Albrecht, M. P.

Falk, Werner

Krammer, P. H.

Serfling, E.

Stricker, K.

English

University of Regensburg Publication Server

Molecular and Cellular Biology of Cytokines, pages 191-196 © 1990 Wiley-Liss, Inc. INTERNALIZATION OF I L - 1 IS REQUIRED FOR IL- 2 GENE ACTIVA-TION IN EL4 THYMOMA CELLS. W. F a l k , M.P. A l b r e c h t , K. S t r i c k e r , *E. S e r f l i n g , and P.H. Krammer. I n s t i t u t e f o r Immunology and G e n e t i c s , DKFZ, D-6900 H e i d e l b e r g , FRG, and I n s t i t u t e f o r V i r o l o g y and Immunobiology, U n i v e r s i t y o f Würzburg, D-8700 Würzburg, FRG INTRODUCTION S e c r e t i o n o f IL-2 and e x p r e s s i o n o f h i g h a f f i n i t y I L -2R a r e r e q u i r e d f o r p r o l i f e r a t i o n o f T c e l l s upon m i t o g e n o r a n t i g e n s t i m u l a t i o n . The c y t o k i n e I L - 1 p l a y s a key r o l e i n i n d u c t i o n o f t h e s e p r o c e s s e s . I t has been d e m o n s t r a t e d t h a t I L - 1 i n d u c e s IL-2 e x p r e s s i o n by normal and n e o p l a s t i c T h e l p e r c e l l s a f t e r s t i m u l a t i o n . ( F a r r a r e t a l . , 1980; K a-s a h a r a e t a l . , 1985) A l s o , I L - 1 i n d u c e s IL-2R e x p r e s s i o n on a h e l p e r T c e l l a f t e r s t i m u l a t i o n o f t h e a n t i g e n r e c e p t o r (Kaye e t a l . , 1984) and on murine t h y m o c y t e s i n t h e p r e -s e n c e o f IL - 2 (Männel e t a l . , 1985). S i m i l a r e f f e c t s c a n be o b s e r v e d w i t h s e l e c t e d s u b c l o n e s o f EL4 thymoma c e l l s i n t h e p r e s e n c e o f PMA (L o w e n t h a l e t a l . , 1986). The mode o f a c t i o n o f I L - 1 i s not known. I L - 1 s t i m u l a t e s cAMP a c c u m u l a -t i o n p o i n t i n g t o an involvememt o f a G p r o t e i n ( C h e d i d e t a l . , 1 9 89). There i s c o n f l i c t i n g e v i d e n c e w i t h r e s p e c t t o t h e PKC pathway. A l s o , i t was r e p o r t e d r e c e n t l y t h a t I L - 1 a c t i v a t e s t h e n u c l e a r f a c t o r KB (Osborn e t a l . , 1989). L i k e many 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 and i t s t r a n s p o r t t o t h e n u c l e u s was r e p o r t e d by M i z e l e t a l . ( 1 9 8 7 ) . Whether 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 a r e s p o n s e t o I L - 1 was not known. By s e l e c t i o n o f two v a r i a n t s o f EL4 t h a t d i f f e r i n t h e i r I L - 1 r e s p o n s i -v e n e s s we show i n t h i s r e p o r t t h a t I L - 1 i n t e r n a l i z a t i o n c o r r e l a t e s w i t h I L - 1 r e s p o n s i v e n e s s and t h a t as a c o n s e -quence t h e I L - 2 p r o moter i s a c t i v a t e d . METHODS The s e l e c t i o n o f EL4 s u b c l o n e s and t h e measurement 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 were d e s c r i b e d by von Hoegen e t a l . (1989) C r o s s l i n k i n g o f t h e IL-1R w i t h l a b e l e d I L - 1 and PAGE was done as d e s c r i b e d (von Hoegen e t a l . , 1 9 8 9 ) . I n t e r n a l i -z a t i o n and s e p a r a t i o n o f membrane and c y t o p l a s m a a s s o c i a t e d I L - 1 was d e s c r i b e d (Von Hoegen e t a l . , 1 9 8 9 ) . N o r t h e r n a n a l y s i s and t h e p r o b e s u s e d were d e s c r i b e d by F a l k e t a l . ( 1 9 8 9 ) . T r a n s f e c t i o n o f EL4 was done by t h e DEAE d e x t r a n