t i v ef r a c t i o n o f to t a l l g G a u t o a n t i b o d y a s I g G 2 a . T h i s s u g g e s t i o n is b a s e d u p o n t h e



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1 8 8 8 MURINE COLLAGEN 11 AUTOIMMUNE ARTHRITISf i n d i n g t h a t ( a ) t h e p e r c e n t I g G 2 a a u t o a n t i b o d y r e s p o n s e d i f f e r e d i n H - 2 c o n -g e n i c s t ra i n s ( T a b l e V , D 2 v s. B 1 0 . D 2 / n ) , t h e r e b y s u g g e s t i n g l i n k ag e t o a l o c u so u t s i d e t h e H - 2 o r M H C , a n d ( b) t h e r e i s a li nk b e t w e e n h i g h - p e r c e n t I g G 2 aa u t o a n t i b o d y r e s p o n s i v e n e s s i n I g - l c D 1 , D 2 , a n d S W R s t r a i n s ( T a b l e V ) . A ni n t e re s t in g c o n s e q u e n c e o f I g h - C g e n e i n v o l v e m e n t m i g h t b e t h e u s e o f Ig -1a l l o t y p e s i n p r e d i c t i n g s u s c e p t i b i l i t y t o a d i s e a s e i n v o l v i n g a p a r t i c u l a r I g Gs u b c l a s s s u c h a s I g G 2 a . F o r e x a m p l e , i n t h e c a se o f a n e x p e r i m e n t a l d i s e a s e l ik eC I I A I A , t h e p r e s e n c e o f th e I g - l c a l l o t y p e w o u l d p r e d i c t h ig h - p e r c e n t I g G 2 aa u t o a n t i b o d y r e s p o n s i v e n e s s a n d t h e p o s s i b i li t y o f d is e a s e . A s s h o w n i n T a b l e V ,h i g h - p e r c en t I g G 2 a a u t o a n t i b o d y r e s p o n s i v en e s s a p p e a r s t o b e a d o m i n a n t t ra it ,s o th a t h i g h - p e r c e n t I g G 2 a a u t o a n t i b o d y r e s p o n s i v e n e ss w o u l d b e e x p e c t e d e v e ni n i n d i v i d u a ls h e t e r o z y g o u s f o r t h e I g - l c a l l o t y p e . O f c o u r s e , a d d i t i o n a l s t u d i e sw i t h a l lo t y p e c o n g e n i c s t r a in s a r e n e e d e d t o d e t e r m i n e i f t h is t r a i t is l i n k e d t othe Igh-C locus , a s we l l a s t o de t e r m ine i f t h i s tr a i t i s a ssoc i a t ed wi th o t he r Ig -1a l l o types ( Ig - l a , d , e , e t c . ) .I n a r e c e n t s t u d y , W o o l e y e t a l. ( 2 3 ) n o t e d t h a t t h e D 1 a n d S W R s t r a in s a r eb o t h H - 2 q a n d a r e e q u a l l y i m m u n o r e s p o n s i v e t o C I I , y e t t h e D 1 s tr a in iss u s c e p t i b l e t o C I I A I A , w h i l e th e S W R s t r a i n i s r e s i st a n t. W e a n a l y z e d t h e I g Ga u t o a n t i b o d y s u b cl a ss re s p o n s e o f t h e S W R s t r ai n a n d , a s s h o w n i n T a b l e V ,t o t a l I g G a u t o a n t i b o d y a n d p e r c e n t I g G 2 a a u t o a n t i b o d y r e s p o n s e s w e r e h i g ha n d e s s e n ti a ll y i d e n ti c a l t o t h o s e o f t h e D 1 s t ra i n . B a s e d u p o n t h e d e m o n s t r a t e dr e s i s t a n c e o f C 5 - d e f i c i e n t m i c e t o p a s s i v e l y t r a n s f e r r e d d i s e a s e ( 6 ) , w e s u g g e s tt ha t r e s i s t ance i n t he S W R s t r a in i s r e l a t e d t o t he f ac t t ha t t h i s s t r a in i s gene t i ca l l yd e f i c i e n t i n C 5 ( 1 5 ) . C 5 i s l i n k e d t o t h e H c l o c u s , w h i c h p r o b a b l y i s n o t w i t h i nt h e M H C ( 2 4 ). T h u s , s u s c e p t i b i l it y t o C I I A I A a p p e a r s t o b e li n k e d t o t w o n o n -M H C g e n e s, o n e t h a t d e t e r m i n e s t h e a v ai la b il it y o f C 5 a n d a n o t h e r w h i c he n s u r e s t h a t a h i gh p e r c e n t o f to t al I g G a u t o a n t i b o d y is a n a p p r o p r i a t e c o m p l e -m e n t a c t i v a t in g s u b cl a ss , a n d t o a n M H C g e n e t h a t d e t e r m i n e s t h e o v e r a l lm a g n i t u d e o f th e I g G a u t o a n t i b o d y r e s p o n s e .T h e d a t a i n T a b l e s I I I a n d V e m p h a s i z e t h a t n o n e o f th e s e g e n e t i c fa c t o r sa l o n e i s s u f f i c i e n t f o r d i s e a s e . T h e 8 o f 1 2 B 6 D 1 m i c e t h a t f a i l e d t o d e v e l o pd i s ea s e w e r e h i g h - p e rc e n t I g G 2 a a u t o a n t i b o d y r e s p o n d e r s , a s w e r e D 2 m i c e.H o w e v e r , i n t h e s e c a s e s , t o t a l I g G a u t o a n t i b o d y l e v e l s w e r e t o o l o w , i . e . , b e l o wt h e l o w e s t I g G 2 a a u t o a n t i b o d y l e v e l a s s o c i a t e d w i t h d i s e a s e . I n t h e c a s e o fn o n a r t h r i t i c B 6 m i c e , to t a l r e s p o n s e s w e r e w i t h i n t h e r a n g e o f a r t h r i t ic B 6 D 1l e v e l s , b u t t h e p e r c e n t I g G 2 a r e s p o n s e w a s t o o l o w t o e n s u r e a d e q u a t e s e r u mI g G 2 a a u t o a n t i b o d y l ev e ls . F in a l ly , t h e r e s i s t a n t S W R s t r a i n i s b o t h a h i g h - to t a la n d a h i g h - p e r c e n t I g G 2 a r e s p o n d e r , b u t i s g e n e t i c a ll y d e f i c ie n t in C 5 .

