LYMPHOKINE RESEARCH Volume 8, Number 3, 1989 Mary Ann Liebert, Inc., Publishers
Activation of T Cells by Interleukin 1 Involves Internalization of Interleukin 1
WERNER F A L K , ILKA VON HÖGEN, and PETER H . K R A M M E R
Institute for Immunology and Genetics, German Cancer Research Center, D-6900 Heidelberg, FRG
ABSTRACT
The c y t o k i n e i n t e r l e u k i n - 1 p l a y s a n i m p o r t a n t r o l e i n t h e i n d u c t i o n o f I L - 2 s e c r e t i o n and I L - 2 r e c e p t o r e x p r e s s i o n . The e v e n t s t h a t f o l l o w b i n d i n g o f I L - 1 t o i t s r e c e p t o r a r e n o t known. We f o u n d t h a t i n a p u r i f i e d T c e l l p o p u l a t i o n ( c o m p r i s i n g t h e L y t2" and L3T4" T c e l l s ) I L - 1 i n t h e a b s e n c e o f a n t i g e n o r m i t o g e n i n d u c e d s t r o n g p r o l i f e r a t i o n and de novo e x p r e s s i o n o f IL-2R l i g h t c h a i n mRNA.
T h i s was a c c o m p a n i e d by h i g h a f f i n i t y I L - 2 b i n d i n g . P r o d u c t i o n o f I L - 2 o r I L - 2 mRNA was n o t d e t e c t e d u n d e r t h e s e c o n d i t i o n s . As a model s y s t e m f o r I L - 1 a c t i o n two EL4 s u b c l o n e s were i s o l a t e d . EL4 5D3 r e s p o n d e d t o I L - 1 by a u g m e n t a t i o n o f PMA i n d u c e d I L - 2 s e c r e t i o n and IL-2R e x p r e s s i o n . EL4D6/76 bound I L - 1 w i t h t h e same a f f i n i t y b u t d i d n o t r e s p o n d . We f o u n d t h a t t h i s l i n e was u n a b l e t o i n t e r n a l i z e s u r f a c e bound I L - 1 . The f i n d i n g s u g g e s t s t h a t i n T c e l l s i n t e r n a l i z a t i o n o f I L - 1 i s r e q u i r e d f o r i t s a c t i v i t y .
INTRODUCTION
I n t e r l e u k i n - 1 ( I L - 1 ) e x e r t s two m a i n e f f e c t s on T c e l l s . T o g e t h e r w i t h m i t o g e n o r a n t i g e n i t i n d u c e s t h e s e c r e t i o n o f i n t e r l e u k i n - 2 ( I L - 2 ) ( 1 ) and t h e e x p r e s s i o n o f I L - 2 r e c e p t o r s ( I L - 2 R ) ( 2 ) . When mouse t h y m o c y t e s were s t i m u l a t e d w i t h I L - 1 i n t h e a b s e n c e o f added m i t o g e n o r a n t i g e n e x p r e s s i o n o f IL-2R was o b s e r v e d ( 3 ) . T h i s e f f e c t was o n l y s e e n i n t h e p r e s e n c e o f I L - 2 and was d e t e r m i n e d by b i n d i n g o f a m o n o c l o n a l a n t i b o d y a g a i n s t t h e IL-2R and by p r o l i f e r a t i o n . We c o n c l u d e d f r o m t h e s e e x p e r i m e n t s t h a t i n t h e absence o f m i t o g e n o r a n t i g e n I L - 1 i n d u c e s o n l y e x p r e s s i o n o f IL-2R and no I L - 2 s e c r e t i o n . To o b t a i n i n s i g h t i n t o t h e mechanism o f t h e s e phenomena we a t t e m p t e d 1. t o i d e n t i f y a p u r i f i e d t a r g e t c e l l p o p u l a t i o n f o r t h i s e f f e c t , and 2. t o e s t a b l i s h a model s y s t e m t o be a b l e t o s t u d y t h e mechanism on a b i o c h e m i c a l and m o l e c u l a r l e v e l .
