LYMPHOKINE RESEARCH Volume 9, Number 4,1990 Mary Ann Liebert, Inc., Publishers
RESEARCH REPORT
Tumor-Induced Tumor Necrosis Factor Production in Macrophages
D.N. MÄNNEL, R. JÄNICKE, U. WESTENFELDER, B. ECHTENACHER, A. KIST, and W. FALK
Institute for Immunology and Genetics, German Cancer Research Center, FRG
ABSTRACT
Tumor-associated tumor n e c r o s i s f a c t o r (TNF) p r o d u c t i o n i n p a t i e n t s as w e l l as a TNF-inducing membrane c o n s t i t u e n t of tumor c e l l s have been r e p o r t e d . In a murine fibrosarcoma model we analyzed TNF p r o d u c t i o n d u r i n g growth of a tumor t r a n s p l a n t . In situ h y b r i d i z a t i o n showed t h a t a g r a d u a l l y i n c r e a s i n g number of c e l l s w i t h i n the tumor t i s s u e became p o s i t i v e f o r TNFmRNA. A l s o , i n s p l e e n c e l l s o f tumor-bearing mice TNFmRNA became more abundant i n l a t e r stages of tumor growth compared t o e a r l y stages. In plasma of these animals, however, TNF a c t i v i t y was not d e t e c t e d at any time even 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 e n d o t o x i n . N e u t r a l i z a t i o n w i t h monoclonal a n t i b o d i e s of endogenous TNF d u r i n g tumor growth d i d not a f f e c t the growth r a t e of the tumor, i n d i c a t i n g t h a t e i t h e r the a n t i b o d i e s d i d not reach the r e l e v a n t TNF p r o d u c t i o n and a c t i o n s i t e s or t h a t endogenously produced TNF d i d not p l a y a s i g n i f i c a n t r o l e i n t h i s tumor model.
INTRODUCTION
TNF i s a product mainly of a c t i v a t e d monocytes/macrophages which b e s i d e s i t s c y t o t o x i c a c t i v i t y in vitro f o r some tumor c e l l s e x e r t s a p l e t h o r a of e f f e c t s on many d i f f e r e n t k i n d s of c e l l types. Due t o numerous b i o l o g i c a l l y important
f u n c t i o n s , TNF i s thought t o p l a y a key r o l e i n r e g u l a t i o n o f the n o n s p e c i f i c host response i n inflammation. Monocytes of cancer p a t i e n t s have been shown t o spontaneously r e l e a s e s i g n i f i c a n t l y higher amounts of TNF i n t o the supernatant when compared t o c o n t r o l s ( 1 ) . A l s o , enhanced l e v e l s of TNF i n serum or plasma of cancer p a t i e n t s have been r e p o r t e d (2,3). In these p a t i e n t s , a f t e r r e s e c t i o n of t h e tumors the TNF l e v e l s r e t u r n e d t o t h a t of c o n t r o l s ( 3 ) .
In a d d i t i o n , we were a b l e t o demonstrate t h a t tumor membrane c o n s t i t u e n t s d i r e c t l y a c t i v a t e human monocytes f o r TNF production ( 4 ) . TNFmRNA e x p r e s s i o n by tumor i n f i l t r a t i n g macrophages has a l s o been r e p o r t e d r e c e n t l y ( 5 ) . In order t o
i n v e s t i g a t e whether TNF p r o d u c t i o n c o r r e l a t e s with tumor growth, we determined TNF i n plasma and TNFmRNA i n tumors and spleens of mice i n o c u l a t e d i n t r a d e r m a l l y w i t h fibrosarcoma c e l l s . In a d d i t i o n , t o address the q u e s t i o n o f the r o l e o f the observed tumor-induced TNF the animals were t r e a t e d w i t h anti-murine TNF monoclonal a n t i b o d i e s throughout the experiment.