I n c o n c l u s i o n , s u s c e p t i b i l i t y t o C I I A I A c a n b e r e g a r d e d a s a c o m p l e x i n t e r -a c t io n o f m u l ti p l e M H C a n d n o n - M H C g e n e s th a t c o n t r o l n o t o n l y t h e t o ta lh u m o r a l r e s p o n s e , b u t a l s o t h e q u a l i t y o f t h is r e s p o n s e , e . g . , t h e I g G 2 s u b c l a ssa s w e ll as t h e a v a i la b i li t y o f m e d i a t o r s o f t h e i n f l a m m a t o r y r e s p o n s e s u c h a sc o m p l e m e n t C 5 .

S u m m a r yA n a l y s i s o f t h e I g G a u t o a n t i b o d y s u b c l a s s r e s p o n s e i n t h e c o l la g e n I I a u t o i m -m u n e a r t h r i t i s ( C I I A I A ) - s u s c e p t i b l e D 1 s t r a i n m i c e r e v e a l e d t h a t t h e o n s e t o f



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W A T S O N A N D T O W N E S 1 8 8 9d i s ea s e w as as s o c ia t e d w i th a p r e d o m i n a n c e o f I g G 2 a a u t o a n t i b o d y . I n a c o m -p a r a t i v e s t u d y , r e s i s t a n c e i n t h e B 6 s t r a i n w a s a s s o c i a t e d w i t h a d e f i c i e n t I g G 2 aa u t o a n t i b o d y r e s p o n s e . B 6 I g G 1 , 2 b , a n d 3 a u t o a n t i b o d y r e s p o n s e s g e n e ra l lyo v e r l a p p e d t h o s e o f a r t h r it i c D 1 m i c e , a n d e s t i m a t e s o f a n t i b o d y c r o s s r e a c t i v i t ya n d a f f i n i ty w e r e s i m i l a r f o r b o t h s t ra i n s. I n c r o s s e s b e t w e e n D 1 a n d B 6 , a r th r i t i sd e v e l o p e d o n l y in t h o s e F j m i c e w i th I g G 2 a a u t o a n t i b o d y r e s p o n s e s a p p r o x i m a t -i n g o r e x c e e d i n g t h o s e i n a r t h r i t i c D 1 m i c e . A d d i t i o n a l s t u d i e s w i t h B 6 a n d B 1 0s t ra in s s u g g e s t e d a n a l t e r n a t e r o l e f o r t h e I g G 2 b a u t o a n t i b o d y r e s p o n s e .

