DANIELA N. MÄNNEL,' WERNER F A L K , ' and HINNAK N O R T H O F F

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LYMPHOKINE RESEARCH Volume 6, Number 3, 1987 Mary Ann Liebert, Inc., Publishers

RESEARCH REPORT

Endotoxic Activities of Tumor Necrosis Factor Independent of IL1 Secretion by

Macrophages / Monocytes

DANIELA N. MÄNNEL,' WERNER F A L K , ' and HINNAK N O R T H O F F

²

1 Institute of Immunology and Genetics, German Cancer Research Center, 6900 Heidelberg, FRG

2DRK Blutspendezentrale, 7900 Ulm, FRG

ABSTRACT

R e c o m b i n a n t t u m o r n e c r o s i s f a c t o r (TNF) h a d h y p o t h e r m i c a c t i v i t y i n v i v o . I n t r a v e n o u s i n j e c t i o n o f TNF r e s u l t e d i n a h y p o t h e r m i c r e a c t i o n o f m i c e w i t h i n 3 t o 6 h o u r s . T h i s r e a c t i o n was n o t t h e r e s u l t o f i n t e r l e u k i n 1 ( I L 1 ) r e l e a s e from m a c r o p h a g e s / m o n o c y t e s . P e r i t o n e a l e x u d a t e c e l l c u l t u r e s f r o m e n d o t o x i n l o w r e s p o n d e r m i c e o r human p e r i p h e r a l m o n o n u c l e a r l e u k o c y t e c u l t u r e s d i d n o t g e n e r a t e IL1 a c t i v i t y i n t h e s u p e r n a t a n t a f t e r e x p o s u r e t o TNF. The a d d i t i o n o f i n t e r f e r o n - T C I F N ^ Y ) o r p r e e x p o s u r e t o IFN~Y and t h e n s t i m u l a t i o n w i t h TNF d i d a l s o n o t r e s u l t i n IL1 s e c r e t i o n . No IL1 i n h i b i t o r was g e n e r a t e d and TNF d i d n o t i n t e r f e r e w i t h t h e IL1 t e s t s y s t e m s . T h e r e f o r e , we c o n c l u d e t h a t t h e h y p o t h e r m i c a c t i v i t y o f TNF i s n o t m e d i a t e d v i a t h e i n d u c t i o n o f I L 1 p r o d u c t i o n by mono- n u c l e a r p h a g o c y t e s .

INTRODUCTION

TNF h a s been d e m o n s t r a t e d a s t h e m e d i a t o r r e s p o n s i b l e f o r t h e t u m o r n e c r o s i s phenomenon w h i c h o c c u r s a f t e r i n j e c t i o n o f e n d o t o x i n i n t o tumor b e a r i n g h o s t s

( 1 ) . The c e l l u l a r s o u r c e o f TNF a r e a c t i v a t e d macrophages w h i c h produce TNF a f t e r s t i m u l a t i o n w i t h b a c t e r i a l l i p o p o l y s a c c h a r i d e (LPS) ( 2 ) . W i t h t h e a v a i l a b i l i t y o f r e c o m b i n a n t TNF (3) a wide s p e c t r u m o f b i o l o g i c a l a c t i v i t i e s f o r TNF h a s b e e n d e s c r i b e d b e s i d e s i t s t u m o r c y t o t o x i c e f f e c t . I t a l s o became o b v i o u s t h a t TNF s h a r e s a number o f b i o l o g i c a l f u n c t i o n s w i t h i n t e r l e u k i n 1 ( I L 1 ) (M) a n d t h a t i t m e d i a t e s e n d o t o x i n e f f e c t s ( 5 ) . The q u e s t i o n was a s k e d whether t h e e n d o t o x i c e f f e c t s o f TNF were a consequence o f IL1 i n d u c t i o n o r w h e t h e r t h e y w e r e d i r e c t TNF e f f e c t s .

I n d u c t i o n o f f e v e r i n r a b b i t s s e r v e s as a q u a n t i t a t i v e d e t e r m i n a t i o n o f m i n u t e amounts o f e n d o t o x i n . The mechanism o f t h i s v e r y s e n s i t i v e f e v e r t e s t i n c l u d e s t h e p r o d u c t i o n o f p r o s t a g l a n d i n s o f t h e E s e r i e s v i a i n d u c t i o n o f endogenous IL1 ( 4 ) . I n c o n t r a s t t o r a b b i t s , mice and r a t s r e a c t w i t h h y p o t h e r m i a upon e n d o t o x i n i n j e c t i o n when t h e y a r e k e p t a t room t e m p e r a t u r e ( 6 ) . T h i s e n d o t o x i n - i n d u c e d h y p o t h e r m i a i s p r o b a b l y n o t d u e t o c h a n g e s o f t h e t h e r m o r e g u l a t i o n i n t h e h y p o t h a l a m u s b u t r a t h e r a t o x i c e f f e c t o n t h e b l o o d v e s s e l s . I n t h i s s t u d y we d e m o n s t r a t e t h e i n d u c t i o n o f h y p o t h e r m i a i n mice u p o n TNF i n j e c t i o n s i m i l a r t o

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t h e e n d o t o x i n - i n d u c e d h y p o t h e r m i a . We i n v e s t i g a t e d whether t h i s e n d o t o x i n - 1 i k e f u n c t i o n o f TNF i s a d i r e c t e f f e c t o f TNF o r w h e t h e r i t i s m e d i a t e d v i a I L 1 p r o d u c t i o n by macrophages.