A s u b p o p u l a t i o n o f L y t2" and L3T4" t h y m o c y t e s (DNT) s t r o n g l y r e s p o n d e d t o t h e c o m b i n a t i o n o f b o t h c y t o k i n e s i n a s y n e r g i s t i c f a s h i o n . We f o u n d t h a t t h e main e f f e c t o f I L - 1 was t o s t i m u l a t e de novo e x p r e s s i o n o f h i g h a f f i n i t y IL-2R. S i n c e t h e m u r i n e thymoma EL4 c a n r e s p o n d t o I L - 1 by a u g m e n t a t i o n o f I L - 2 s e c r e t i o n and IL-2R e x p r e s s i o n (4) we s u b c l o n e d EL4 and s e l e c t e d two s u b c l o n e s t h a t d i f f e r e d i n r e s p o n s i v e n e s s t o I L - 1 w i t h r e s p e c t t o b o t h p r o p e r t i e s . U s i n g c l o n e s we show t h a t i n t e r n a l i z a t i o n o f I L - 1 c o r r e l a t e s w i t h I L - 1 a c t i v i t y .
MATERIALS AND METHODS.
C e l l C u l t u r e s : C e l l s were c u l t u r e d i n RPMI 1640 c o n t a i n i n g 52 FCS and 50 /*l/ml g e n t a m y c i n a t 37°C i n a h u m i d i f i e d atmosphere o f 52 C 02 i n a i r . Thymocytes h a d 1 0 2 FCS and 3 x l O_ 5M 2 - m e r c a p t o e t h a n o l i n a d d i t i o n .
P r e p a r a t i o n o f DNT: DNT were p r e p a r e d f r o m thymuses o f 4-week o l d C3H m i c e by a n t i b o d y and complement t r e a t m e n t (5).The m i x t u r e i n c l u d e d a n t i b o d i e s a g a i n s t t h e a n t i g e n s L3T4, L y t2, I ak, a n d MAC-1. The y i e l d was 11 o f t o t a l t h y m i c c e l l s They were 80-902 Thy-1 p o s i t i v e and 45-502 IL-2R p o s i t i v e .
P r o l i f e r a t i o n A s s a y s : P r o l i f e r a t i o n was a s s e s s e d by u s i n g s t a n d a r d methods a s d e s c r i b e d ( 5 ) .
Recombinant C y t o k i n e s : Human (hu) r I L - l o was k i n d l y p r o v i d e d by D r s . A. S t e r n and P. L o m e d i c o , Hoffmann-LaRoche, N u t l e y , N J , USA. The s p e c i f i c a c t i v i t y was 5xl06TJ/mg p r o t e i n d e t e r m i n e d by t h e l y m p h o c y t e a c t i v a t i n g f a c t o r a s s a y . P u r i f i e d human r e c o m b i n a n t I L - 2 f r o m E. c o l i was k i n d l y s u p p l i e d by C e t u s C o r p o r a t i o n , E m e r y v i l l e , USA.
A s s a y f o r I L - 2 R e c e p t o r E x p r e s s i o n : A f f i n i t y p u r i f i e d mab 7D4 ( 6 ) was r a d i o i o d i n a t e d u s i n g t h e c h l o r a m i n T method. C e l l s were washed and r e s u s p e n d e d i n 90 pi b i n d i n g medium (RPMI 1640, 0.006M Hepes, IX BSA, 0.12 N a - A z i d e ) . mab 7D4 was added i n 10 / i l b i n d i n g medium t o a f i n a l c o n c e n t r a t i o n o f 4 pg/ml. Bound r e a d i o a c t i v i t y was d e t e r m i n e d a s d e s c r i b e d ( 7 ) .
RNA E x t r a c t i o n a n d H y b r i d i z a t i o n P r o c e d u r e s : C y t o p l a s m i c RNA was p r e p a r e d by t h e method o f C h e l e y and A n d e r s o n ( 8 ) . RNA f o r N o r t h e r n a n a l y s i s was p r e p a r e d by t h e g u a n i d i n e - i s o t h i o c y a n a t e / c e s i u m c h l o r i d e method. H y b r i d i z a t i o n s were done i n t h e p r e s e n c e o f d e x t r a n s u l p h a t e and formamide a t 42°C. The p r o b e f o r t h e mouse I L - 2 was g e n e r o u s l y p r o v i d e d by D r . A. S c h i m p l (Würzburg, FRG) and c o n t a i n s t h e c o m p l e t e exon 4 o f t h e I L - 2 gene ( 9 ) . The probe f o r t h e mouse IL-2R was g e n e r o u s l y p r o v i d e d by D r . T. M a l e k ( M i a m i , F L , U S A ) . I t r e p r e s e n t s t h e E c o R I - P s t l f r a g m e n t o f t h e IL-2R cDNA ( c l o n e p m I L - 2 p r l ) ( 1 0 ) .
