Tumor-Induced Tumor Necrosis Factor Production in ... - Core

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 necrosis f a c t o r (TNF) production i n p a t i e n t s as w e l l as a TNF-inducing membrane constituent of tumor c e l l s have been reported. In a murine fibrosarcoma model we analyzed TNF production during 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 that 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 spleen c e l l s of 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 detected at any time even a f t e r s t i m u l a t i o n with b a c t e r i a l endotoxin. N e u t r a l i z a t i o n with monoclonal antibodies of endogenous TNF during 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 that e i t h e r the a n t i b o d i e s d i d not reach the relevant TNF production and a c t i o n s i t e s or that endogenously produced TNF d i d not play 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 besides 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 exerts 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 kinds of c e l l types. Due to 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 play a key r o l e i n r e g u l a t i o n of 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 to 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). Also, 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 reported (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 the tumors the TNF l e v e l s returned to that of c o n t r o l s (3). In a d d i t i o n , we were able to demonstrate that 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 expression by tumor i n f i l t r a t i n g macrophages has also been reported r e c e n t l y (5). In order to i n v e s t i g a t e whether TNF production 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 with fibrosarcoma c e l l s . In a d d i t i o n , t o address the question of the r o l e of the observed tumor-induced TNF the animals were t r e a t e d with anti-murine TNF monoclonal antibodies 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 at the 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) with 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 O CFS1 c e l l s i n 50pi PBS were injected intradermally into the flanks o f C3H mice (Staatl. 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 spleens 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 during 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 labeled with P by the 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 with a human /?-actin cDNA probe (a 560 bp S a l 1-Eco RI fragment of /?-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 the 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 evaluated by normalization using 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 frozen 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 at -70°C u n t i l the preparation 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 described r e c e n t l y (4). TNF Bioassav and ELISA; Blood was c o l l e c t e d from the r e t r o o r b i t a l plexus at i n d i c a t e d times. Plasma was prepared and stored 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 the L929 bioassay (7) ( i n the presence o f actinomycin 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 of 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 of the spleens o f these r a t s with P3.X63.Ag8.653. One clone (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 vivo, 20 pg V l q given i p were able 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 submitted). For t h e T N F - s p e c i f i c ELISA, p l a t e s were coated with 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 of the 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 applied. The enzymatic activity after reaction with s t r e p t a v i d i n - p e r o x i d a s e and tetramethylbenzidine (Sigma) as peroxidase s u b s t r a t e was determined at 5 t o 20 min. S e n s i t i v i t y o f the 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 determination was performed using a 4 day p r o l i f e r a t i o n assay with the 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 (Knoll AG, Ludwigshafen, FRG) and peroxidase-coupled goat anti-rabbit 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 with anti-Mac1 a n t i b o d i e s according t o the s u p p l i e r ' s p r o t o c o l (Boehringer, Mannheim, FRG). 6

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Mice r e c e i v e d 10 CFS1 c e l l s intradermally and blood was drawn r e g u l a r l y during tumor growth beginning on day 6. No TNF was d e t e c t a b l e i n t h e plasma o f these tumor b e a r i n g animals at any time neither i n the TNF bioassay nor i n a TNFspecific ELISA system. Even after an i n j e c t i o n of b a c t e r i a l endotoxin (S.minneaota LPS, Sigma, lOpg/animal, i p ) 2 hours before 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 of 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 present, but the 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 during tumor growth (data not shown). mRNA from t h e spleens 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 increased during tumor growth and was highest a t the l a t e s t time point (day 24) o f the experiment ( F i g . l ) . A peak of 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 expression i n the spleen during an e a r l y stage o f tumor growth.