MATERIALS AND METHODS

M i c e

M a l e C3H/He o r C3H/HeJ m i c e 4-8 w e e k s o f a g e w e r e e i t h e r o b t a i n e d f r o m Z e n t r a l i n s t i t u t für V e r s u c h s t i e r k u n d e , Hannover, F.R.G. o r from B o m h o l t g a r d L t d . , Ry, Denmark o r from t h e J a c k s o n L a b o r a t o r i e s , B a r H a r b o r , M a i n e , U.S.A.

Reagents

P u r i f i e d r e c o m b i n a n t human TNF was g e n e r o u s l y s u p p l i e d by t h e BASF AG, L u d w i g s h a f e n , F.R.G. The p r e p a r a t i o n c o n t a i n e d l e s s t h a n 1 . 3ng e n d o t o x i n p e r mg p r o t e i n . Recombinant human IFN-T was s u p p l i e d by Dr. C a r l Thomae GmbH, B i b e r a c h , F.R.G. H i g h l y p u r i f i e d r e c o m b i n a n t human I L 2 f r o m E. c o l i was o b t a i n e d from 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 , C a l i f o r n i a , U.S.A. F o r s t a n d a r d p u r p o s e s human I L 1ß was s e m i p u r i f i e d as d e s c r i b e d r e c e n t l y ( 7 ) . L i p o p o l y s a c c h a r i d e p r e p a r a t i o n s w e r e e i t h e r d e r i v e d f r o m S. m o n t e v i d e o SH94 p r e p a r e d a c c o r d i n g t o t h e p h e n o l w a t e r e x t r a c t i o n method f o l l o w e d by t h e p h e n o l - c h l o r o f o r m p e t r o l e t h e r p r o c e d u r e (8) o r from S. t y p h . 0901 ( D i f c o , FRG). G l u t a r a l d e h y d e - f i x e d S t a p h y l o c o c u s a u r e u s c e l l s ( P a n s o r b i n , C a l b i o c h e m , B e h r i n g D i a g n o s t i c s , L a J o l l a , C a l i f o r n i a ) were used as 0.1% (w/v) s u s p e n s i o n .

Temperature D e t e r m i n a t i o n

R e c t a l t e m p e r a t u r e were measured u s i n g an e l e c t r o n i c t e m p e r a t u r e probe (Haake DT-10, K a r l s r u h e , F.R.G.). Groups o f f i v e mice were k e p t i n cages w i t h o u t m a k i n g s p e c i a l a r r a n g e m e n t s t o keep t h e a n i m a l s warm d u r i n g t h e e x p e r i m e n t .

C u l t u r e Medium

The c u l t u r e medium used was RPMI 1640 ( G i b c o ) w i t h 10$ h e a t i n a c t i v a t e d f e t a l c a l f serum (Seromed, B i o c h r o m KG, B e r l i n , F.R.G.). The s p e c i a l b a t c h o f FCS u s e d was p r e v i o u s l y t e s t e d f o r l a c k o f any IL1 i n d u c i n g a c t i v i t y a s d e s c r i b e d ( 9 ) .

IL1 G e n e r a t i n g C u l t u r e s

P e r i t o n e a l e x u d a t e c e l l s ( 2 x 1 06/ m l ) f r o m e i t h e r C3H/He o r C3H/HeJ mice were o b t a i n e d 18 h a f t e r i n j e c t i o n o f 1ml PBS i . p . T h e y w e r e c u l t u r e d w i t h t h e i n d i c a t e d a g e n t s i n c u l t u r e medium f o r 24 h o u r s . Human mononuclear c e l l s were p r e p a r e d from b u f f y c o a t s (ACD s t a b i l i z e r ) o f n o r m a l b l o o d u n i t s by F i c o l l - H y - p a q u e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n ( 1 0 ) . The c e l l s were i n c u b a t e d a t 3 x 1 06

c e l l s / m l a t 37° C f o r 1.5 h i n c u l t u r e f l a s k s ( F a l c o n , B e c t o n D i c k i n s o n ) and n o n a d h e r e n t c e l l s w e r e r e m o v e d by w a s h i n g t h e c e l l s 3 t i m e s w i t h 37°C c u l t u r e medium. F l a s k s w i t h t h e a d h e r e n t c e l l s a t t a c h e d were p l a c e d on i c e f o r 20 m i n u t e s and a d h e r e n t c e l l s r e c o v e r e d by v i g o r o u s l y p i p e t t i n g w i t h i c e c o l d c u l t u r e medium. The a d h e r e n t c e l l s were i m m e d i a t e l y s e e d e d a n d c u l t u r e d a t 3 x 1 05/ m l i n m i c r o t i t e r p l a t e s ( F a l c o n ) i n t h e p r e s e n c e o f r e a g e n t s as i n d i c a t e d . A l t e r n a t i v e - l y , t h e y were i n c u b a t e d a t 1 x 1 0⁶/ m l i n 5 0 m l p o l y p r o p y l e n t u b e s ( F a l c o n ) a t a n a n g l e o f 45° f o r 24 h i n t h e p r e s e n c e o r absence o f IFNVf (100U/ml) o r TNF as i n d i c a t e d . T h e r e a f t e r t h e y were washed 3 t i m e s and seeded a t 3 x 1 0⁵/ m l i n m i c r o - t i t e r p l a t e s ( F a l c o n ) and i n c u b a t e d f o r f u r t h e r 24 h i n t h e p r e s e n c e o f LPS ( S . t y p h . ). A f t e r t h e i n d i c a t e d t i m e s s u p e r n a t a n t s f r o m t h e macrophage/ monocyte c u l t u r e s were c o l l e c t e d f o r IL1 d e t e r m i n a t i o n .