I n i n b r e d s t r a i n s w i t h k n o w n g e n e t i c b a c k g r o u n d s , a d i s s o c i a t i o n b e t w e e n t h em a g n i t u d e o f th e t o t a l I g G a u t o a n t i b o d y r e s p o n s e a n d t h e p e r c e n t o f to t a l a sI g G 2 a w a s d e m o n s t r a t e d . T h e H - 2 q , I g - l c D 1 s t r a in w a s a h i g h - t o ta l a n d h i g h -p e r c e n t I g G 2 a r e s p o n d e r , w h i l e t h e H - 2 d , I g - l c D 2 s t ra i n w a s a l o w - t o ta l b u th i g h - p e r c e n t I g G 2 a r e s p o n d e r . T h e H - 2 b , I g - l b B 6 s tr a in w a s a lo w - t ot a l a n dl o w - p e r c e n t I g G 2 a r e s p o n d e r , w h i le t h e H - 2 b / q , I g - l b / c ( B 6 D 1 )F ~ h y b r i d w a s al o w - to t a l b u t h i g h - p e r c e n t I g G 2 a r e s p o n d e r . A f u r t h e r d i ss o c ia ti o n b e t w e e nh i g h - p e r c e n t I g G 2 a a u t o a n t i b o d y r e s p o n s i v e n e s s a n d t h e H - 2 h a p i o t y p e w a sd e m o n s t r a t e d b y th e H - 2 c o n g e n i c B 1 0 . D 2 / n ( H - 2 d , I g - l b ) s t ra i n, i n w h i c h al o w - p e r c e n t I g G 2 a r e s p o n s e w a s o b s e r v e d t o d i f f e r f r o m t h e D 2 s t ra in . H i g h -p e r c e n t I g G 2 a a u t o a n t i b o d y r e s p o n s i v e n e ss a ls o a p p e a r e d t o b e in h e r i t e d a s ad o m i n a n t t r a i t b a s e d u p o n h i g h r e s p o n s e s i n a ll ( B 6 D 1 )F ~ h y b r i d s a n d b a c k c r o s s e st o D 1 . T h e s e f i n d i n g s s u g g e s t t h a t t h e H - 2 h a p l o t y p e i s i n v o l v e d i n t h e t o t a l I g Ga u t o a n t i b o d y r e s p o n s e b u t t h a t t h e r e l a t iv e f r a c t io n o f th e t o t a l re s p o n s e a sI g G 2 a is i n d e p e n d e n t o f t h e H - 2 h a p l o t y p e a n d p o s s ib l y r e l a t e d t o I g h - C g e n e s.

C 5 - d e f i c ie n t S W R ( H - 2 q , I g - l c ) m i c e w e r e f o u n d t o h a v e a h i g h t o t a l a u t o a n -t i b o d y r es p o n s e t o m o u s e C I I a n d I g G 2 a c o m p a r a b l e t o ar t h ri t ic D 1 m i c e , b u tt h e s e m i c e d i d n o t d e v e l o p a r t h r i t i s .

B a s e d u p o n t h e s e o b s e r v a t i o n s , w e c o n c l u d e t h a t s u s c e p t i b i li t y t o C I I A I Ar e q u i r e s t h e i n t e r a c t i o n o f m u l t i p l e g e n e s , b o t h m a j o r h i s t o c o m p a t i b i l ty c o m p l e x( M H C ) a n d n o n - M H C , w h i c h i n fl u e n c e t h e m a g n i t u d e ( to t al I gG ) a n d t h e q u al it y( I g G s u b c la s s ) o f t h e a u t o i m m u n e r e s p o n s e a n d t h e a v a il a b il i ty o f a p p r o p r i a t em e d i a t o r s ( C 5 ) t o i n i t i a t e t h e i n f l a m m a t o r y r e a c t i o n .Received or publication 15July 1985.

