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Possibility of a direct pyrogenic effect of muramyl dipeptide
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7
INDUCTION OF ADJUVANT ARTHRITIS S . N a g a o a n d A. T a n a k a Department
of Biochemistry,
IN R A T S
Shimane
BY MURAMYL
Medical
DIPEPTIDE
University,
Izumo
(MDP) 693,
Japan
Tubercle bacilli and their adjuvant-active c o m p o n e n t s s u c h as w a x D, peptidoglycans, a n d t h e i r f r a g m e n t s i n d u c e a d j u v a n t a r t h r i t i s . A m o n g the peptidoglycan fragments hitherto tested, disaccharide-heptapeptided i s a c c h a r i d e w a s f o u n d to be t h e s m a l l e s t a r t h r i t o g e n i c s t r u c t u r e a n d t h e m i n i m a l s t r u c t u r e in the b a c t e r i a l c e l l w a l l s r e s p o n s i b l e for t h e i r a d j u v a n t activity, muramyl dipeptide (MDP), h a s b e e n r e p o r t e d n o t to b e a r t h r i t o g e n i c . W e h a v e f o u n d t h a t M D P d i d c a u s e a d j u v a n t a r t h r i t i s in W K A rats, w h e n i n c o r p o r a t e d in a w a t e r - i n - o i l e m u l s i o n c o n s i s t i n g o f an e q u a l v o l u m e o f Incomplete Freund's Adjuvant of Difco Laboratories and phosphate buffered s a l i n e . W h e n , h o w e v e r , i n c o r p o r a t e d in the s a m e t y p e o f e m u l s i o n p r e p a r e d b y D r a c k e o l a n d A r l a c e l A, M D P d i d n o t i n d u c e t h e a r t h r i t i s , s u g g e s t i n g an i m p o r t a n c e o f a r o l e p l a y e d b y the v e h i c l e f o r t h e d e v e l o p m e n t of t h e d i s e a s e . A s i g n i f i c a n t c o r r e l a t i o n e x i s t e d b e t w e e n the s w e l l i n g o f the i n j e c t i o n s i t e s a n d t h e d e v e l o p m e n t o f the d i s e a s e . In c o n t r a s t to g r a n u l o m a f o r m a t i o n b y MDP, t h e i n d u c t i o n o f a r t h r i t i s b y M D P s e e m s to r e q u i r e T c e l l s , since M D P d i d n o t i n d u c e t h e a r t h r i t i s in n e o n a t a l l y t h y m e c t o m i s e d W K A rats. M D P w a s a c t i v e as a d j u v a n t in t h e s e W K A r a t s b u t w e c o u l d n o t d e t e c t a n y a n t i g e n i c i t y o f M D P in t h e s e W K A rats, w h e n M D P w a s i n j e c t e d in t h e s a m e w a y as for a r t h r i t i s i n d u c t i o n . T h e p r e s e n t d a t a i n d i c a t e t h a t M D P is arthritogenic. T h i s m a y b e o f i m p o r t a n c e , b e c a u s e , f i r s t l y , it s u g g e s t s t h a t a n y a t t e m p t s to a p p l y M D P to h u m a n s s h o u l d be d o n e c a u t i o u s l y , and, s e c o n d l y , it w i l l c o n t r i b u t e to o u r u n d e r s t a n d i n g of t h e p a t h o g e n e s i s of t h e adjuvant disease.
P O S S I B I L I T Y OF A D I R E C T P Y R O G E N I C E F F E C T OF M U R A M Y L D I P E P T I D E . G. R i v e a u z K. M a s e k t M. P a r a n t and L. C h e d i d G r o u p e de R e c h e r c h e n°31 du C . N . R . S . , I m m u n o p h a r m a c o l o g i e exp4rimentale, I n s t i t u t P a s t e u r , 7 5 7 2 4 P a r i s cedex 15, F r a n c e . M u r a m y l d i p e p t i d e (MDP) is a low m o l e c u l a r w e i g h t s y n t h e t i c a d j u v a n t j a n a l o g of a s t r e p t o c o c c a l p e p t i d o g l y c a n s u b u n i t . It r e p r e s e n t s the m i n i m a l s t r u c t u r e e s s e n t i a l for e l i c i t i n g the a d j u v a n t e f f e c t a s c r i b e d in F r e u n d ' s c o m p l e t e a d j u v a n t . M D P was l i k e w i s e shown to be the m i n i m a l r e q u i r e m e n t for p e p t i d o glycan-induced c h a n g e s in t e m p e r a t u r e . T h e fever p a t t e r n o b s e r v e d in the r a b b i t a f t e r an i n t r a v e n o u s i n j e c t i o n of M D P is s i m i l a r to the b i p h a s i c resp o n s e s i n d u c e d by b a c t e r i a l p y r o g e n s . H o w e v e r , i n t r a c e r e b r o v e n t r i c u l a r (ICV) administration of M D P e l i c i t e d a m o n o p h a s i c f e v e r w i t h a p e a k b e t w e e n 3 and 4 hr f o l l o w i n g i n j e c t i o n , and by this r o u t e the m i n i m a l p y r o g e n i c dose ( 2 . 