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40-i

-f 1 I 1 1 I I 1 I ! 0 3 6 9 12 15 18 21 24 27

HOURS AFTER INJECTION

FIGURE 1. H y p o t h e r m i a i n M i c e a f t e r E n d o t o x i n o r TNF I n j e c t i o n .

C3H/He (open s y m b o l s ) o r C3H/HeJ ( s o l i d s y m b o l s ) mice were i n j e c t e d i n t r a v e n o u s l y w i t h e i t h e r 1 0 0 y g o f L P S f r o m S. m o n t e v i d e o ( c i r c l e s ) o r w i t h 4 0 y g o f TNF

( s q u a r e s ) i n 5 0 y l o f PBS. The r e c t a l t e m p e r a t u r e was measured b e f o r e t h e i n j e c - t i o n and a t 1,3,6 and 24 h a f t e r i n j e c t i o n and e x p r e s s e d as mean ± S.D. o f g r o u p s o f 5 t o 7 m i c e , a) C3H/He mice i n j e c t e d w i t h TNF d i e d a f t e r 6 h o u r s .

IL1 A s s a y Systems

Thymocytes ( 0 . 5 ~ 1 x 1 06) from C3H/HeJ mice were c u l t u r e d i n a volume o f 0.1ml i n t h e p r e s e n c e o f PHA/M (50yg/ml) ( S e r v a , München, F.R.G.) w i t h s e r i a l d i l u t i o n s o f t h e s u p e r n a t a n t s f o r 72 h. F o r F i g . 2 B i n s t e a d o f PHA r e c o m b i n a n t I L 2 was used as c o s t i m u l a t o r ( 7 ) . P r o l i f e r a t i o n was d e t e r m i n e d by a 4h p u l s e a t t h e e n d o f t h e c u l t u r e p e r i o d w i t h 1.0yCi o f m e t h y l - ( 3 H ) - t h y m i d i n e (3(H)~TdR, s p e c i f i c a c t i v i t y 50 Ci/mmol, Amersham I n t e r n a t i o n a l L t d . , Amersham U.K.) u n l e s s o t h e r w i s e s t a t e d . IL1 t i t e r s a r e g i v e n as t h e f i n a l d i l u t i o n s o f t h e s u p e r n a t a n t s w h i c h cause cpm 2 . 5 - f o l d h i g h e r t h a n background. U n i t s o f s e m i p u r i f i e d human I L 1 ß c o r r e s p o n d t o t h e d i l u t i o n w h i c h c a u s e s 1/2 maximal p r o l i f e r a t i o n ( F i g . 2 ) .

RESULTS TNF-Induced H y p o t h e r m i a

I n j e c t i o n o f p u r i f i e d l i p o p o l y s a c c h a r i d e (LPS) i n t o L P S - s e n s i t i v e mice l e a d s t o h y p o t h e r m i a when t h e a n i m a l s a r e k e p t a t room t e m p e r a t u r e . T h i s r e a c t i o n i s a t y p i c a l e n d o t o x i n e f f e c t and c a n e a s i l y be measured. V a l u e s o f body t e m p e r a t u r e o f C3H/He mice a t d i f f e r e n t t i m e s a f t e r i . v . a p p l i c a t i o n o f LPS a r e shown i n F i g . 1 . W i t h i n 6 h o u r s t h e t e m p e r a t u r e d r o p p e d by 3 t o 5°C and r e c l i n e d t h e r e - a f t e r . A s i m i l a r drop i n t e m p e r a t u r e was measured a f t e r a p p l i c a t i o n o f r e c o m b i - n a n t TNF ( F i g . 1 ) . T h i s h y p o t h e r m i c e f f e c t a f t e r TNF i n j e c t i o n was not due t o c o n t a m i n a t i n g LPS, s i n c e c o n c e n t r a t i o n s o f LPS e q u i v a l e n t t o t h e c o n t a m i n a t i o n w e r e u n a b l e t o i n d u c e s u c h a r e a c t i o n ( d a t a n o t s h o w n ) . A l s o , a s i g n i f i c a n t h y p o t h e r m i c r e a c t i o n was i n d u c e d w i t h TNF i n L P S - l o w r e s p o n d e r C3H/HeJ m i c e i n w h i c h L P S i t s e l f d i d n o t i n d u c e h y p o t h e r m i a . T h i s i n d i c a t e d t h a t t h e h y p o t h e r m i c r e a c t i o n a f t e r a p p l i c a t i o n o f t h e TNF p r e p a r a t i o n was n o t m e d i a t e d by LPS. I t h a s b e e n shown i n a s e p a r a t e s t u d y t h a t t h e m e d i a t i o n o f t y p i c a l e n d o t o x i n e f f e c t s - i n c l u d i n g h y p o t h e r m i a - by TNF i n v i v o i s a dose dependent, i m p o r t a n t f u n c t i o n o f TNF ( 1 1 ) .

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RECIPROCAL DILUTION RECIPROCAL DILUTION OF TNF (10/ug/ml)

FIGURE 2. Lack o f I L 1 - A c t i v i t y by TNF

A) T h y m o c y t e s ( 5 x 1 05) f r o m C3H/HeJ m i c e were c u l t u r e d i n t h e p r e s e n c e o f a s u b o p t i m a l c o n c e n t r a t i o n o f m i t o g e n and d i f f e r e n t d i l u t i o n s o f e i t h e r IL1 (6U/ml) ( O ) o r TNF (10ng/ml) ( o ) i n 0.2ml volume. 3H TdR u p t a k e was measured i n a 16h p u l s e .