R e f e r e n c e s1. Trentham, D. E. , A. S. Townes, and A. H. Kang. 1977. Autoimmuni ty to type I Icollagen: an e xperim ental m odel o f arthrit is. J . Exp. Med. 146:857.2. C ourte nay, J. S., M. J. D ailman, A . D. Dyan, A. Martin, and B. Mosedale. 1980.Imm unisation against hete rolog ous type II collagen induces arthrit is in mice. Nature

(Lond.). 283:666.3. Morgan, K., R. B. Clague, M. J. Shaw, S. A. Firth, T . M . Tw ose, and P. J. L. H olt .1981. Nat ive type I I ca l logen-induced arthr i ti s in the ra t. Th e effec t of complem entdeple t ion by cobra venom fac tor . Arthritis Rheum. 24:1356.4. Stuar t , J . M . , K. Tomod a, T . J . Yoo, A. S. Tow nes, and A. H. Kang. 1983. Serumtransfer of collagen-induced arthrit is. II. Identification and localization of autoanti-bod y to type I I col lagen in don or an d rec ipient ra ts . Arthritis Rheum. 26:1237.5. Kerw ar, S. S., M. E. Englert , R. A. McReynolds, M . J. L andes, J. M . Lloyd, A. L.



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1 8 9 0 M U R I N E C O L L A G E N I I A U T O I M M U N E A R T H R I T I SO ro n s k y , a n d F . J . Wi l s o n . 1 9 8 3 . T y p e I I c o l l a g e n - in d u c e d a r th r i t i s . S tu d ie s w i thp u r i f i e d a n t i c o ll a g e n i m m u n o g l o b u l i n . Ar t h r i ti s R h e u m . 2 6 :1 1 2 0 .

6 . Wa t s o n , W. C . , A . S . T o w n e s , P . S . B ro w n , a n d J . A . P i t c o c k . 1 9 8 5 . In v i vo a n d i nvi tro e v id e n c e o f a ro l e fo r c o m p le m e n t C 5 in ty p e I I c o l l a g e n - in d u c e d a r th r i ti s . Clin.Res . 3 3 :7 9 4 A . (A b s t r . )

7 . Wo o le y , P . H . , H . S . L u th ra , J . M, S tu a r t , a n d C . S . D a v id . 1 9 8 1 . T y p e I I c o l l a g en -in d u c e d a r th r i t i s i n m ic e . I . Ma jo r h i s to c o m p a t ib i l i t y c o m p le x ( I r e g io n ) l i n k a g e a n da n t i b o d y c o r r e l a t e s . J . Ex p . M e d . 1 5 4 :6 8 8 .

8 . H u s e , A . , P . H . W o o le y , H . S . L u th ra , J . M . S tu a r t , a n d C . S . D a v id . 1 9 8 4 . N e wre c o m b in a n t s c o n f i rm m a p p in g o f s u sc e p t ib il i t y to ty p e I I c o l l a g e n - in d u c e d a r th r i t i sto th e I -A s u b re g io n in m ic e . Fed. Proc. 4 3 :1 8 2 0 (A b s t r . 2 3 5 8 ) .

9 . N i l ss o n , U . R ., a n d H . J . M u l l e r -E b e rh a rd . 1 9 6 7 . D e f i c i e n c y o f t h e fi f t h c o m p o n e n to f c o m p l e m e n t i n m i c e w i th a n i n h e r i t e d c o m p l e m e n t d e f ec t . J . Exp . Med . 125:1 .

1 0 . S tu a r t , J . M . , M. A . C re m e r , S . N . D ix i t, A . H . K a n g , a n d A , S . T o w n e s . 1 9 7 9 .C o l l a g e n - in d u c e d a r th r i t i s i n r a t s . C o m p a r i s o n o f v i t r e o u s a n d c a r t i l a g e -d e r iv e dco l lagens . Archr i t i s Rheum. 2 2 :3 4 7 .

1 1 . V o l l e r , A . , D . E . B id w e l l, a n d A . B a r t l e t t . 1 9 7 6 . E n z y m e im m u n o a s s a y s in d i a g n o s t i cm e d i c i n e. T h e o r y a n d p r a c ti c e . B u l l . W H O . 53:55 .

1 2 . E y , P . L . , S . J . P ro w s e , a n d C . R . J e n k in . 1 9 7 8 . I s o l a t io n o f p u re Ig G I , Ig G 2 a , a n dI g G 2 b i m m u n o g l o b u l i n s f r o m m o u s e s e r u m u s i n g p r o t e i n A - S e p h a r o s e . I m m u n o c h e m -istry. 15:429 .

1 3 . A v ra m e a s , S. , T . T e r n y n c k , a n d J . L . G u e s d o n . 1 9 7 8 . C o u p l in g o f e n z y m e s toa n t ib o d ie s a n d a n t ig e n s . S c a n d . J . I m m u n o l . 8:7.