MATERIALS AND METHODS
Tumor C e l l s : Methylcholanthrene-induced fibrosarcoma c e l l s CFS1 were generated on a C3H mouse and kept a t t h e German Cancer Research Center. The c e l l s were grown as s i n g l e c e l l s i n t i s s u e c u l t u r e i n RPMI 1640 (Gibco) w i t h 10% FCS. The c e l l s were washed i n phosphate b u f f e r e d s a l i n e (PBS) and l x l O6 CFS1 c e l l s i n 50pi PBS were i n j e c t e d i n t r a d e r m a l l y i n t o t h e f l a n k s o f C3H mice ( S t a a t l . V e r s u c h s t i e r a n s t a l t , Hannover, F.R.6.).
Northern B l o t A n a l y s i s ; Cytoplasmic RNA was prepared from t h e s p l e e n s o f i n d i v i d u a l mice a t d i f f e r e n t times d u r i n g tumor growth, e l e c t r o p h o r e s e d i n 1%
agarose-formaldehyde g e l s . RNA was t r a n s f e r r e d t o nylon f i l t e r s and h y b r i d i z e d as p r e v i o u s l y d e s c r i b e d ( 4 ) . A TNFcDNA probe (a 750 bp Eco Rl-fragment o f t h e coding r e g i o n o f human TNFcDNA) was l a b e l e d with 3 2P by t h e random primer method. A f t e r autoradiography t h e f i l t e r s were s t r i p p e d and h y b r i d i z e d w i t h a human /?-actin cDNA probe (a 560 bp S a l 1-Eco RI fragment o f /?-actin cDNA). The h y b r i d i z a t i o n s i g n a l s were q u a n t i f i e d by scanning t h e o p t i c a l d e n s i t y o f t h e autoradiograms.
The TNFmRNA s i g n a l s were e v a l u a t e d by n o r m a l i z a t i o n u s i n g t h e r e s p e c t i v e a c t i n mRNA s i g n a l s .
In Situ H y b r i d i z a t i o n ; Tumor t i s s u e was f r o z e n i n l i q u i d n i t r o g e n immediately a f t e r e x c i s i o n and kept a t -70°C u n t i l t h e p r e p a r a t i o n o f 5mm c r y o s e c t i o n s . F i x a t i o n and h y b r i d i z a t i o n was performed as d e s c r i b e d r e c e n t l y ( 4 ) .
TNF B i o a s s a v and ELISA; Blood was c o l l e c t e d from t h e r e t r o o r b i t a l plexus a t i n d i c a t e d times. Plasma was prepared and s t o r e d a t -20°C u n t i l t e s t e d f o r TNF a c t i v i t y i n t h e L929 b i o a s s a y (7) ( i n t h e presence o f a c t i n o m y c i n D) and i n a s p e c i f i c ELISA f o r murine TNF. A monoclonal r a t ant i murine TNF antibody was generated by immunization o f r a t s with p u r i f i e d n a t u r a l murine TNF and f u s i o n o f the s p l e e n s o f t h e s e r a t s with P3.X63.Ag8.653. One c l o n e (Vlq) was s e l e c t e d which n e u t r a l i z e d murine TNF. lpg V l q Ig n e u t r a l i z e d 80 pg o f recombinant murine TNF
(rmTNF, K n o l l AG, Ludwigshafen, FRG). In v i v o , 20 pg V l q g i v e n i p were a b l e t o p r o t e c t mice from LPS-induced l e t h a l shock (Echtenacher, B. e t a l . , manuscript s u b m i t t e d ) . F o r t h e T N F - s p e c i f i c ELISA, p l a t e s were coated w i t h p u r i f i e d V l q
(10pq/ml). A f t e r i n c u b a t i o n o f t h e coated w e l l s with s e r i a l d i l u t i o n s o f t h e t e s t samples b i o t i n y l a t e d p r o t e i n A - p u r i f i e d p o l y c l o n a l r a b b i t anti-rmTNF ( K n o l l AG, Ludwigshafen, FRG) Ig (ISpq/ml) was a p p l i e d . The enzymatic a c t i v i t y a f t e r r e a c t i o n w i t h s t r e p t a v i d i n - p e r o x i d a s e and t e t r a m e t h y l b e n z i d i n e (Sigma) as p e r o x i d a s e s u b s t r a t e was determined a t 5 t o 20 min. S e n s i t i v i t y o f t h e ELISA was 1 ng/ml f o r rmTNF.
