IMWNOLOGICAL COMMUNICATIONS, 8 ( 4 ) , 457-468
(1979)
THE EFFECT OF PERITONEAL NACROPlIAGES FROM M I C E INJECTED WITH LEISHMANIA BRAZILIENSIS ON THE I N VITRO GROWTH I F TUMOR CELLS Fernando l l e r i n o and -1uan L u i s Departainento d e M e d i c i n a E x p e r i m e n t i l , I n s t i t u t o Venezolano d e I n v e s t i g a c i o n e s C i e n t i f i c i s , A p a r t a d o 1 8 2 7 , Caracas, Venezuela
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
"Abstract" Leishmania b r a z i l i e n s i s r e p o r t e d l y i s c a p a b l e of p r o d u c i n g a r e d u c t i o n i n t h e g r o w t h of s o l i d and a s c i t e s m u r i n e l e u k e m i a a d lymphomas. The p o s s i b i l i t y t h a t t h e i n h i b i t o r y e f f e c t on t h e a s c i t e s tumor was produced by t h e a c t i v a t i o n of t h e macrophage p e r i t o n e a l c e l l p o p u l a t i o n was e x p l o r e d . I t w a s o b s e r v e d t h a t a d h e r e n t c e l l s from t h e p e r i t o n e a l c a v i t y of b r a z i l i e n s i s i n j e c t e d m i c e c a u s e d a marked i n h i b i t o r v e f € e c t on t h e i n v i t r o growth of t h e 6C3HED lymphoma and EL-4 l e u k e m i a c e l l s . T h i s e f f e c t w a s d e p e n d e n t on t h e d e g r e e of t h e macrophage a c t i v a t i o n , w a s n o t produced by s u p e r n a t a n t s from c u l t u r e s of a c t i v a t e d macrophages, and i t seems t h a t conIn a d d i t i o n t o t a c t c e l l between t a r g e t and e f f e c t o r c e l l s i s n e c e s s a r y . I,. b r a z i l i e n s i s : b e i n g c y t o s t a t i c , t h i s e f f e c t was a l s o c y t o t - o x i c . T h e a c t i v a t e d macrophages w e r e a l s o c a p a b l e of s u p p r e s s i n g t h e m u l t i p l i c a t i o n of normal c e l l s i n d u c e d by m i t o g e n , b u t t h i s w a s n o t o b s e r v e d i f t h e c e l l s were a l r e a d y u n d e r g o i n g m u l t i p l i c a t i o n . A s i m i l a r c y t o s t a t i c e f f e c t cm t h e tumor c e l l s was o b s e r v e d t o be produced by t h e p e r i t o n e a l non-adherent c e l l p o p u l a t i o n of t h e L. b r a z i l i e n s i s i n j e c t e d mice.
I t h a s been w e l l e s t a b l i s h e d t h a t p e r i t o n e a l macrophages o b t a i n e d . from a n i m a l s i n j e c t e d w i t h b a c i l l u s Calmette-Guei-in
(BCC:)
,
chemicals, o r tliose
from a n i m a l s c h r o n i c a l l y i n f e c t e d by p a r a s i i - e s i n h i b i t , o r g r e a t l y r e d u c e ,
-
i n v i v o a n d i n v i t r o tumor g r o w t h ( 1 , 2 , 3 ) .
._
P r e v i o u s s t u d i e s have shown t h a t t h e a d m i n i s t r a t i o n of a n o n - p a t h o g e n i c s t r a i n o f Leishmania b r a z i l i e n s i s i n f l u e n c e d t h e g r o w t h of a s c i t i c and s o l i d lymphoma and l e u k e m i a ( 4 ) .
I t was a l s o d e m o n s t r a t e d t h a t a c t - i v a t e d macro-
p h a g e s which c a u s e d a marked i n h i b i t o r y e f f e c t on t.he i n v i t r o growth of s u c h tumors (5) w e r e p r e s e n t i n the p e r i t o n e a l c a v j t y of t h e mice p r e v i o u s l y in.jected w i t h t h e p a r a s i t e .