B) T h y m o c y t e s ( 5 x 1 05) from C3H/HeJ mice were c u l t u r e d i n t h e p r e s e n c e o f I L 2 (25U/ml) w i t h o u t IL1 ( O ) , w i t h 0.013U/ml ( • ) , 0.93U/ml ( • ) , 9U/ml ( O ) o f I L 1 , and d i f f e r e n t d i l u t i o n s o f TNF O O y g / m l ) . 3H TdR u p t a k e was measured i n a 6h p u l s e .

IL1 Serum L e v e l s a f t e r TNF I n j e c t i o n

S i n c e I L 1 i s a w e l l known m e d i a t o r o f L P S - i n d u c e d f e v e r i n r a b b i t s , L P S - i n - duced h y p o t h e r m i a i n mice c o u l d a l s o be m e d i a t e d by endogenous p r o d u c t i o n o f I L 1 . T h e r e f o r e , t h e p o s s i b i l i t y was t e s t e d whether TNF i n j e c t i o n i n d u c e d IL1 p r o d u c - t i o n i n the a n i m a l s . No s i g n i f i c a n t IL1 a c t i v i t y was d e t e c t e d i n s e r a o f e i t h e r L P S - s e n s i t i v e o r L P S - i n s e n s i t i v e mice c o l l e c t e d a t d i f f e r e n t t i m e s ( 1 h , 3h, 6h and 24h) a f t e r TNF (40ug) a p p l i c a t i o n . Only i n a few LPS s e n s i t i v e a n i m a l s l i t t l e IL1 was d e t e c t e d 6 h a f t e r LPS (200yg) i n j e c t i o n . TNF a c t i v i t y was s t i l l d e t e c t a - b l e i n serum 6h a f t e r i n j e c t i o n o f TNF ( d a t a n o t shown). T h i s o b s e r v a t i o n a g r e e d v e r y w e l l w i t h o u r e a r l i e r f i n d i n g s a n d d a t a f r o m o t h e r l a b o r a t o r i e s , w h i c h showed t h a t serum l e v e l s o f i n t e r l e u k i n 1 can o n l y be measured a f t e r L P S i n j e c - t i o n i n m i c e w h i c h h a v e a h i g h l y a c t i v a t e d m o n o n u c l e a r p h a g o c y t e system (R.

Urbaschek e t a l . , u n p u b l i s h e d r e s u l t s ) . I n f l u e n c e o f TNF on IL1 T e s t Systems

The o b s e r v a t i o n t h a t no IL1 was m e a s u r a b l e i n t h e s e r a o f t h e TNF i n j e c t e d a n i m a l s even though TNF a c t i v i t y was s t i l l p r e s e n t i n d i c a t e d t h a t TNF h a d no I L 1 e f f e c t i n t h e t h y m o c y t e c o s t i m u l a t o r a s s a y s y s t e m . To t e s t whether TNF had any e f f e c t i n t h e IL1 a s s a y o r whether i t c o u l d mask IL1 a c t i v i t y TNF was t i t r a t e d i n t o t h e t h y m o c y t e c o s t i m u l a t o r a s s a y . TNF d i d n o t s u b s t i t u t e f o r IL1 i n t h i s c l a s s i c a l I L 1 t e s t s y s t e m ( F i g . 2 A ) . A l s o , i n a s e c o n d t e s t s y s t e m f o r I L 1 a c t i v i t y i n w h i c h p r o l i f e r a t i o n o f murine t h y m o c y t e s was i n d u c e d by s i m u l t a n e o u s a d d i t i o n o f I L 2 p l u s I L 1 ( 7 ) , TNF was u n a b l e t o s u b s t i t u t e o r m o d u l a t e I L 1 a c t i v i t y ( F i g . 2 B ) .

F a i l u r e o f TNF t o Induce IL1 P r o d u c t i o n i n v i t r o

To f u r t h e r examine whether TNF c o u l d induce. IL1 a c t i v i t y i n m a c r o p h a g e s , I L 1 g e n e r a t i o n i n v i t r o was t e s t e d . M u r i n e p e r i t o n e a l e x u d a t e c e l l s ( P E C ) were c u l t u r e d i n d i f f e r e n t c o n c e n t r a t i o n s o f r e c o m b i n a n t TNF and the s u p e r n a t a n t s were t e s t e d f o r I L 1 a c t i v i t y . Low amounts o f IL1 a c t i v i t y were g e n e r a t e d i n s u p e r n a - t a n t s o f PEC from L P S - s e n s i t i v e mice a f t e r 24 h o u r s ( T a b l e 1A). PEC f r o m L P S - i n - s e n s i t i v e C3H/HeJ m i c e , h o w e v e r , d i d n o t r e l e a s e IL1 i n t o t h e s u p e r n a t a n t upon

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TNF e x p o s u r e . However, macrophages o f C3H/HeJ mice were a b l e t o p r o d u c e IL1 when s t i m u l a t e d w i t h S t a p h , a u r e u s as shown i n T a b l e 1B. TNF c o m p l e t e l y l o s t the c y t o t o x i c a c t i v i t y a f t e r h e a t i n g a t 100°C f o r 10 m i n u t e s ( d a t a not shown). T h i s h e a t e d m a t e r i a l s t i l l i n d u c e d t h e same t i t e r . o f IL1 a c t i v i t y i n t h e C3H/He m a c r o p h a g e c u l t u r e s as t h e u n t r e a t e d TNF. T h i s i n d i c a t e d t h a t the IL1 a c t i v i t y was n o t i n d u c e d by b i o l o g i c a l l y a c t i v e TNF b u t r a t h e r by a h e a t r e s i s t a n t c o n t a m i n a t i o n of t h e TNF p r e p a r a t i o n .