I n t e r l e u k i n 6 (IL61 Assay; The IL6 d e t e r m i n a t i o n was performed u s i n g a 4 day p r o l i f e r a t i o n assay w i t h t h e IL6-dependent hybridoma B9 ( 8 ) .
Immunohistochemistrv: C e l l - a s s o c i a t e d TNF i n tumor s e c t i o n s was determined with a p r o t e i n A - p u r i f i e d r a b b i t anti-rmTNF Ig ( K n o l l AG, Ludwigshafen, FRG) and p e r o x i d a s e - c o u p l e d goat a n t i - r a b b i t antibody (Dianova, Hamburg, FRG). As s u b s t r a t e 3-amino-9 e t h y l c a r b a z o l e (AEC, Sigma) was used. The presence o f macrophages/monocytes was determined by s t a i n i n g o f tumor s e c t i o n s w i t h anti-Mac- 1 a n t i b o d i e s a c c o r d i n g t o t h e s u p p l i e r ' s p r o t o c o l (Boehringer, Mannheim, FRG).
RESULTS
Mice r e c e i v e d 106 CFS1 c e l l s i n t r a d e r m a l l y and b l o o d was drawn r e g u l a r l y d u r i n g tumor growth b e g i n n i n g on day 6. No TNF was d e t e c t a b l e i n t h e plasma o f t h e s e tumor b e a r i n g animals a t any time n e i t h e r i n t h e TNF b i o a s s a y nor i n a TNF- s p e c i f i c ELISA system. Even a f t e r an i n j e c t i o n o f b a c t e r i a l endotoxin
(S.minneaota LPS, Sigma, lOpg/animal, i p ) 2 hours b e f o r e b l e e d i n g t h e mice, no TNF was found i n t h e plasma. Low l e v e l s o f i n t e r l e u k i n 6 (IL6) were d e t e c t a b l e i n the tumor-bearing animals from day 17 on a f t e r tumor i m p l a n t a t i o n . In plasma o f animals which had r e c e i v e d b a c t e r i a l endotoxin IL6 was always p r e s e n t , but t h e c o n c e n t r a t i o n d i d not change s i g n i f i c a n t l y d u r i n g tumor growth (data n o t shown).
mRNA from t h e s p l e e n s o f tumor-bearing mice was prepared and analyzed i n Northern b l o t s . The s i g n a l s p e c i f i c f o r TNFmRNA i n c r e a s e d d u r i n g tumor growth and was h i g h e s t a t t h e l a t e s t time p o i n t (day 24) o f t h e experiment ( F i g . l ) . A peak o f TNFmRNA on day 8 i n d i c a t e d a t r a n s i e n t r i s e i n TNF e x p r e s s i o n i n t h e s p l e e n d u r i n g an e a r l y stage o f tumor growth.
20000-,
6 8 10 13 15 17 20 22 24 Time After Tumor Implantation (Days)
FIGURE 1: TNFmRNA Expression i n Spleens of Tumor bearing Mice a t D i f f e r e n t Time P o i n t s a f t e r Tumor Implantation. The spleens of the mice were removed a t t h e i n d i c a t e d times and TNFmRNA determined i n Northern b l o t a n a l y s i s . The TNFmRNA s i g n a l from spleens of non tumor bearing mice had a d e n s i t y o f 6400.
In situ h y b r i d i z a t i o n of TNFmRNA i n tumor t i s s u e r e v e a l e d very few TNF p o s i t i v e c e l l s at e a r l y stages of tumor growth. The number of c e l l s p o s i t i v e f o r TNFmRNA i n c r e a s e d g r a d u a l l y from day 6 on (tested on days 6, 8, 10, 13, IS, 11, 20, 22 and 24) and was maximal a t the l a t e s t time p o i n t t e s t e d . F i g . 2 d e p i c t s t y p i c a l TNFmRNA s i g n a l s i n tumor t i s s u e from day 8 and day 20 of tumor growth.