5 x i 0 -7 DM) was l o w e r e d of about 2 0 0 , O 0 0 - f o l d . N e i t h e r the p e p t i d e nor the s u g a r m o i e t y of M D P i n d u c e d any f e b r i l e r e s p o n s e at the d o s a g e level of iOODg s h o w i n g that m u r a m y l d i p e p t i d e r e p r e s e n t s the m i n i m a l a c t i v e s t r u c t u r e . T h e p r e s e n c e of t r a n s f e r a b l e c i r c u l a t i n g e n d o g e n o u s p y r o g e n was c l e a r l y shown in the b l o o d of r a b b i t s m a d e f e b r i l e by an i n t r a v e n o u s i n j e c t i o n of MDP. P l a s m a t r a n s f e r e x p e r i m e n t s a f t e r ICV a d m i n i s t r a t i o n f a i l e d to d e m o n s t r a t e any p y r o g e n i c a c t i v i t y in the b l o o d t a k e n from d o n o r s at v a r i o u s time. M o r e over, p r e v i o u s t r e a t m e n t w i t h n i t r o g e n m u s t a r d did not m o d i f y the f e b r i l e r e s p o n s e to MDP, but i n h i b i t e d p r o d u c t i o n of c i r c u l a t i n g e n d o g e n o u s p y r o g e n . By c o n t r a s t , i n d o m e t h a c i n did not a f f e c t in vitro or in vi~o p r o d u c t i o n of p y r o g e n by r a b b i t cells a l t h o u g h it b l o c k e d f e b r i l e r e s p o n s e s to IV or ICV a d m i n i s t r a t i o n of MDP. T h i s t r e a t m e n t , h o w e v e r , did not d e c r e a s e , and even e n h a n c e d i m m u n o s t i m u l a n t p r o p e r t i e s of MDP. T h e s e r e s u l t s c o n f i r m that p y r o g e n i c i t y is not e s s e n t i a l for s t i m u l a n t p r o p e r t i e s of MDP, and show that even in the a b s e n c e of e n d o g e n o u s p y r o g e n M D P can i n d u c e a f e b r i l e r e s p o n s e .
7
INDUCTION OF ADJUVANT ARTHRITIS S . N a g a o a n d A. T a n a k a Department
of Biochemistry,
IN R A T S
Shimane
BY MURAMYL
Medical
DIPEPTIDE
University,
Izumo
(MDP) 693,
Japan
Tubercle bacilli and their adjuvant-active c o m p o n e n t s s u c h as w a x D, peptidoglycans, a n d t h e i r f r a g m e n t s i n d u c e a d j u v a n t a r t h r i t i s . A m o n g the peptidoglycan fragments hitherto tested, disaccharide-heptapeptided i s a c c h a r i d e w a s f o u n d to be t h e s m a l l e s t a r t h r i t o g e n i c s t r u c t u r e a n d t h e m i n i m a l s t r u c t u r e in the b a c t e r i a l c e l l w a l l s r e s p o n s i b l e for t h e i r a d j u v a n t activity, muramyl dipeptide (MDP), h a s b e e n r e p o r t e d n o t to b e a r t h r i t o g e n i c . W e h a v e f o u n d t h a t M D P d i d c a u s e a d j u v a n t a r t h r i t i s in W K A rats, w h e n i n c o r p o r a t e d in a w a t e r - i n - o i l e m u l s i o n c o n s i s t i n g o f an e q u a l v o l u m e o f Incomplete Freund's Adjuvant of Difco Laboratories and phosphate buffered s a l i n e . W h e n , h o w e v e r , i n c o r p o r a t e d in the s a m e t y p e o f e m u l s i o n p r e p a r e d b y D r a c k e o l a n d A r l a c e l A, M D P d i d n o t i n d u c e t h e a r t h r i t i s , s u g g e s t i n g an i m p o r t a n c e o f a r o l e p l a y e d b y the v e h i c l e f o r t h e d e v e l o p m e n t of t h e d i s e a s e . A s i g n i f i c a n t c o r r e l a t i o n e x i s t e d b e t w e e n the s w e l l i n g o f the i n j e c t i o n s i t e s a n d t h e d e v e l o p m e n t o f the d i s e a s e . In c o n t r a s t to g r a n u l o m a f o r m a t i o n b y MDP, t h e i n d u c t i o n o f a r t h r i t i s b y M D P s e e m s to r e q u i r e T c e l l s , since M D P d i d n o t i n d u c e t h e a r t h r i t i s in n e o n a t a l l y t h y m e c t o m i s e d W K A rats. M D P w a s a c t i v e as a d j u v a n t in t h e s e W K A r a t s b u t w e c o u l d n o t d e t e c t a n y a n t i g e n i c i t y o f M D P in t h e s e W K A rats, w h e n M D P w a s i n j e c t e d in t h e s a m e w a y as for a r t h r i t i s i n d u c t i o n . T h e p r e s e n t d a t a i n d i c a t e t h a t M D P is arthritogenic. T h i s m a y b e o f i m p o r t a n c e , b e c a u s e , f i r s t l y , it s u g g e s t s t h a t a n y a t t e m p t s to a p p l y M D P to h u m a n s s h o u l d be d o n e c a u t i o u s l y , and, s e c o n d l y , it w i l l c o n t r i b u t e to o u r u n d e r s t a n d i n g of t h e p a t h o g e n e s i s of t h e adjuvant disease.