method. Cat a c t i v i t y was measured 48h a f t e r t r a n s f e c t i o n by c o n v e n t i o n a l methods. The c o n s t r u c t i o n o f t h e p l a s m i d s u s e d f o r t r a n s f e c t i o n was d e s c r i b e d by S e r f -l i n g e t a l . ( 1 9 8 9 ) . RESULTS AND DISCUSSION. I n an a t t e m p t t o d e t e r m i n e whether I L - 1 i n t e r n a l i z a -t i o n was r e q u i r e d f o r a c t i v i t y o f I L - 1 on T c e l l s s u b c l o n e s o f EL4 t h a t d i f f e r e d i n t h e i r r e s p o n s e t o I L - 1 were s e l e c -t e d by l i m i t i n g d i l u t i o n . The p a r a m e t e r s u s e d f o r d i s t i n c -t i o n o f t h e c l o n e s were t h e p r o d u c t i o n o f I L - 2 p r o t e i n i n t h e c e l l s u p e r n a t a n t and t h e e x p r e s s i o n o f t h e IL-2R on t h e c e l l s u r f a c e . One s e l e c t e d s u b c l o n e , EL4 5D3, r e s p o n d e d t o I L - 1 whereas t h e o t h e r one, EL4D6/76, showed no I L - 1 r e -sponse. The c e l l s were s t i m u l a t e d w i t h e i t h e r PMA o r w i t h PMA + I L - 1 . The r a t i o s o f PMA + IL-l/PMA ( S I ) s t i m u l a t i o n w i t h r e s p e c t t o b o t h p a r a m e t e r s were d e t e r m i n e d . The S I f o r IL-2R was 5.8 f o r EL4 5D3 and 1.0 f o r EL4D6/76. C o r r e s p o n -d i n g l y , t h e S I f o r I L - 2 was 3.2 f o r EL4 5D3 and 1.0 f o r EL4D6/76. To a s c e r t a i n t h a t t h i s d i f f e r e n c e i n r e s p o n s i v e n -e s s was n o t s i m p l y due t o a l a c k o f IL-1R i n t h e n o n r e s p o n -s i v e c l o n e EL4D6/76 IL-1R b i o c h e m i c a l c h a r a c t e r i s t i c s were a n a l y z e d by S c a t c h a r d a n a l y s i s . No d i f f e r e n c e was f o u n d between t h e two c l o n e s (Kd = 1.2-2.5xlO" 1 0, 2300-2400 r e c e p -t o r s / c e l l ) . C r o s s l i n k i n g o f t h e r e c e p t o r w i t h i t s l a b e l l e d l i g a n d and PAGE a l s o r e v e a l e d no d i f f e r e n c e i n molecuî^x mass (85 kD) . B o t h c l o n e s r esponded t o PMA w i t h p r o d u c t i o n o f I L - 2 and e x p r e s s i o n o f IL-2R s u g g e s t i n g no m a j o r d i f f e -r e n c e i n t h e PKC p a t h w a y . l t was shown t h a t i n I L - 1 r e -s p o n s i v e c e l l s I L - 1 i s i n t e r n a l i z e d and t r a n s p o r t e d t o t h e n u c l e a r membrane ( M i z e l e t a l . , 1 9 8 7 ) . To i n v e s t i g a t e whe-t h e r t h e d e f e c t o f EL4D6/76 would be ba s e d on i t s i n c a -I t was c l e a r from t h i s and s i m i l a r e x p e r i m e n t s t h a t t h e a d d i t i o n o f I L - 1 t o t h e r e a c t i o n d i d n o t r e s u l t i n a s h i f t e d p r o d u c t i o n o f IL-2mRNA. The e f f e c t o f I L - 1 was t o i n c r e a s e t h e amount o f IL-2mRNA a t a g i v e n t i m e a f t e r s t i -m u l a t i o n . I n e x p e r i m e n t s aimed a t d e t e r m i n i n g t h e h a l f l i f e o f t h i s mRNA we c o u l d a l s o f i n d no d i f f e r e n c e due t o t h e e f f e c t o f I L - 1 . The most r e a s o n a b l e e x p l a n a t i o n o f t h e s e d a t a was t h a t I L - 1 d i d not a f f e c t p o s t t r a n s c r i p t i o n a l e v e n t s i n EL4 5D3. The i n c r e a s e i n IL-2mRNA c o u l d t h e r e f o r e be e x p l a i n e d b e s t by t h e a s s u m p t i o n t h a t I L - 1 e x e r t e d i t s e f f e c t on t h e l e v e l o f t r a n