L Y M P H O K I N E R E S E A R C H Volume 8, Number 3,1989 Mary Ann Liebert, Inc., Publishers
Induction of Monokine Production by Tumor Cells
D.N. M A N N E L and R. JANICKE
Institut filr Immunologic und Genetik Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-6900 Heidelberg, FRG
ABSTRACT
C e l l s o f t h e p r o e r y t h r o m y e l o i d c e l l l i n e K562 and o f t h e T c e l l l i n e J u r k a t were a b l e t o i n d u c e an i n c r e a s e i n TNF-, I L l a - , and ILl/?-mRNA e x p r e s s i o n i n human p e r i p h e r a l b l o o d d e r i v e d monocytes in vitro. The a c t i v a t i n g p r i n c i p l e o f t h e s e tumor c e l l s was a s s o c i a t e d w i t h f r a c t i o n s o f membrane p r e p a r a t i o n s o f d i s t i n c t m o l e c u l a r mass, 32-38 kD f o r J u r k a t c e l l s and 46-54 kD f o r K562 c e l l s , r e s p e c t i v e l y . A t l e a s t p a r t o f t h e a c t i v a t i n g c o n s t i t u e n t seemed t o be p r o t e i n i n n a t u r e . I s o l a t e d membrane p r e p a r a t i o n s o f b o t h c e l l t y p e s i n d u c e d p r o d u c t i o n and s e c r e t i o n o f TNF. S t i m u l a t i o n o f monocytes w i t h t h e v i a b l e tumor c e l l s l e d t o TNF r e l e a s e o n l y when J u r k a t c e l l s were u s e d . V i a b l e K562 c e l l s i n d u c e d enhanced TNFmRNA e x p r e s s i o n b u t seemed t o a b s o r b s o l u b l e TNF f r o m t h e s u p e r n a t a n t .
INTRODUCTION
I t i s a g e n e r a l l y o b s e r v e d phenomenon t h a t monocytes/macrophages a r e a b l e t o d i s t i n g u i s h between n e o p l a s t i c c e l l s and n o r m a l c e l l s in vitro. The macrophages/monocytes d e s t r o y tumor c e l l s s e l e c t i v e l y as a r e s u l t o f a c t i v a t i o n . One means o f tumor c e l l k i l l i n g by a c t i v a t e d macrophages i s t h e p r o d u c t i o n and u t i l i z a t i o n o f tumor n e c r o s i s f a c t o r (TNF) as a c y t o t o x i c e f f e c t o r m o l e c u l e t o w h i c h some tumor c e l l s i n c o n t r a s t t o n o r m a l c e l l s a r e e x q u i s i t e l y s e n s i t i v e . The q u e s t i o n a r o s e w h e t h e r s u c h a phenomenon t a k e s a l s o p l a c e in vivo and w h e t h e r t h e tumor c e l l s c a n d i r e c t l y a c t i v a t e macrophages/monocytes f o r monokine p r o d u c t i o n . S e v e r a l r e p o r t s have documented an enhanced c a p a b i l i t y o f TNF p r o d u c t i o n by p e r i p h e r a l b l o o d m o n o n u c l e a r c e l l s o f c a n c e r p a t i e n t s ( 1 ) , enhanced TNF l e v e l s i n serum o r p l a s m a o f c a n c e r p a t i e n t s ( 2 , 3 ) , and enhanced c y t o s t a t i c a c t i v i t y o f monocytes f r o m c a n c e r p a t i e n t s ( 4 , 5 ) . We i n v e s t i g a t e d i n an in vitro s y s t e m w h e t h e r tumor c e l l s f r o m two c e l l l i n e s , K562 and J u r k a t , were c a p a b l e o f d i r e c t l y a c t i v a t i n g human p e r i p h e r a l b l o o d monocytes f o r TNF-, i n t e r l e u k i n 1 (IL1) o-, and ILl/?-mRNA e x p r e s s i o n , and TNF r e l e a s e .
MATERIALS AND METHODS
T i s s u e C u l t u r e C o n d i t i o n s : A l l c e l l l i n e s and t h e monocytes were c u l t u r e d i n RPMI
1640 (GIBCO), s u p p l e m e n t e d w i t h 10% h e a t i n a c t i v a t e d FCS (GIBCO) and 50/ig/ml g e n t a m y c i n (GIBCO). K562, and J u r k a t were r e g u l a r l y t e s t e d f o r mycoplasma c o n t a m i n a t i o n and a l s o f o r t h e absence o f v i r a l s t r u c t u r e s . Human p e r i p h e r a l b l o o d monocytes were i s o l a t e d f r o m b l o o d o f h e a l t h y d o n o r s by F i c o l l - P a q u e ( P h a r m a c i a ) 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 and t h e p e r i p h e r a l b l o o d m o n o n u c l e a r l e u c o c y t e s ( P B L ) were washed t w i c e w i t h RPMI 1640. M o n o c y t e s were i s o l a t e d by a d h e r e n c e on p l a s t i c f o r 1 h. The r e m a i n i n g c e l l s were >95% monocytes as d e t e r m i n e d by d i f f e r e n t i a l s t a i n i n g .
R e a g e n t8; F i x e d Staphylococcus aureus (Staph, aureus) c e l l s ( P a n s o r b i n , C a l b i o c h e m ) 10/xg/ml o r 5. minnesota l i p o p o l y s a c c h a r i d e ( L P S ) ( D i f c o L a b o r a t o r i e s ) lO^g/ml was u s e d f o r monocyte s t i m u l a t i o n .