I o d i n a t i o n o f h u r I L - l a : P u r i f i e d h u r I L - l a was l a b e l e d w i t h 125I by u t i l i z i n g t h e Enzymobead r e a g e n t a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s ( B i o R a d Richmond, CA). B i o l o g i c a l a c t i v i t y o f t h e i o d i n a t e d m a t e r i a l was 80 t o 902 o f t h e o r i g i n a l m a t e r i a l ( d a t a n o t shown).
B i n d i n g o f L a b e l e d C y t o k i n e s t o C e l l s : C e l l s were washed a n d i n c u b a t e d w i t h l a b e l e d c y t o k i n e s a s d e s c r i b e d ( 5 ) . T c e l l s were i n c u b a t e d f o r 2h a t 37°C i n f r e s h medium p r i o r t o b i n d i n g . 1 2 5I - I L - 2 was p u r c h a s e d f r o m NEN (NEX 229, D r e i e i c h , FRG) o r Amersham ( I M 197, F r a n k f u r t , FRG). The b i n d i n g r e a c t i o n was t e r m i n a t e d by c e n t r i f u g i n g t h e c e l l s t h r o u g h a n o i l m i x t u r e . S p e c i f i c b i n d i n g i s r e p r e s e n t e d a s t h e d i f f e r e n c e i n cpm bound by c e l l s w i t h and w i t h o u t n o n l a b e l e d c y t o k i n e .
I n t e r n a l i z a t i o n o f 1 2 5I - r I L - l a : 4 x l 06 c e l l s and 8 U o f 1 2 5I - r I L - l o were p r e i n c u b a t e d i n 500 pi RPMI 1640 c o n t a i n i n g 52 FCS a t 8°C f o r 1 h and t h e n t r a n s f e r r e d t o 37°C.
Samples were removed a t t h e i n d i c a t e d t i m e s . C e l l s were p e l l e t e d by c e n t r i f u g a t i o n a n d r e s u s p e n d e d i n 500 pi pH 3 b u f f e r e d medium. A f t e r i n c u b a t i o n on i c e f o r 5 m i n t o remove s u r f a c e bound r a d i o a c t i v i t y c e l l s were c e n t r i f u g e d a g a i n and l y s e d i n 200 plA l y s i s b u f f e r (10 mM T r i s H C l , pH 7.4; 150 mM N a C l ; IX T r i t o n X-114; 2mM PMSF; 22 I s o p r o p a n o l ) . A f t e r 10 m i n i n c u b a t i o n on i c e t h e n u c l e a r and c y t o p l a s m i c f r a c t i o n s were s e p a r a t e d by c e n t r i f u g a t i o n a t lOOOOxg a t 8°C f o r 10 m i n . R a d i o a c t i v i t y was d e t e r m i n e d i n a gamma c o u n t e r .
RESULTS AND DISCUSSION
We were prompted t o i n v e s t i g a t e DNT f o r a I L - 1 r e s p o n s e i n t h e a b s e n c e o f m i t o g e n o r a n t i g e n b e c a u s e i t was p u b l i s h e d t h a t DNT c a r r y r e c e p t o r s f o r I L - 1 and r e s p o n d t o I L - 1 i n t h e p r e s e n c e o f m i t o g e n s ( 1 1 ) . We knew f r o m o u r p r e v i o u s e x p e r i m e n t s t h a t t h e r e was a l o w p e r c e n t a g e o f I L - l - r e s p o n d e r c e l l s among t h y m o c y t e s . T h e r e f o r e , DNT were p r e p a r e d f r o m thymuses o f a d u l t m i c e and t e s t e d f o r a s y n e r g i s t i c r e s p o n s e t o t h e c o m b i n a t i o n o f I L - 1 and I L - 2 . I t was shown t h a t t h e s y n e r g i s t i c e f f e c t i n t h e p r e s e n c e o f s a t u r a t i n g c o n c e n t r a t i o n s o f I L - 2 was I L - 1 - dose dependent ( 5 ) . N e i t h e r I L - 1 n o r I L - 2 a l o n e h a d any e f f e c t on g r o w t h .