457 Copyright 0 1979 by Marcel Dekker, Inc. All Rights Reserved Neither this w o r k n o r dny p d r t may he reproduced o r transmitted in any f o r m o r by a n y means. electronic or mechanical, mcludmy phoiocopylng. microfilming, a n d recording, or by any information storage and retrieval s y s t e m , without permission in writing f r o m t h e publisher.
MERINO AND LUIS
458
T h i s r e p o r t d e s c r i b e s t h e e f f e c t of c e l l s from t h e p e r i t o n e a l c a v i t y of mice i n j e c t e d w i t h
&.
b r a z i l i e n s i s on t h e growth of tumor c e l l s a s w e l l
a s s u g g e s t i o n s about t h e p o s s i b l e mechanisms.
M a t e r i a l s and Methods
Mice of t h e s t r a i n s C3H/HeJ and C57BL/6J w e r e o b t a i n e d from The Jackson Laboratory (Bar Harbor, Maine, USA) and maintained i n o u r animal colony. Food and water were a d m i n i s t e r e d ad l i b i t u m .
The a s c i t e s tumors used were
t h e 6C3HEU lymphoma, and t h e lymphoblastic leukemia EL-4.
Both tumors were
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
o b t a i n e d from D r . F. Milgrom ( S t a t e U n i v e r s i t y of New York, Buffalo) and maintained i n syngenic mice by weekly t r a n s f e r s . The s t r a i n of
L.
b r a z i l i e n s i s used was o r i g i n a l l y i s o l a t e d from a
human c a s e of l e i s h m a n i a s i s and has been maintained by t h e Department of C e l l Biology, School of S c i e n c e s , Universidad C e n t r a l de Venezuela i n v i t r o i n Davis' medium f o r more than 500 passages. I n our experiments, t h i s s t r a i n was maintained i n NCTC 135 medium ( G I B C O , USA) supplemented w i t h 5% f e t a l c a l f serum.
Leishmania growth h a s
been maintained f o r over one y e a r i n t h i s medium and no pathogenic e f f e c t was seen when t h e s e p a r a s i t e s were i n j e c t e d i n mice ( 4 ) . In _ v i t_ ro _
s t u d i e s on t h e e f f e c t of p e r i t o n e a l macrophages on t h e growth
of a s c i t e s tumor c e l l s were performed as d e s c r i b e d by D r o l l e r and Remington I n b r i e f , p e r i t o n e a l exudate c e l l s from mice i n j e c t e d w i t h I._.
(6).
bra-
z i l i e n s i s and u n i n o c u l a t e d c o n t r o l mice were h a r v e s t e d i n h e p a r i n i z e d medium 199.
Cells ( 2 ~ 1 0 ~ were ) p l a t e d i n g l a s s t e s t t u b e s and incubated
a t 37°C i n an atmosphere of 5% COz.
A f t e r two hours, t h e non-adherent
c e l l s were removed by washing t h e t u b e s t h r e e times w i t h medium 199, and
lo5
a s c i t e s tumor c e l l s were then added t o t h e t u b e s c o n t a i n i n g t h e adherent
cells.
T h i s p r o p o r t i o n of c e l l s was always used u n l e s s o t h e r w i s e s t a t e d .
Before being added, t h e tumor c e l l s were p l a t e d i n P e t r i d i s h e s f o r two hours.
The non-adherent
c e l l s were t h e n removed, and used f o r t h e t e s t .
Tumor c e l l growth was e v a l u a t e d by determining t h e i n c o r p o r a t i o n of t r i t i a t e d
459
,SE.lSHMANIA BRAZILIENSIS AND TUMOR CELL GROWTH t.hymidine (3€I-TdR,
s p e c i f i c a c t i v i t y 1 . 9 Ci/mfl, N e w Iln;%landN u c l e a r , U S A ) .
One p C i of t h e i s o t o p e w a s added t o e a c h t u h e and t h e t u b e s were i n c u b a t e d f o r 24 h o u r s .