Human and mouse TNF a r e n o t c o m p l e t e l y h o m o l o g o u s ( 1 2 ) . I n o r d e r t o t e s t whether a p o s s i b l e IL1 i n d u c t i o n m i g h t be a s p e c i e s - s p e c i f i c a c t i v i t y o f TNF, e n r i c h e d human p e r i p h e r a l b l o o d a d h e r e n t c e l l s ( m o s t l y monocytes) were exposed t o graded amounts of r e c o m b i n a n t human TNF i n the p r e s e n c e o r absence of LPS. TNF by i t s e l f d i d n o t i n d u c e I L 1 p r o d u c t i o n i n t h e s e c u l t u r e s ( T a b l e 2 ) . T h i s was not d i f f e r e n t i n the p r e s e n c e of IFN-Y w h i c h i s known t o e n h a n c e L P S - i n d u c e d IL1 s e c r e t i o n by human m o n o c y t e s ( 1 3 ) . F i n a l l y , TNF 10ug -3pg/ml d i d n o t i n f l u e n c e t h e t i t e r o f IL1 w h i c h was i n d u c e d by s t i m u l a t i o n w i t h L P S . T h e s e d a t a showed t h a t n e i t h e r IL1 s e c r e t i o n i t s e l f n o r any d e t e c t a b l e i n h i b i t o r y a c t i v i t y was i n d u c e d by TNF.

M o n o c y t e s from d i f f e r e n t human d o n o r s s e c r e t e w i d e l y d i f f e r i n g amounts of IL1 upon s t i m u l a t i o n w i t h a g i v e n dose o f LPS. N e v e r t h e l e s s , e n r i c h e d human monocytes r e p r e s e n t a s e n s i t i v e s y s t e m f o r m o n i t o r i n g IL1 p r o d u c t i o n ( 9 ) . T a b l e 3 shows t h a t L P S - s t i m u l a t e d e n r i c h e d m o n o c y t e s r e l e a s e d h i g h e r t i t e r s o f IL1 t h a n h o m o l o g o u s m o n o n u c l e a r c e l l s c o n t a i n i n g c o m p a r a b l e amounts of a d h e r e n t c e l l s . IFN-Y can f u r t h e r enhance IL1 t i t e r s . The d a t a d e s c r i b e d a b o v e o b t a i n e d w i t h I F N - Y c o n t a i n i n g m o n o c y t e - e n r i c h e d c u l t u r e s do not e x c l u d e the p o s s i b i l i t y t h a t TNF m i g h t i n f l u e n c e monocyte IL1 s e c r e t i o n v i a an i n d i r e c t r o u t e i m p l i c a t i n g l y m p h o c y t e s . T h e r e f o r e , t h e e f f e c t o f TNF on the IL1 s e c r e t i o n by mononuclear c e l l s was t e s t e d . A g a i n , TNF 10yg ^3pg/ml d i d n e i t h e r i n d u c e any IL1 s e c r e t i o n by i t s e l f , n o r d i d i t a f f e c t IL1 p r o d u c t i o n by mononuclear l e u c o c y t e s when t h e s e c e l l s were s t i m u l a t e d w i t h LPS (1pg t o 10ng/ml) ( d a t a not shown).

P e r i p h e r a l human m o n o c y t e s , when kept i n c u l t u r e f o r 24 h r s i n t h e absence o f s t i m u l a n t s , l o s e t h e i r s e n s i t i v i t y t o LPS as measured by i n d u c t i o n of I L 1 . I t has b e e n shown t h a t t h i s p r o c e s s can be p r e v e n t e d o r d e l a y e d by IFN-Y (13,1*0. The p o s s i b i l i t y was t e s t e d whether TNF might i n t e r f e r e w i t h t h i s i n v i t r o d i f f e r e n t i a

TABLE 1

IL1 Induction i n Murine P e r i t o n e a l Exudate C e l l Cultures

I L 1

(

^{t i t e r}

) D —

s t i m u l u s3 C3H/He c e l l s C3H/HeJ c e l l s

A) LPS 10yg 24 <4 TNF 10yg 6 <4 1]jg 4 <4 0.1yg <4 <4 none <4 <4 B) LPS 50yg 64 <4

TNF 10ug₃₂ <4

i n a c t i v a t e d TNF 10ug 32 <4

S t a p h , a u r e u s >512 256 none 16 <4

a P e r i t o n e a l e x u d a t e c e l l s ( 2 x 1 06/ m l ) o f e i t h e r C3H/He o r C3H/HeJ mice were c u l t u r e d i n t h e p r e s e n c e o f e i t h e r LPS (S. m o n t e v i d e o SH94, 10 o r 5 0 u g / m l ) , TNF ( 0 . 1 - 1 O y g / m l as i n d i c a t e d ) , h e a t i n a c t i v a t e d TNF w h i c h was kept a t 100°C f o r 10 m i n u t e s (10yg/ m l ) , S t a p h , a u r e u s c e l l s ( P a n s o r b i n 0.1% (w/v)) o r no s t i m u l u s .

b IL1 a c t i v i t y i n t h e s u p e r n a t a n t was d e t e r m i n e d i n t h e thymocyte c o s t i m u l a t o r a s s a y .