I n o t h e r e x p e r i m e n t s ( n o t shown) we showed that, t h e p r o l i f e r a t i v e r e s p o n s e was dependent on I L - 2 ( t h e r e s p o n s e was i n h i b i t e d t o 902 by mab a g a i n s t t h e I L - 2 R )
1 2 3 4 5 6 7
1 2 3 4 5 6 7Figure I i K i n e t i c s of IL-2 and IL-2R mRNA. DNT were c u l t u r e d w i t h I L - 1 (10 U/ml) and I L - 2 (50 U/ml) f o r 1 t o 3 d a y s . C y t o p l a s m i c RNA f r o m s u c h c e l l s was b l o t t e d i n s e r i a l t w o f o l d d i l u t i o n s and p r o b e d w i t h p r o b e s f o r t h e l i g h t c h a i n o f t h e I L - 2R a n d I L - 2 . The l e f t p a n e l o f F i g . 2 shows t h a t t h e r e was r e l a t i v e l y l i t t l e I L - 2R mRNA on day 0 ( l a n e 3 ) , no d e t e c t a b l e mRNA on day 1 ( l a n e 4) a n d d r a m a t i c a l l y i n c r e a s i n g amounts on days 2 and 3 ( l a n e s 5 a n d 6 ) .
and t h a t t h i s i n h i b i t i o n was c o u n t e r a c t e d by I L - 1 ( 5 ) . I t was a l s o shown t h a t a n 18h p u l s e w i t h I L - 1 was s u f f i c i e n t t o i n d u c e m a x i m a l p r o l i f e r a t i o n s u g g e s t i n g a d i f f e r e n t i a t i o n s i g n a l g i v e n by I L - 1 . ( 5 ) . These d a t a l e d t o t h e c o n c l u s i o n t h a t I L - 1 o n l y s t i m u l a t e d IL-2R e x p r e s s i o n b u t n o t I L - 2 s e c r e t i o n i n DNT. T h i s h y p o t h e s i s was c o n f i r m e d on t h e mRNA l e v e l and by I L - 2 b i n d i n g .
The c o n t r o l s i n c l u d e d i n t h e d o t b l o t were RNA f r o m a n I L - 2 - d e p e n d e n t c e l l l i n e ( l a n e 1 ) , RNA f r o m PMA s t i m u l a t e d EL4 ( l a n e 2 ) , and RNA f r o m t h e s t i m u l a t e d macrophage c e l l l i n e P388 D l ( l a n e 7 ) . The r i g h t p a n e l o f F i g . 2 shows t h a t I L - 2 mRNA u n d e r t h e s e c o n d i t i o n s was u n d e t e c t a b l e i n DNT. T h i s f i n d i n g was c o r r o b o r a t e d by t h e b i n d i n g o f 1 2 5I - I L - 2 under h i g h a f f i n i t y c o n d i t i o n s . DNT were c u l t u r e d i n I L - 1 p l u s I L - 2 f o r 4 days and e a c h a n a l i q u o t was removed f o r b i n d i n g . 6 x l 05 c e l l s p e r a s s a y w e l l were i n c u b a t e d i n 20nM 1 2 5I - I L - 2 a n d t h e amount o f r a d i o a c t i v i t y bound was d e t e r m i n e d . I L - 2 b i n d i n g e x a c t l y f o l l o w e d t h e p a t t e r n o f IL-2R mRNA e x p r e s s i o n . S p e c i f i c cpm bound on days 0, 1, 2, and 3 were 1501, 132, 2712, a n d 3482, r e s p e c t i v e l y . S c a t c h a r d a n a l y s i s p e r f o r m e d on day 3 o f c u l t u r e showed t h a t t h e I L - 1 i n d u c e d de novo e x p r e s s e d r e c e p t o r was o f h i g h a f f i n i t y ( KD 23 t o 38 pM) ( 5 ) . We c o n c l u d e d f r o m t h e s e e x p e r i m e n t s t h a t I L - 1 i n t h e a b s e n c e o f m i t o g e n s o n l y s t i m u l a t e s h i g h a f f i n i t y IL-2R e x p r e s s i o n . F o r t h e s e c r e t i o n o f I L - 2 by T c e l l s a second s i g n a l l i k e a m i t o g e n i s r e q u i r e d . I n o u r e x p e r i m e n t a l s y s t e m i t m i g h t be p o s s i b l e t h a t a second s i g n a l f o r IL-2R e x p r e s s i o n i s p r o v i d e d by I L - 2 . I t i s known t h a t I L - 2 u p r e g u l a t e s i t s own r e c e p t o r ( 1 2 ) . I t was i m p o s s i b l e i n o u r s y s t e m t o s t u d y t h e e f f e c t o f e i t h e r c y t o k i n e a l o n e s i n c e t h e c e l l s d i d n o t s u r v i v e w i t h o u t I L - 2 . Whether t h e p75 c h a i n o f t h e IL-2R was a l s o r e g u l a t e d c a n n o t be answered by o u r e x p e r i m e n t s . There i s good e v i d e n c e , however, f o r t h e i t s c o n s t i t u t i v e e x p r e s s i o n t h y m o c y t e s ( 1 3 ) .