TNF A s s a y ; S u p e r n a t a n t s o f t h e a d h e r e n t c e l l f r a c t i o n o f 4 x l 06 m o n o n u c l e a r l e u c o c y t e s p e r ml were t e s t e d i n t h e TNF s p e c i f i c ELISA s y s t e m as d e s c r i b e d r e c e n t l y ( 6 ) .
C y t o b l a s t P r e p a r a t i o n . Membrane P r e p a r a t i o n and E l e c t r o e l u t i o n f r o m SDS-PAGE:
E n u c l e a t e d c e l l s ( c y t o b l a s t s ) were p r e p a r e d as d e s c r i b e d ( 7 ) . Tumor c e l l membranes were p r e p a r e d by h o m o g e n i s a t i o n o f t h e c e l l s i n lOmM s o d i u m p h o s p h a t e b u f f e r pH 7.4, c o n t a i n i n g ImM M g C l , 30mM N a C l , ImM DTT, 0.005mM PMSF, and 0.1/xg/ml DNase, and s u b s e q u e n t c e n t r i f u g a t i o n t h r o u g h a 41Z s u c r o s e g r a d i e n t . The membrane p r e p a r a t i o n s were s e p a r a t e d on a 12.5X SDSPAGE, and p o r t i o n s o f t h e g e l s c o r r e s p o n d i n g t o d i f f e r e n t s i z e f r a c t i o n s were e l e c t r o e l u t e d i n 50mM T r i s , 0.38M g l y c i n e and 0.1Z d e o x y c h o l a t e f o r 1 h a t 100 V o l t .
mRNA D e t e c t i o n : RNA e x t r a c t i o n f o r d o t b l o t s was p e r f o r m e d by i s o l a t i n g t h e RNA by t h e g u a n i d i n e - H C l method ( 8 ) and b l o t t i n g t h e RNA o n t o n y l o n f i l t e r s (GENOFIT, H e i d e l b e r g , F.R.G.). F o r N o r t h e r n a n a l y s i s t h e RNA was p r e p a r e d u s i n g t h e g u a n i d i n e - t h i o c y a n a t e method ( 9 ) . A f t e r s e p a r a t i o n on 1Z a g a r o s e - f o r m a l d e h y d g e l s t h e RNA was t r a n s f e r r e d t o n y l o n f i l t e r s and h y b r i d i z e d as d e s c r i b e d ( 1 0 ) . F o r h y b r i d i z a t i o n t h e f o l l o w i n g p r o b e s were u s e d : 425 bp P s t I f r a g m e n t o f t h e n o n t r a n s l a t e d 3 ' r e g i o n o f human TNFcDNA (BASF, L u d w i g s h a f e n , F.R.G.), 460 bp human I L i a EcoRI-BamHl cDNA f r a g m e n t ( p 3 - I L l a ) o f t h e c o d i n g r e g i o n , 530 bp human Ihlp BamHl-Ndel cDNA f r a g m e n t ( p l l - I L l / ? ) o f t h e c o d i n g r e g i o n ( b o t h by U. G u b l e r , Hoffmann LaRoche, N u t l e y , New J e r s e y ) , 560 bp human / ? - a c t i n S a l l - E c o R l f r a g m e n t o f t h e cDNA as d e s c r i b e d ( 1 1 ) . The p r o b e s were l a b e l e d by t h e random p r i m e r method as d e s c r i b e d r e c e n t l y ( 1 2 ) .
RESULTS AND DISCUSSION
M o n o c y t e s i s o l a t e d f r o m b l o o d o f h e a l t h y d o n o r s e x p r e s s b a s e l i n e l e v e l s o f TNFmRNA and ILlmRNA when c u l t u r e d f o r 2 h o u r s a f t e r i s o l a t i o n . The mRNA e x p r e s s i o n was m a r k e d l y enhanced when t h e monocytes were c o c u l t u r e d w i t h e i t h e r K562 o r J u r k a t c e l l s . C o c u l t u r e w i t h n o n a d h e r e n t c e l l s f r o m a d i f f e r e n t b l o o d donor was u n a b l e t o a c t i v a t e t h e monocytes e x l c u d i n g a l l o g e n e i c d e t e r m i n a n t s as a g e n e r a l s t i m u l u s ( F i g . l ) . C y t o b l a 9 t s o f K562 c e l l s o r p u r i f i e d membrane p r e p a r a t i o n s o f b o t h tumor c e l l l i n e s were e q u a l l y a b l e t o i n d u c e i n c r e a s e d TNF-, I L l a - and ILl/?-mRNA e x p r e s s i o n .
Dot b l o t a n a l y s i s f o r gene a c t i v a t i o n by d i f f e r e n t s i z e f r a c t i o n s o f t h e tumor c e l l membrane p r e p a r a t i o n s r e v e a l e d a c t i v a t i n g c o n s t i t u e n t s w i t h r e l a t i v e m o l e c u l a r mass o f 32-38 kD f o r J u r k a t c e l l s and 46-54 kD f o r K562 c e l l s ( F i g . 2 ) . E q u i v a l e n t f r a c t i o n s o f membrane p r e p a r a t i o n s f r o m a l l o g e n e i c n o n a d h e r e n t p e r i p h e r a l b l o o d l e u c o c y t e s d i d n o t i n d u c e TNF-, I L l a - o r ILl/?-mRNA e x p r e s s i o n . The a c t i v a t i n g a c t i v i t y i n tumor c e l l membrane f r a c t i o n s was d e s t r o y e d when t h e f r a c t i o n s were t r e a t e d w i t h p r o t e a s e s ( d a t a n o t shown), i n d i c a t i n g a p r o t e i n s t r u c t u r e r e s p o n s i b l e f o r a c t i v i t y .