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TABLE 2

Influence of TNF on the IL1 A c t i v i t y Generated i n Monocyte Cultures - _ IL1 ( t i t e r )

A d d i t i o n t o t h e c u l t u r e s³

TNF/ml ₀ IFN-Y LPS

10 yg

<4 <4 64

1 yg

a a 64

0.1 yg

a a

³²

10 ng

a <4 64

1 ng

a <4 64

0.1 ng

<4 a 64

0 . 0 3 ng

a <4 64

0 . 0 0 3 n g

<4 <4 64

none

a a 64

a IL1 a c t i v i t y i n s u p e r n a t a n t s of a d h e r e n t human p e r i p h e r a l mononuclear l e u c o c y t e c u l t u r e s ( 3 x 1 0⁵/ m l ) was d e t e r m i n e d i n t h e thymocyte c o s t i m u l a t o r a s s a y a f t e r 24 h. The c u l t u r e s c o n t a i n e d t h e i n d i c a t e d amounts o f TNF i n t h e p r e s e n c e o r absence o f e i t h e r IFN-Y (300 U/ml) o r LPS from S . t y p h . (100ng/ m l ) .

a t i o n p r o c e s s i n a s i m i l a r way a s I F N - Y . T a b l e 4 shows t h a t t h i s was n o t t h e c a s e . P r e i n c u b a t i o n f o r 24 h a t 37°C of e n r i c h e d monocytes l e d t o n e a r l y c o m p l e t e a b r o g a t i o n o f IL1 p r o d u c t i o n d u r i n g a s u b s e q u e n t 24 h r s L P S - s t i m u l a t i o n p e r i o d ( c o l u m n B ) . P r e i n c u b a t i o n a t 4°C had no s u c h e f f e c t , i . e . l e f t IL1 i n d u c i b i l i t y i n t a c t . P r e i n c u b a t i o n a t 37°C i n t h e p r e s e n c e o f IFN-Y was a l s o a b l e t o s u s t a i n o p t i m a l IL1 i n d u c i b i l i t y . I n c o n t r a s t , TNF 10yg -0.1ng/ml c o u l d n o t s u s t a i n IL1 i n d u c i b i l i t y , nor d i d i t a d v e r s e l y a f f e c t IL1 p r o d u c t i o n by r e s p o n s i v e monocytes.

M o n o c y t e v i a b i l i t y was not i n f l u e n c e d by i n c u b a t i o n a t 37°C and t h e r e was no IL1 i n h i b i t o r d e t e c t a b l e i n s u p e r n a t a n t s o f c u l t u r e d c e l l s ( d a t a not shown).

DISCUSSION

TNF i s a t y p i c a l m e d i a t o r of e n d o t o x i n e f f e c t s ( 5 , 1 1 ) . I t i n d u c e s a number o f symptoms s e e n i n a n i m a l s w i t h b a c t e r i a l i n f e c t i o n s or o t h e r m e t a b o l i c d i s o r d e r s . The i d e n t i t y o f TNF w i t h c a c h e c t i n and t h e knowledge of c a c h e c t i n f u n c t i o n s h e d some l i g h t on t h e m o l e c u l a r m e c h a n i s m o f t h e m e t a b o l i c d e r a n g e m e n t s e e n i n c a c h e c t i c a n i m a l s ( 1 5 ) . C a c h e c t i n i s a m e d i a t o r r e s p o n s i b l e f o r w e i g h t l o s s and

TABLE 3

IL1 A c t i v i t y Generated i n D i f f e r e n t Culture Systems IL1 ( t i t e r )

c e l l s3 LPS (ng/ml)

100 1 0.1 0.01 0.001 0

MNL 8 4 4 <4 <4 <4

a d h e r e n t c e l l s 32 16 16 4 <4 <4

a d h e r e n t c e l l s

+ IFN-Y 32 32 16 16 8 <4

a P e r i p h e r a l human mononuclear l e u c o c y t e s ( 2 x 1 06/ m l ) c o n t a i n i n g 15% monocytes o r the a d h e r e n t c e l l f r a c t i o n ( 3 x 1 05/ m l ) c o n t a i n i n g 90^95% m o n o c y t e s w e r e c u l t u r e d w i t h o r w i t h o u t human I F N - Y (300 U/ml) and t h e i n d i c a t e d amount o f LPS from S. t y p h . N u m b e r s g i v e n i n t h e T a b l e r e p r e s e n t t i t e r s of IL1 measured i n 24 h r s s u p e r n a t a n t s by t h e thymocyte c o s t i m u l a t o r a s s a y .

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TABLE M

Influence of TNF preculture of human monocytes on the IL1 a c t i v i t y generated by LPS IL1 ( t i t e r )

A d d i t i o n t o the a f t e r p r e c u l t u r e

c u l t u r e s3 A B C

none 24h 37°C 24h 4°C

TNF 10 ug/ml 256 4 256

1 ug/ml 256 4 256

100 ng/ml 256 4 256

10 ng/ml 256 4 256

1 ng/ml 256 4 256

0.1 ng/ml 256 M 256

Y-IFN 100 U/ml 256 256 256

0 256 4 256

a P e r i p h e r a l human a d h e r e n t mononuclear l e u c o c y t e s ( 3 x 1 05/ m l ) were c u l t u r e d w i t h t h e i n d i c a t e d amount o f TNF o r human IFN-Y (100U/ m l ) . The c u l t u r e s w e r e s t i m u - l a t e d w i t h LPS from S . t y p h . (10ng/ml) e i t h e r i m m e d i a t e l y a t the o n s e t o f c u l t u r e s