The mechanism o f a c t i o n o f I L - 1 i n T c e l l s i s n o t known. L i k e o t h e r g r o w t h f a c t o r s I L - 1 i s r a p i d l y i n t e r n a l i z e d ( 1 4 ) . To s t u d y e a r l y e v e n t s i n I L - 1 t r i g g e r e d T c e l l r e s p o n s e s we c l o n e d by l i m i t i n g d i l u t i o n two EL4 c e l l l i n e s t h a t d i f f e r e d i n t h e i r I L - 1 r e s p o n s e . EL4 5D3 r e s p o n d e d t o I L - 1 w i t h a u g m e n t a t i o n o f I L - 2 s e c r e t i o n a n d IL-2R e x p r e s s i o n whereas EL4D6/76 d i d n o t r e s p o n d t o I L - 1 . C e l l s o f b o t h l i n e s were s t i m u l a t e d a t 1 06/ m l w i t h PMA (10ng/ml) a n d PMA p l u s I L - 1 (10U/ml). A f t e r 24h o f c u l t u r e I L - 2 t i t e r s were measured i n t h e s u p e r n a t a n t and IL-2R e x p r e s s i o n on t h e c e l l s was d e t e r m i n e d . I n EL4 5D3 I L - 1 augmented IL-2R e x p r e s s i o n (measured by b i n d i n g o f l a b e l e d 7D4 a n t i - I L - 2 R a n t i b o d y ) by a f a c t o r o f 5.8 a n d I L - 2 s e c r e t i o n by 3.2. These numbers v a r i e d between e x p e r i m e n t s (range 2 - 1 0 ) . No I L - l - i n d u c e d i n c r e a s e i n b o t h p a r a m e t e r s was o b s e r v e d i n EL4D6/76. B o t h
• #
l i n e s p r o d u c e d c o m p a r a b l e amounts o f I L - 2 (220 U/ml v e r s u s 130 U/ml when s t i m u l a t e d w i t h PMA) a n d bound a c o m p a r a b l e amount o f a n t i b o d y (1.300 cpm/105
c e l l s v e r s u s 1.929 cpm). I t was shown b e f o r e t h a t I L - 1 b i n d s t o s p e c i f i c c e l l s u r f a c e r e c e p t o r s ( 1 5 ) . The most o b v i o u s e x p l a n a t i o n , t h e r e f o r e , f o r t h i s o b s e r v e d l a c k o f I L - 1 r e s p o n s i v e n e s s o f c l o n e EL4D6/76 was a m i s s i n g I L - 1 R on t h i s c e l l . T h i s q u e s t i o n was i n v e s t i g a t e d by d e t e r m i n i n g number and a f f i n i t y o f IL-1R on b o t h c l o n e s by e v a l u a t i o n o f S c a t c h a r d p l o t s o f 1 2 5I - I L - 1 b i n d i n g t o c e l l s . I t was f o u n d t h a t EL4D6/76 and EL4 5D3 e x p r e s s e d 2300 t o 2400 c e l l s u r f a c e I L - 1 r e c e p t o r s p e r c e l l , w i t h a n a p p a r e n t d i s s o c i a t i o n c o n s t a n t i n t h e range o f 1.2 - 2 . 5 x l O "1 0M . T h i s d i s s o c i a t i o n c o n s t a n t i s i n good agreement w i t h t h e d a t a p u b l i s h e d f o r EL4 by K i l i a n e t a l . ( 1 6 ) . A l s o , no d i f f e r e n c e s were f o u n d i n a f f i n i t y c r o s s - l i n k i n g e x p e r i m e n t s w i t h 1 2 5I - I L - 1 ( 7 ) . B o t h c l o n e s showed a band a t 80 - 85 kD w h i c h i s c o n s i s t e n t w i t h p u b l i s h e d d a t a ( 1 7 ) .
M i z e l e t a l . (14) have shown t h a t l a b e l e d I L - 1 i s t r a n s p o r t e d t o t h e n u c l e u s i n EL4 and S w i s s 3T3 c e l l s . To t e s t t h e p o s s i b i l i t y t h a t EL4D6/76 m i g h t have l o s t t h e c a p a c i t y t o i n t e r n a l i z e I L - 1 , we m o n i t o r e d t h e u p t a k e o f 1 2 5I - r I L - l a by t h e s e c e l l s o v e r a t i m e p e r i o d o f 6 h .