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8 10 13 15 17 2 0 2 2 24 Time After Tumor Implantation (Days)

FIGURE 1: TNFmRNA Expression i n Spleens of Tumor bearing Mice at D i f f e r e n t Time Points a f t e r Tumor Implantation. The spleens of the mice were removed at the 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 density of 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 revealed 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 increased gradually from day 6 on (tested on days 6, 8, 10, 13, IS, 11, 20, 22 and 24) and was maximal at the l a t e s t time point 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 quantify, t h i s gradual increase of c e l l s showing TNFmRNA expression was highly reproducible i n d i f f e r e n t sets of

FIGURE 2: In Situ TNFmRNA Expression i n Tumor Tissue. Tumor t i s s u e s from day 8 (a) and day 20 (b) a f t e r tumor implantation were hybridized 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 negative f o r TNFmRNA expression and the c e l l s were not able t o induce TNFmRNA expression 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 the 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 the 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 the tumor t i s s u e s f o r the presence of monocytes/macrophages a l s o revealed an i n c r e a s i n g number o f Mac-1-positive c e l l s during tumor growth. The number of phagocytes detected by s t a i n i n g , however, exceeded the number o f c e l l s with TNFmRNA s i g n a l considerably• A f t e r d e t e c t i o n o f tumor-induced endogenous TNF expression, we attempted t o determine t h e r o l e of t h i s TNF f o r tumor growth. Therefore, the animals received an i p i n j e c t i o n of murine TNF-neutralizing antibodies together with 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 the growth curves of the tumors i n untreated and anti-TNF-treated 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 the diameter o f t h e tumors at any time became obvious. A l s o , t h e body weight of the animals on day 17 a f t e r tumor implantation d i d not vary between the two groups (15.6±1.3g f o r untreated versus 15.7±0.9g f o r anti-TNF-treated mice). Thus, a p p l i c a t i o n of V l q antibodies had no obvious e f f e c t on tumor growth i n t h i s mouse model.

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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 the day of tumor implantation and every t h i r d day t h e r e a f t e r . Tumor diameters were measured i n the untreated group (O) and the anti-TNF-treated group (A) on t h e i n d i c a t e d days and are given with standard d e v i a t i o n s .

DISCUSSION The production of TNF during tumor growth in vivo as p r e d i c t e d by the l i t e r a t u r e (1-5) was v e r i f i e d i n the tumor model described above. Not only d i d tumor i n f i l t r a t i n g macrophages synthesize TNF but a l s o c e l l s i n the spleens of these tumor bearing 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 that 1. p h y s i c a l contact o f tumor c e l l s with macrophages induced l o c a l production o f TNF and 2. t h a t t h e tumortransplant induced a status o f inflammation leading to systemic a c t i v a t i o n o f macrophages/monocytes. The high TNFmRNA expression i n spleens a f t e r day 20 of tumor growth could 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 detected i n the tumor-bearing animals i s i n agreement with a recent p u b l i c a t i o n (7) but i n contrast t o the observed enhanced TNF l e v e l s i n cancer p a t i e n t s (2,3).

This could 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 or by the s e n s i t i v i t y of the TNF assays used i n these experiments. Detection of 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 the high p r o t e i n content of the t e s t samples or due t o the presence of i n h i b i t o r s and/or soluble r e c e p t o r s . T h i s could a l s o be the reason f o r the f a i l u r e t o detect TNF i n the plasma of tumor bearing mice a f t e r endotoxin administration. This observation 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 administration. The presence of IL6 a t l a t e r stages of tumor growth might be an i n d i r e c t sign of the presence of a c t i v a t e d macrophages because TNF belongs t o the best inducers f o r IL6 production (7). Tumor burden was shown t o be a s e n s i t i z i n g factor f o r detrimental e f f e c t s of TNF or 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 tumorbearing mice was rather unexpected. Although soluble TNF was not detected, 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 during tumor growth, the mice were treated with n e u t r a l i z i n g antibodies t o murine TNF. The concentration of antibodies i n the serum was high enough t o completely neutralize endotoxin-induced TNF released i n lethal shock situations (Echtenacher, B. et a l . , manuscript submitted) at any time of the 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 the tumor growth or t o the behavior of the animals. I t i s conceivable that t r a n s p l a n t a t i o n of such a large number of chemically induced tumor c e l l s simply overwhelms the primary defense system i n which TNF i s meant t o play 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 that the antibodies d i d not get t o the r e l e v a n t s i t e of TNF production and - a c t i o n and, therefore, were i n e f f e c t i v e . The e l u c i d a t i o n of the r o l e of endogenous TNF i n tumor growth obviously 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 Heidelberg, FRG.