( A ) , a f t e r 24h o f p r e c u l t u r e a t 37°C ( B ) , o r a f t e r 24h o f p r e c u l t u r e a t 4°C ( C ) . IL1 a c t i v i t y was d e t e r m i n e d i n t h e s u p e r n a t a n t s 24h a f t e r LPS s t i m u l a t i o n i n t h e thymocyte c o s t i m u l a t o r a s s a y .

w a s t i n g o f t h e b o d y ' s e n e r g y r e s e r v e s a p p a r e n t l y due t o r e d u c e d l i p o p r o t e i n l i p a s e p r o d u c t i o n and f u n c t i o n ( 1 6 ) . P a s s i v e i m m u n i z a t i o n w i t h a n t i b o d i e s t o TNF c a n s h i f t t h e LD50 o f b a c t e r i a l e n d o t o x i n i n mice t o h i g h e r doses o f LPS, t h u s i n d i c a t i n g t h e i n v o l v e m e n t of T N F / c a c h e c t i n i n t h e l e t h a l e f f e c t s o f e n d o t o x i n ( 1 7 ) . A l s o , t h e t y p i c a l p a t t e r n o f p l a s m a enzyme l e v e l s w h i c h i s seen a f t e r e n d o t o x i n a p p l i c a t i o n can be o b t a i n e d w i t h TNF i n j e c t i o n s ( 1 1 ) . S i n c e TNF i s n o t t h e o n l y m e d i a t o r r e l e a s e d i n an o r g a n i s m a f t e r e n d o t o x i n e x p o s u r e i t i s impor- t a n t t o i n v e s t i g a t e whether a l l e n d o t o x i n e f f e c t s can be m e d i a t e d by one s i n g l e m e d i a t o r l i k e TNF. A l t e r n a t i v e l y , s e v e r a l d i f f e r e n t m e d i a t o r s c o u l d be i n d u c e d w h i c h t o g e t h e r a r e r e s p o n s i b l e f o r t h e e n d o t o x i c r e a c t i o n o r d i f f e r e n t m e d i a t o r s c o u l d be e l i c i t e d w i t h i d e n t i c a l o r s i m i l a r f u n c t i o n s .

The h y p o t h e r m i c r e a c t i o n o f mice a f t e r e n d o t o x i n a p p l i c a t i o n i s a t y p i c a l i n v i v o e f f e c t o f LPS w h i c h c a n e a s i l y be m e a s u r e d . TNF i n j e c t i o n o f mice a l s o i n d u c e d h y p o t h e r m i a ( F i g . 1 ) . T h i s h y p o t h e r m i c r e a c t i o n was n o t t h e r e s u l t o f c o n t a m i n a t i n g LPS i n the TNF p r e p a r a t i o n s i n c e the h y p o t h e r m i a was a l s o i n d u c e d by TNF i n LPS low r e s p o n d e r C3H/HeJ a n i m a l s . M e d i a t i o n of t h e f e v e r r e a c t i o n o f LPS i n most s p e c i e s i s s u p p o s e d l y due t o i n d u c t i o n o f I L 1 - r e l e a s e i n macrophages ( 4 ) . P u r i f i e d r e c o m b i n a n t IL1 i s p y r o g e n i c i n r a b b i t s and q u a l i f i e s as endogenous p y r o g e n ( 4 ) . The same has been d e s c r i b e d f o r TNF ( 1 8 ) . Because of t h e o v e r l a p p i n g e f f e c t s o f IL1 and TNF i t seemed i m p o r t a n t t o us t o d e t e r m i n e w h e t h e r t h e s e TNF e f f e c t s w e r e d i r e c t e f f e c t s o f TNF or whether they were m e d i a t e d v i a the endogenous i n d u c t i o n o f IL1 p r o d u c t i o n . The o b s e r v a t i o n t h a t TNF d i d n o t i n t e r f e r e w i t h IL1 t e s t systems n e i t h e r on the l e v e l o f IL2 p r o d u c t i o n nor on t h e l e v e l o f I L 2 r e c e p t o r i n d u c t i o n i n t h y m o c y t e s made i t p o s s i b l e t o m e a s u r e IL1 w i t h o u t h a v i n g t o remove TNF from the t e s t samples.

We d i d n o t d e t e c t IL1 a c t i v i t y i n the serum o f a n i m a l s 1 h t o 24 h a f t e r t h e y h a d r e c e i v e d l a r g e amounts o f TNF and showed a s t r o n g h y p o t h e r m i c r e a c t i o n . PEC c u l t u r e s from LPS r e s p o n d e r mice s t i m u l a t e d w i t h graded amounts o f TNF p r o d u c e d l o w a m o u n t s o f IL1 a c t i v i t y , e s p e c i a l l y when background l e v e l s o f IL1 a c t i v i t y were a l r e a d y seen w i t h o u t s t i m u l a t i o n . These o b s e r v a t i o n s s u p p o r t the n o t i o n t h a t TNF m i g h t i n d u c e a f a c t o r ( s ) w h i c h s y n e r g i z e s w i t h IL1 i n t h e c o s t i m u l a t o r a s s a y ( 1 9 ) . But a l s o a b i o l o g i c a l l y i n a c t i v e , h e a t i n a c t i v a t e d TNF p r e p a r a t i o n was s t i l l a b l e t o i n d u c e t h e same IL1 r e s p o n s e . IL1 p r o d u c t i o n was not i n d u c e d when c u l t u r e s c o n t a i n i n g p e r i t o n e a l macrophages o f LPS-low r e s p o n d e r C3H/HeJ mice were s t i m u l a t e d w i t h t h e same .amounts o f TNF. A d d i t i o n o f i n d o m e t h a c i n t o b l o c k p r o s t a g l a n d i n p r o d u c t i o n t h a t i s known t o i n h i b i t I L 1 p r o d u c t i o n d i d a l s o n o t l e a d t o h i g h e r IL1 a c t i v i t y ( d a t a not shown).