P O S S I B I L I T Y OF A D I R E C T P Y R O G E N I C E F F E C T OF M U R A M Y L D I P E P T I D E . G. R i v e a u z K. M a s e k t M. P a r a n t and L. C h e d i d G r o u p e de R e c h e r c h e n°31 du C . N . R . S . , I m m u n o p h a r m a c o l o g i e exp4rimentale, I n s t i t u t P a s t e u r , 7 5 7 2 4 P a r i s cedex 15, F r a n c e . M u r a m y l d i p e p t i d e (MDP) is a low m o l e c u l a r w e i g h t s y n t h e t i c a d j u v a n t j a n a l o g of a s t r e p t o c o c c a l p e p t i d o g l y c a n s u b u n i t . It r e p r e s e n t s the m i n i m a l s t r u c t u r e e s s e n t i a l for e l i c i t i n g the a d j u v a n t e f f e c t a s c r i b e d in F r e u n d ' s c o m p l e t e a d j u v a n t . M D P was l i k e w i s e shown to be the m i n i m a l r e q u i r e m e n t for p e p t i d o glycan-induced c h a n g e s in t e m p e r a t u r e . T h e fever p a t t e r n o b s e r v e d in the r a b b i t a f t e r an i n t r a v e n o u s i n j e c t i o n of M D P is s i m i l a r to the b i p h a s i c resp o n s e s i n d u c e d by b a c t e r i a l p y r o g e n s . H o w e v e r , i n t r a c e r e b r o v e n t r i c u l a r (ICV) administration of M D P e l i c i t e d a m o n o p h a s i c f e v e r w i t h a p e a k b e t w e e n 3 and 4 hr f o l l o w i n g i n j e c t i o n , and by this r o u t e the m i n i m a l p y r o g e n i c dose ( 2 . 5 x i 0 -7 DM) was l o w e r e d of about 2 0 0 , O 0 0 - f o l d . N e i t h e r the p e p t i d e nor the s u g a r m o i e t y of M D P i n d u c e d any f e b r i l e r e s p o n s e at the d o s a g e level of iOODg s h o w i n g that m u r a m y l d i p e p t i d e r e p r e s e n t s the m i n i m a l a c t i v e s t r u c t u r e . T h e p r e s e n c e of t r a n s f e r a b l e c i r c u l a t i n g e n d o g e n o u s p y r o g e n was c l e a r l y shown in the b l o o d of r a b b i t s m a d e f e b r i l e by an i n t r a v e n o u s i n j e c t i o n of MDP. P l a s m a t r a n s f e r e x p e r i m e n t s a f t e r ICV a d m i n i s t r a t i o n f a i l e d to d e m o n s t r a t e any p y r o g e n i c a c t i v i t y in the b l o o d t a k e n from d o n o r s at v a r i o u s time. M o r e over, p r e v i o u s t r e a t m e n t w i t h n i t r o g e n m u s t a r d did not m o d i f y the f e b r i l e r e s p o n s e to MDP, but i n h i b i t e d p r o d u c t i o n of c i r c u l a t i n g e n d o g e n o u s p y r o g e n . By c o n t r a s t , i n d o m e t h a c i n did not a f f e c t in vitro or in vi~o p r o d u c t i o n of p y r o g e n by r a b b i t cells a l t h o u g h it b l o c k e d f e b r i l e r e s p o n s e s to IV or ICV a d m i n i s t r a t i o n of MDP. T h i s t r e a t m e n t , h o w e v e r , did not d e c r e a s e , and even e n h a n c e d i m m u n o s t i m u l a n t p r o p e r t i e s of MDP. T h e s e r e s u l t s c o n f i r m that p y r o g e n i c i t y is not e s s e n t i a l for s t i m u l a n t p r o p e r t i e s of MDP, and show that even in the a b s e n c e of e n d o g e n o u s p y r o g e n M D P can i n d u c e a f e b r i l e r e s p o n s e .