1 4 . V la d u t iu , A . O . 1 9 8 2 . A u t o im m u n e th y ro id it i s : c o n v e r s io n o f l o w - re s p o n d e r m ic e toh i g h - r e s p o n d e r s b y c y c l o p h o s p ha m i d e . Cl in . Exp . Immuno l . 4 7 :6 8 3 .

1 5. E r i c k s o n , R . P ., D . K . T a c h ib a n a , L . A . H e rz e n b e rg , a n d L . T . R o s e n b e rg . 1 9 6 4 . As i ng l e g e n e c o n t r o l l in g h e m o l y t i c c o m p l e m e n t a n d a s e r u m a n t i g en i n t h e m o u s e . J .I m m u n o l . 9 2 :6 1 1 .

1 6 . F a rk a s , A . I ., G . A . Me d g y e s i , G . F u s t, K . Mik lo s , a n d J . G e rg e ly . 1 9 8 2 . Im m u n o g e n -i c ity o f a n t ig e n c o m p le x e d w i th a n t ib o d y . I . R o le o f d i f f e re n t i s o ty p e s. Immuno logy .4 5 :4 8 3 .

1 7. E y , P. L . , G . J . R u s s e ll - J on e s , a n d C . R . J e n k in . 1 9 8 0 . I s o ty p e o f m o u s e Ig G 1 .E v i d e n c e f o r ' n o n - c o m p l e m e n t - f i x i n g ' Ig G 1 a n t i b o d ie s a n d c h a r a c te r i z a ti o n o f t h e i rc a p a c i ty to in t e r f e re w i th Ig G 2 s e n s i t i z at io n o f t a rg e t r e d b lo o d c e ll s fo r l ys is b yc o m p l e m e n t . M o l . I m m u n o l . 17:699 .

18 . F ir th , S . A. , K. Mo rgan , H . B. Evans , and P . J . L . H ol t . 1984 . Ig G subc lasses inc o l l a g e n - in d u c e d a r th r i t i s i n th e r a t . Immuno l . Le t t . 7:243 .

1 9. E z e k o w i tz , R . A . B ., M. B a m p to n , a n d S . G o rd o n . 1 9 8 3 . Ma c ro p h a g e a c t iv a t io ns e le c tiv e ly e n h a n c e s e x p re s s io n o f F c r e c e p to r s fo r Ig G 2 a . J . Exp . Med . 1 5 7 :8 0 7 .

2 0 . Wo o le y , P . H . , H . S . L u th ra , A . H u s e , a n d C . S . D a v id . 1 9 8 5 . Im m u n o g e n e t i cre g u la t io n o f t h e im m u n e re s p o n s e to c h ic k ty p e I I c o l l a g e n in m ic e . Fed. Proc.4 4 :9 5 5 (A b s t r . 3 1 8 0 ) .2 1 . R o s e n b e rg , Y . J . 1 9 8 2 . I s o ty p e -s p e c i f i c T c e ll r e g u la t io n o f im m u n o g lo b u l in e x p re s -s ion . I m m u n o l . Re v . 6 7 :3 3 .

2 2 . M c D e v i t t , H . O . , a n d B . B e n a c e r ra f . 1 9 6 9 . G e n e t i c c o n t ro l o f s p ec i f ic im m u n ere s p o n s e s . Ad v . I m m u n o l . 11:31 .

2 3 . Wo o le y , P . H . , H . S . L u th ra , A . H u s e , a n d C . S . D a v id . 1 9 8 5 . R e g u la t io n o fs u s c e p t ib i l i t y to ty p e I I c o l l a g e n - in d u c e d a r th r i t i s (C IA ) b y g e n e s m a p p in g e x te r io r



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W AT S ON AND T OW NE S 1 8 9 1t o t h e m a j o r h i s t o c o mp a t i b i li t y c o m p l e x ( H- 2 ) i n mi c e. Arthritis Rheum. 2 8 : $ 4 2 ( A b s t r.170).

2 4 . He r z e n b e r g , L . A . , D . K . T a c h i b a n a , L . A . He r z e n b e r g , a n d L . T . Ro s e n b e r g . ! 9 6 3 .A g e n e l o c u s c o n c e r n e d wi t h h e mo l y t i c c o mp l e me n t i n Mu s mu s c u l u s . Genetics.4 8 : 7 1 1 .


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Published December 1, 1985