s c r i p t i o n o f t h e I L - 2 gene. We t h e r e f o r e d e c i d e d t o i n v e s t i g a t e t h e e f f e c t o f I L - 1 on t h e mouse I L - 2 p r o m o t e r . V a r i o u s c o n s t r u c t s were p r e p a r e d t h a t a l l c o n t a i n e d t h e CAT r e p o r t e r gene i n c l u d i n g t h e Tk promo-t e r . D i f f e r e n t segments o f t h e mouse IL- 2 p r o m o t e r were c l o n e d 5' o f t h e Tk p r omoter i n t h e s e c o n s t r u c t s . pILCAT 0 c o n t a i n e d t h e segment from -7 t o -2000, pILCAT 1 c o n t a i n e d t h e segment from -7 t o -483, and pILCAT 2/1+ c o n t a i n e d t h e segment from -7 t o -293. The p o s i t i v e c o n t r o l f o r t r a n s f e c -t i o n was pTkCAT 21A c o n t a i n i n g t h e MSV e n h a n c e r , t h e nega-t i v e c o n t r o l was pBLCAT 2 c o n t a i n i n g no e n hancer ( S e r f l i n g e t a l . , 1 9 8 9 ) . These c o n s t r u c t s were used t o t r a n s f e c t t h e I L - 1 r e s p o n s i v e c e l l EL4 5D3 by t h e DEAE d e x t r a n method. 48h a f t e r t r a n s f e c t i o n t h e c e l l s were s t i m u l a t e d w i t h I L - 1 o r PMA a l o n e o r w i t h t h e c o m b i n a t i o n o f t h e 2 r e a g e n t s . A f -t e r o v e r n i g h t s t i m u l a t i o n t h e s u p e r n a t a n t s were removed f o r t h e measurement o f t h e I L - 2 p r o d u c e d . The c e l l p e l l e t was l y s e d t o d e t e r m i n e t h e CAT a c t i v i t y . TABLE 1. I L - 1 a c t i v a t e s t h e mouse IL-2 p r o m o t e r i n t r a n s -f e c t e d EL4 5D3 P l a s m i d - I L - 1 PMA PMA+IL-1 SI Cat S I I L - 2 pTkCat21A 12 13.5 18.1 18.7 1 .03 4.0 pBLCat2 <1 <1 1 .0 1 . 0 1 .0 3.5 p I L C a t O 1 .4 2.8 8.1 13.6 1.7 5.0 p I L C a t l 1 <1 5.3 9.7 1.8 3.0 p I L C a t 2 / l + 1 <1 5 . 0 12.1 2.4 5.2 The numbers d e p i c t % a c e t y l a t i o n . They r e p r e s e n t t h e mean o f 4 e x p e r i m e n t s . B o l d numbers r e p r e s e n t s t i m u l a t i o n i n d e x ( S I ) . S I e q u a l s PMA+IL-1/ PMA s t i m u l a t i o n . S I f o r I L - 2 de-t e r m i n a t i o n i s t h e r a t i o o f t h e u n i t s I L - 2 p r o d u c e d u n d e r t h e r e s p e c t i v e c o n d i t i o n s . EL4 5D3 c e l l s were t r a n s f e c t e d w i t h t h e g i v e n p l a s m i d s and s t i m u l a t e d a f t e r 48h w i t h t h e g i v e n s t i m u l i . The T a b l e shows t h a t I L - 1 a c t i v a t e s t h e I L - 2 p r o m o t e r . A l l t r a n s f e c t e d c e l l s r e s p o n d e d t o I L - 1 w i t h i n c r e a s e d I L - 2 p r o d u c t i o n i r r e s p e c t i v e o f t h e p l a s m i d used showing t h a t no p l a s m i d i n t e r f e r e d w i t h i n d u c t i o n o f t h e endogenous I L - 2 gene and t h a t t h e t r a n s f e c t i o n s had no t o x i c e f f e c t on t h e c e l l s . The p o s i t i v e c o n t r o l pTkCAT 21A c o n f e r r e d c o n s t i t u -t i v e e x p r e s s i o n whereas t h e n e g a t i v e c o n t r o l pBLCAT 2 c o n -f e r r e d no s i g n i f i c a n t CAT e x p r e s s i o n whether o r n o t t h e c e l l s were s t i m u l a t e d . A l l p l a s m i d s t h a t c o n t a i n e d segments o f t h e I L - 2 p r o m o t e r showed s t i m u l a t i o n - d e p e n d e n t CAT and I L - 2 e x p r e s s i o n . I n a l l c a s e s t h i s e x p r e s s i o n was enhanced by I L - 1 . The s m a l l e s t fragment ( i n p l