7
0-1 , , , , ! | r 0 1 2 3 4 5 6
Time (hr)
Figure.2t K i n e t i c s of i n t e r n a l i z a t i o n of IL-1. R a d i o a c t i v i t y a c c u m u l a t e d i n t h e c y t o p l a s m i c f r a c t i o n o f EL4 5D3 c e l l s a n d r e a c h e d a p l a t e a u a f t e r 3 t o 4 h . I n c o n t r a s t , t h e r e was no s i g n i f i c a n t i n c r e a s e o f r a d i o a c t i v i t y i n t h e c o r r e s p o n d i n g c y t o p l a s m i c f r a c t i o n o f EL4D6/76.
When t h e same e x p e r i m e n t was c a r r i e d o u t i n t h e p r e s e n c e o f PMA. no q u a l i t a t i v e d i f f e r e n c e was d e t e c t e d . Thus, i n t e r n a l i z a t i o n o f I L - 1 c o r r e l a t e s w i t h i t s a c t i v i t y i n EL4 c e l l s . Whether i n t e r n a l i z a t i o n o f I L - 1 o r i t s t r a n s p o r t t o t h e c y t o p l a s m a n d n u c l e u s a r e n e c e s s a r y f o r a r e s p o n s e o f a c e l l t o I L - 1 i s n o t known. N e v e r t h e l e s s , t h i s h y p o t h e s i s i s s u p p o r t e d by o u r e x p e r i m e n t s showing t h a t c h l o r o q u i n e (10/ig/ml) i n h i b i t e d I L - 1 i n d u c e d I L - 2 s e c r e t i o n o f EL4 5D3 c e l l s (5 x 1 05/ m l ) by 40 t o 70Z. I t m i g h t t h e r e f o r e be p o s s i b l e t h a t I L - 1 h a s t o r e a c t w i t h a n u c l e a r r e c e p t o r a s p o s t u l a t e d by M i z e l e t a l . ( 1 4 ) . I f I L - 1 does n o t i n f l u e n c e any p o s t t r a n s l a t i o n a l p a r a m e t e r s what i s i t s a c t i v i t y ? A f i r s t i n d i c a t i o n was o b t a i n e d f r o m a a n a l y s i s o f mRNA p r o d u c t i o n f o r I L - 2 a n d I L - 2 R . C e l l s were i n d u c e d w i t h I L - 1 a n d PMA o r PMA a l o n e f o r 24 and 48 h a n d c y t o p l a s m i c RNA e x t r a c t s were p r o b e d f o r I L - 2 and IL-2R mRNA, r e s p e c t i v e l y .
T o g e t h e r w i t h p r e l i m i n a r y d a t a showing t h a t t h e h a l f l i f e o f I L - 2 mRNA was n o t d i f f e r e n t when EL4 5D3 c e l l s were s t i m u l a t e d w i t h PMA o r PMA p l u s I L - 1 we c o n c l u d e d t h a t t h e e f f e c t o f I L - 1 was most l i k e l y due t o i n c r e a s e d t r a n s c r i p t i o n o f I L - 2 and IL-2R g e n e s . I t i s t o t a l l y unknown how I L - 1 a f f e c t s gene t r a n s c r i p t i o n a n d d e t a i l e d i n v e s t i g a t i o n o f I L - 1 i n d u c e d I L - 2 and IL-2R gene
28 S - 18 S -
1 2
Figure 3s Northern b l o t of stimulated EL4 5D3 c e l l s . A f t e r 24 h o f i n d u c t i o n EL4 5D3 showed a s i g n i f i c a n t amount o f I L - 2 s p e c i f i c mRNA i n d u c e d by PMA ( l a n e 1 ) . T h i s amount was g r e a t l y i n c r e a s e d when I L - 1 i n a d d i t i o n was p r e s e n t d u r i n g s t i m u l a t i o n ( l a n e 2 ) . The same was t r u e f o r IL-2R mRNA ( n o t shown).
t r a n s c r i p t i o n w i l l be n e c e s s a r y t o c l a r i f y t h e pathway o f I L - 1 dependent a c t i v a t i o n o f T c e l l s .
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Im Neuenheimer F e l d 280 D 6900 H e i d e l b e r g
FRG