Though s u b j e c t i v e and d i f f i c u l t t o q u a n t i f y , t h i s gradual i n c r e a s e o f c e l l s showing TNFmRNA expression was h i g h l y r e p r o d u c i b l e i n d i f f e r e n t s e t s of
FIGURE 2: In Situ TNFmRNA Expression i n Tumor T i s s u e . Tumor t i s s u e s from day 8 (a) and day 20 (b) a f t e r tumor implantation were h y b r i d i z e d with a TNF-RNA probe.
The photographs d i s p l a y the same t i s s u e area x 250 as l i g h t and dark f i e l d photograph, r e s p e c t i v e l y .
h y b r i d i z a t i o n s and tumor growth experiments. CFS1 tumor c e l l s themselves were n e g a t i v e f o r TNFmRNA e x p r e s s i o n and t h e c e l l s were not a b l e t o induce TNFmRNA e x p r e s s i o n i n murine p e r i t o n e a l exudate macrophages in vitro. Immunhistochemical s t a i n i n g o f t h e tumor s e c t i o n s with a p o l y c l o n a l anti-rmTNF immunoglobulin f r a c t i o n supported t h e r e s u l t s from in situ h y b r i d i z a t i o n s showing s i m i l a r numbers o f TNF p r o t e i n - c o n t a i n i n g c e l l s (data not shown). S t a i n i n g o f t h e tumor t i s s u e s f o r t h e presence o f monocytes/macrophages a l s o r e v e a l e d an i n c r e a s i n g number o f M a c - 1 - p o s i t i v e c e l l s d u r i n g tumor growth. The number o f phagocytes d e t e c t e d by s t a i n i n g , however, exceeded t h e number o f c e l l s w i t h TNFmRNA s i g n a l c o n s i d e r a b l y •
A f t e r d e t e c t i o n o f tumor-induced endogenous TNF e x p r e s s i o n , we attempted t o determine t h e r o l e o f t h i s TNF f o r tumor growth. T h e r e f o r e , t h e animals r e c e i v e d an i p i n j e c t i o n o f murine T N F - n e u t r a l i z i n g a n t i b o d i e s t o g e t h e r w i t h t h e tumor t r a n s p l a n t and every t h i r d day t h e r e a f t e r . F i g * 3 shows t h e growth curves o f t h e tumors i n u n t r e a t e d and a n t i - T N F - t r e a t e d mice. No s i g n i f i c a n t d i f f e r e n c e i n t h e diameter o f t h e tumors a t any time became obvious. A l s o , t h e body weight o f t h e animals on day 17 a f t e r tumor i m p l a n t a t i o n d i d not vary between t h e two groups
(15.6±1.3g f o r u n t r e a t e d versus 15.7±0.9g f o r a n t i - T N F - t r e a t e d m i c e ) . Thus, a p p l i c a t i o n o f V l q a n t i b o d i e s had no obvious e f f e c t on tumor growth i n t h i s mouse model.
12-
2-
0 J 1 1 1 1 1 1 1
6 S 10 12 14 16 18
Time After Tumor Implantation (days)
FIGURE 3: Tumor Growth i n Untreated and Anti-TNF-Treated Mice. In one group each animal r e c e i v e d 100/¿g i n 0.3ml V l q hybridoma supernatant i p on t h e day o f tumor i m p l a n t a t i o n and every t h i r d day t h e r e a f t e r . Tumor diameters were measured i n t h e u n t r e a t e d group (O) and t h e a n t i - T N F - t r e a t e d group (A) on t h e i n d i c a t e d days and are g i v e n w i t h standard d e v i a t i o n s .
DISCUSSION
The p r o d u c t i o n o f TNF d u r i n g tumor growth in vivo as p r e d i c t e d by t h e l i t e r a t u r e (1-5) was v e r i f i e d i n t h e tumor model d e s c r i b e d above. Not o n l y d i d tumor i n f i l t r a t i n g macrophages s y n t h e s i z e TNF but a l s o c e l l s i n t h e spleens o f these tumor b e a r i n g animals became p o s i t i v e f o r TNFmRNA. T h i s i n d i c a t e d t h a t 1.