a s m i d pILCAT 2/1+) s t i l l c o n t a i n e d an element o f t h e I L - 2 p r o m o t e r r e a c t i v e t o I L - 1 . I n summary, we deduce from t h e above d a t a t h e f o l -l o w i n g h y p o t h e s i s . I n t e r a c t i o n o f I L - 1 w i t h i t s r e c e p t o r i s n o t s u f f i c i e n t f o r i n d u c t i o n o f a r e s p o n s e . I L - 1 needs t o be i n t e r n a l i z e d and may t h e n i n t e r a c t w i t h a n u c l e a r r e c e p -t o r s i m i l a r t o t h e a c t i o n o f s t e r o i d hormons. The i n t e r a c -t i o n o f I L - 1 w i t h i t s n u c l e a r r e c e p t o r l e e d s t o t h e a c t i v a -t i o n o f I L - 1 s p e c i f i c n u c l e a r b i n d i n g p r o t e i n s w h i c h i n t u r n a c t i v a t e t h e IL-2 promoter r e s u l t i n g i n t h e p r o c u t i o n o f IL-2mRNA. REFERENCES C h e d i d M, S h i r a k a w a F, N a y l o r P, M i z e l S B ( 1 9 8 9 ) . S i g n a l t r a n s d u c t i o n pathway f o r I L - 1 . I n v o l v e m e n t o f a p e r t u s s i s t o x i n s e n s i t i v e G T P - b i n d i n g p r o t e i n i n t h e a c t i v a t i o n o f a d e n y l a t e c y c l a s e . J Immunol.142: 4301-4306. F a l k W, Männel D N, Dar j e s H, Krammer P K ( 1 9 8 9 ) . I L - 1 i n -d u ces h i g h a f f i n i t y I L -2 r e c e p t o r e x p r e s s i o n o f CD4~8" t h y m o c y t e s . J . Immunol. 143: 513-517. F a r r a r J J , M i z e l S B, F u l l e r - F a r r a r J , F a r r a r W L, H i l f i -k e r M L ( 1 9 8 0 ) . Macrophage i n d e p e n d e n t a c t i v a t i o n o f h e l -p e r T c e l l s . I . P r o d u c t i o n o f i n t e r l e u k i n 2. J . Immunol. 125: 793-798. K a s a h a r a T, M ukaida N, Hatake K f M o t o y o s h i K, Kawai T, S h i o r i - N a d a n o K ( 1 9 8 5 ) . I n t e r l e u k i n 1 ( I L 1) dependent p a b i l i t y t o i n t e r n a l i z e I L - 1 c e l l s o f t h e two c l o n e s were i n c u b a t e d w i t h l a b e l e d I L - 1 a t 37°C and t h e k i n e t i c s o f c y -t o s o l - a s s o c i a t e d I L - 1 was d e t e r m i n e d . We f o u n d t h a t EL4 5D3 i n t e r n a l i z e d I L - 1 r e a c h i n g p l a t e a u l e v e l s w i t h i n 3 h whe-r e a s EL4D6/76 d i d n o t i n t e r n a l i z e any I L - 1 i n t o t h e c y t o -s o l . ( V o n Hoegen e t a l . , 1 9 8 9 ) We c o n c l u d e d f rom t h e s e d a t a t h a t i n t e r n a l i z a t i o n o f I L - 1 i s a p r e r e q u i s i t e f o r a r e -sponse t o I L - 1 . T h i s i n t e r p r e t a t i o n was s u p p o r t e d by t h e f a c t t h a t c h l o r o q u i n e a t a c o n c e n t r a t i o n o f 10 A*g/ml i n -h i b i t e d t h e I L - 1 i n d u c e d i n c r e a s e i n I L - 2 p r o d u c t i o n by 68.5% a t a c e l l c o n c e n t r a t i o n o f 5 x l 0 5 / w e l l and by 42% a t a c e l l c o n c e n t r a t i o n o f 1 0 6 / w e l l . The n e x t q u e s t i o n was whether I L - 1 had i t s e f f e c t on t h e l e v e l o f t r a n s l a t i o n o r t r a n s c r i p t i o n . To t h i s end t h e IL - 2 mRNA l e v e l s were m o n i t o r e d a t d i f f e r e n t t i m e s a f t e r s t i m u l a t i o n w i t h e i t h e r PMA o r PMA + I L - 1 . ( S t i m u l a t i o n w i t h I L - 1 a l o n e d i d n e v e r have any e f f e c t on I L - 2 p r o d u c -t i o n ) . T o t a l RNA was p r e p a r e d and e l e c t r o p h o r e s e d i n a 1% a g a r o s e g e l under d e n a t u r i n g c o n d i t i o n s and t h e N o r t h e r n b l o t was p r o b e d w i t h a probe s p e c i f i c f o r I L - 2 . 