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I n c u l t u r e systems w i t h human c e l l s no IL1 p r o d u c t i o n was i n d u c e d by TNF under a v a r i e t y o f c u l t u r e c o n d i t i o n s . S p e c i a l c a r e was t a k e n t o use r e a g e n t s i . e . f e t a l c a l f serum i n t h e s e systems w h i c h were t e s t e d p r e v i o u s l y t o show t h a t t h e y w e r e f u n c t i o n a l l y L P S - f r e e and d i d n o t i n d u c e any d e t e c t a b l e background produc- t i o n ( 9 ) . C u l t u r e s o f e n r i c h e d monocytes c o u l d n o t be i n d u c e d "by TNF i n a w i d e d o s e r a n g e t o p r o d u c e I L 1 . S u c h c u l t u r e s o f e n r i c h e d m o n o c y t e s r e s p o n d w i t h m e a s u r a b l e IL1 p r o d u c t i o n t o as l i t t l e as 1pg LPS/ml, depending on t h e s e n s i t i v e t y o f t h e i n d i v i d u a l donor ( 9 ) . The s e n s i t i v i t y f o r LPS can be f u r t h e r i n c r e a s e d by IFN-Y. S t i l l , even i n t h e p r e s e n c e o f IFN-Y, TNF c o u l d n o t i n d u c e IL1 s e c r e - t i o n . On t h e o t h e r hand, TNF d i d n o t i n d u c e any IL1 i n h i b i t o r , as w e l l , nor d i d i t i n t e r f e r e w i t h L P S - i n d u c e d IL1 p r o d u c t i o n i n any s e n s e .

A l t h o u g h u n p u r i f i e d mononuclear c e l l s a r e u s u a l l y l e s s e f f e c t i v e i n g e n e r a t i n g IL1 t h a n e q u a l amounts o f p u r i f i e d monocytes, i t c o u l d n o t be e x c l u d e d t h a t TNF m i g h t s t i m u l a t e I L 1 s e c r e t i o n i n d i r e c t l y by a c t i n g on l y m p h o c y t e s . The lympho- c y t e s m i g h t s e c r e t e an IL-1 i n d u c i n g a c t i v i t y i n r e s p o n s e t o TNF. T h i s p o s s i b i l i ^ - t y c a n , h o w e v e r , be d i s m i s s e d , s i n c e u n p u r i f i e d mononuclear c e l l s were a l s o not i n d u c e d by TNF t o produce I L 1 .

I F N - Y i s known t o s u s t a i n t h e I L 1 - i n d u e i b i l i t y by LPS i n monocyte c u l t u r e s a f t e r p r e c u l t u r e a t 3 7 ° C TNF had no c o m p a r a b l e e f f e c t on m o d u l a t i o n o f IL1 s e c r e t i o n . T h u s , u n d e r a v a r i e t y o f s t i m u l a t i o n p r o t o c o l s TNF d i d n e i t h e r enhance nor r e d u c e t h e IL1 a c t i v i t y g e n e r a t e d by monocytes w i t h i n 24 h.

A l l our e x p e r i m e n t a l e v i d e n c e p o i n t s t o a d i r e c t IL1 ^-independent a c t i v i t y o f TNF when a p p l i e d i n v i v o . Our o b s e r v a t i o n s , t h e r e f o r e , s u p p o r t t h e d a t a o f D i n a r e l l o e t a l . who showed a d i r e c t p y r o g e n i c e f f e c t o f TNF i n r a b b i t s ( 1 8 ) . D i f f e r e n t f i n d i n g s h a v e b e e n r e p o r t e d t o the q u e s t i o n whether TNF i n d u c e s IL1 p r o d u c t i o n i n monocyte/macrophage c u l t u r e s ( 1 9 , 2 0 , 2 1 ) . The d i f f e r e n t r e s u l t s by H o f f m a n n (19) can be e x p l a i n e d by t h e f a c t t h a t i n t h e s e s t u d i e s c e l l - a s s o c i a t e d IL1 a c t i v i t y and not r e l e a s e d IL1 was measured.

The a c t i v a t i o n o f murine thymocytes f o r I L 2 p r o d u c t i o n and p r o l i f e r a t i o n seems t o be t h e s p e c i f i c a c t i o n o f I L 1 and n o t s h a r e d by TNF i n s p i t e o f t h e w i d e o v e r l a p o f t h e b i o l o g i c a l a c t i v i t i e s o f the two macrophage p r o d u c t s . A c c o r d i n g t o t h e p r e s e n t e d d a t a TNF does not i n d u c e s t i m u l a t i o n f o r I L 1 s e c r e t i o n by mono^

c y t e s / m a c r o p h a g e s . T h e r e f o r e , t h e e f f e c t s seen a f t e r e n d o t o x i n a p p l i c a t i o n i n v i v o seem t o be t h e sum and t h e d i r e c t c o l l a b o r a t i v e a c t i o n o f s e v e r a l m e d i a t o r s l i k e TNF and IL1 . These m e d i a t o r s a r e most l i k e l y i n d u c e d a t t h e same t i m e and n o t s e q u e n t i a l l y by each o t h e r .

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Address r e p r i n t r e q u e s t s t o : D.N. Manne1

I n s t i t u t e o f Immunology and G e n e t i c s German Cancer R e s e a r c h C e n t e r D-6900 H e i d e l b e r g

F.R.G.