p h y s i c a l c o n t a c t o f tumor c e l l s with macrophages induced l o c a l p r o d u c t i o n o f TNF and 2. t h a t t h e tumortransplant induced a s t a t u s o f inflammation l e a d i n g t o systemic a c t i v a t i o n o f macrophages/monocytes. The high TNFmRNA e x p r e s s i o n i n spleens a f t e r day 20 o f tumor growth c o u l d a l s o be based on t h e c e n t r a l n e c r o s i s i n t h e tumors which was r e g u l a r l y observed a t l a t e r stages o f tumor growth. The f a c t t h a t no plasma TNF was d e t e c t e d i n t h e tumor-bearing animals i s i n agreement w i t h a r e c e n t p u b l i c a t i o n (7) but i n c o n t r a s t t o t h e observed enhanced TNF l e v e l s i n cancer p a t i e n t s (2,3).
T h i s c o u l d e i t h e r be explained by the tumor type used f o r t r a n s p l a n t a t i o n o r by the s e n s i t i v i t y o f t h e TNF assays used i n these experiments. D e t e c t i o n o f TNF i n b i o l o g i c a l f l u i d s l i k e plasma i s sometimes d i f f i c u l t due t o t h e high p r o t e i n content o f t h e t e s t samples or due t o the presence o f i n h i b i t o r s and/or s o l u b l e r e c e p t o r s . T h i s c o u l d a l s o be the reason f o r t h e f a i l u r e t o d e t e c t TNF i n the plasma o f tumor b e a r i n g mice a f t e r endotoxin a d m i n i s t r a t i o n . T h i s o b s e r v a t i o n i s i n c o n t r a s t t o non-tumor bearing mice where c i r c u l a t i n g TNF can r e g u l a r l y be found a f t e r endotoxin a d m i n i s t r a t i o n . The presence o f IL6 a t l a t e r stages o f tumor growth might be an i n d i r e c t s i g n of the presence o f a c t i v a t e d macrophages because TNF belongs t o t h e best inducers f o r IL6 p r o d u c t i o n ( 7 ) . Tumor burden was shown t o be a s e n s i t i z i n g f a c t o r f o r d e t r i m e n t a l e f f e c t s o f TNF o r endotoxin a p p l i c a t i o n ( 9 ) . Therefore, the f a i l u r e t o detect any TNF r e l e a s e i n tumor- b e a r i n g mice was r a t h e r unexpected. Although s o l u b l e TNF was not d e t e c t e d , TNF p r o t e i n and TNFmRNA was c l e a r l y expressed i n macrophages i n tumor t i s s u e and TNFmRNA was expressed i n spleen.
In an attempt t o c l a r i f y the r o l e of t h i s endogenously produced TNF d u r i n g tumor growth, t h e mice were t r e a t e d with n e u t r a l i z i n g a n t i b o d i e s t o murine TNF.
The c o n c e n t r a t i o n o f a n t i b o d i e s i n the serum was high enough t o completely n e u t r a l i z e endotoxin-induced TNF r e l e a s e d i n l e t h a l shock s i t u a t i o n s
(Echtenacher, B. e t a l . , manuscript submitted) at any time o f t h e experiment.
T h i s treatment, however, d i d not have any obvious e f f e c t i n regard t o t h e tumor growth o r t o t h e behavior of the animals. I t i s conceivable t h a t t r a n s p l a n t a t i o n of such a l a r g e number of chemically induced tumor c e l l s simply overwhelms t h e primary defense system i n which TNF i s meant t o p l a y a r o l e as p h y s i o l o g i c a l mediator. A more simple explanation would be t h a t t h e a n t i b o d i e s d i d not get t o the r e l e v a n t s i t e o f TNF production and - a c t i o n and, t h e r e f o r e , were i n e f f e c t i v e . The e l u c i d a t i o n o f t h e r o l e of endogenous TNF i n tumor growth o b v i o u s l y needs t o be i n v e s t i g a t e d i n more r e f i n e d t e s t systems.
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Address r e p r i n t requests t o : D.N. Männel I n s t i t u t e f o r Immunology and Genetics German Cancer Research Center
Im Neuenheimer F e l d 280 6900 H e i d e l b e r g , FRG.