1 2 3 4 5 6 F i g u r e 1. K i n e t i c s o f IL-2mRNA p r o d u c t i o n . EL4 5D3 c e l l s were s t i m u l a t e d w i t h PMA ( l a n e s 1, 3, 5) o r w i t h PMA + I L - 1 ( l a n e s 2, 4, 6).RNA from s t i m u l a t e d c e l l s was p r e p a r e d a f -t e r 8h( l a n e s 1, 2 ) ; 24h ( l a n e s 3, 4 ) ; and a f t e r 48h ( l a n e s 5, 6 ) . The N o r t h e r n b l o t was pr o b e d w i t h an I L - 2 p r o b e . l y m p h o k i n e p r o d u c t i o n by human l e u k e m i c T c e l l l i n e HSB.2 s u b c l o n e s . J . Immunol. 134: 1682-1689. Kaye J , G i l l i s S, M i z e l S B, Shevach E M, Ma l e k T R, D i n a -r e l l o C A f Lachmann L B, Janeway C A (1 9 8 4 ) . Growth o f a c l o n e d h e l p e r T c e l l l i n e i n d u c e d by a m o n o c l o n a l a n t i -body s p e c i f i c f o r t h e a n t i g e n r e c e p t o r : i n t e r l e u k i n 1 i s r e q u i r e d f o r t h e e x p r e s s i o n o f r e c e p t o r s f o r i n t e r l e u k i n 2. J . Immunol. 133: 1339-1345. L o w e n t h a l J W, C e r r o t i n i J C, MacDonald H R ( 1 9 8 6 ) . I n t e r -l e u k i n 1 dependent i n d u c t i o n o f b o t h i n t e r l e u k i n 2 s e c r e -t i o n and i n t e r l e u k i n 2 r e c e p t o r e x p r e s s i o n by thymoma c e l l s . J . Immunol. 137: 1226-1231. Männel D N, M i z e l S B, D i a m a n t s t e i n T, F a l k W ( 1 9 8 5 ) . I n -d u c t i o n o f i n t e r l e u k i n 2 r e s p o n s i v e n e s s i n t h y m o c y t e s by s y n e r g i s t i c a c t i o n o f i n t e r l e u k i n 1 and i n t e r l e u k i n 2. J . Immunol. 134: 3108-3110. M i z e l S B, K i l i a n P L, L e w i s J C, P a g a n e l l i K A, C h i z z o n i t e R A ( 1 9 8 7 ) . The i n t e r l e u k i n 1 r e c e p t o r . Dynamics o f i n -t e r l e u k i n 1 b i n d i n g and i n t e r n a l i z a t i o n i n T c e l l s and f i b r o b l a s t s . J . Immunol. 138: 2906-2912. Osborn L, K u n k e l S, N a b e l G J (1989) . Tumor n e c r o s i s f a c t o r a and i n t e r l e u k i n 1 s t i m u l a t e t h e human i m m u n o d e f i c i e n c y v i r u s e n h a n c e r by a c t i v a t i o n o f t h e n u c l e a r f a c t o r «B. P r o c . N a t l . Acad. S e i . USA 86: 2336-2340. S e r f l i n g E, Bathelmäs R, P f e u f f e r I , Schenk B, Z a r i u s S, Swoboda R, M e r c u r i o F, K a r i n M (1989) U b i q u i t o u s and lym-p h o c y t e s p e c i f i c f a c t o r s a r e i n v o l v e d i n t h e i n d u c t i o n o f t h e mouse i n t e r l e u k i n 2 gene i n T l y m p h o c y t e s . EMBO J . 8: 465-473. Von Hoegen I , F a l k W, K o j o u h a r o f f G, Krammer P K ( 1 9 8 9 ) . I n t e r n a l i z a t i o n o f i n t e r l e u k i n 1 ( I L 1) c o r r e l a t e s w i t h I L 1 - i n d u c e d I L 2 r e c e p t o r e x p r e s s i o n and I L 2 s e c r e t i o n o f EL4 thymoma c e l l s . E u r . J . Immunol. 19: 329-334. ACKNOWLEDGEMENT: We t h a n k H e i d i D a r j e s f o r e x p e r t t e c h n i c a l a s s i s t a n c e . 

Internalization of IL-1 is required for IL-2 gene activation in EL4 thymoma cells

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Internalization of IL-1 is required for IL-2 gene activation in EL4 thymoma cells

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