After t h i s pulse period, t h e culture
moving t h e non-adherent
c e l l s and t h e m o u n t of 3Ii-TdK
c e l l s w a s determined i n t r i c h l o r o a c e t i c
t e r m i n a t e d by re-
1.135
i n c o r p o r a t e d by t h e s e
acicl p r e c i p i t geed m a t e r i a l by
c o u n t i n g i n a s c i n t i l l a t i o n l i q u i d f l u i d composed of t - o l u e n e i n a P a c k a r d "ode1 3330 s p e c t r o m e t e r .
L'IIP,
dm-POPOP and
Triplicate cultures were
a l w a y s performed i n e a c h e x p e r i m e n t . M i t o g e n i c r e s p o n s e of s p l e e n l y m p h o c y t e s w a s s t d i e d by c u l t u r i n g
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
;!.5 x l o 5 mononuclear s p l e e n c e l l s (obtainecl by p r e s s i n g s p l e e n s t h r o u g h a s t a i n l e s s s t e e l g a u z e ) i n 0 . 2 m l KPM 1640 medium ( G I ~ B I : : ~ ,USA) s u p p l e m e n t e d
w i t 1 1 p e n i c i l l i n and s t r e p t o n y c i n and 5% f r e s h human s8iirum i n r , i i c r o t i t e r p l a t e s ( F a l c o n 3 0 4 0 ) a t 3 7 O C i n a 5% CO2 a t m o s p h e r e .
Twenty u 1 phytohemag-
g l u t i n i n M ( D i f c o , USA) w e r e added t o e a c h c u l t u r e an(l t h e r e s p o n s e e v a l u a t e d by t h e i n c o r p o r a t i o n o f 3H-TdR
o f . 24 h o u r s .
on d a y 2 of t h e cultun:, by a l l o w i n g a p u l s e
C u l t u r e s w e r e t e r m i n a t e d by c a l l a s p i r a t ion on g l a s s f i k r
f ' i l t e r s (Reeve Angel) i n a MASH I1 h a r v e s t e r (Microbi.':)logical A s s o c i a t e s ) . A f t e r d r y i n g , t h e p l a t e s w e r e c o u n t e d i n s c j n t i l l a t i o n f l u i d composed of PPO, dm-POPOP and t o l u e n e i n a P a c k a r d model 3330 s p e r t r o m e t e r a t 5 7 % efficiency. A l l experiments w e r e performed i n t r i p l . i c a t e .
S t a t i s t i c a l comparisons
w e r e p e r f o r r i e d by S t u d e n t ' s t - t e s t .
Results
k.
The e f f e c t of t h e i n t r a p e r i t o n e a l a d m i n i s t r a t i o n of
b r a z i l i e n s i s on
--
t h e i n v i t r o tumor g r o w t h w a s d e t e r m i n e d a t d i f f e r e n t t i n e i n t e r v a l s a E t e r
t h e a d m i n i s t r a t i o n of l e i s h m a n i a .
A s c a n be s e e n i n T a b l e I , t h e macro-
phages of t h e s e animals e x e r t e d a s t r o n g i n h i b i t o r y e f f e c t on t h e i n v i t r o growth of t h e 6C3HED lymphoma when t h e y were o b t a i n e d cine t o t h r e e d a y s a f t e r t h e i n j e c t i o n , and d e c r e a s e d i n t h e f o l l o w i n g d a y s . was o b t a i n e d when u s i n g EL-4
l e u k e m i a c e l l s as a t a r g e t
A s i m i l a r result
( T a b l e 11).
Since
MWINO AND LUIS
460 TABLE I
t. b r a z i l i e n s i s
E f f e c t of p e r i t o n e a l macrophages of mice i n j e c t e d with on t h e i n v i t r o growth of lymphoma 6C3HED
3H-TdB i n c o r p o r a t i o n (mean 5 S.D.) by 6C3HED c e l l s Tumor c e l l s c u l t u r e d i n t h e presence o f :
P
CPM 60245
t 12645
1
17427
3
21682
5
49134
5 5001 5 7720 2 9124
7
55613
14
58184
Normal macrophages
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Macrophages taken a t day a f t e r i n j e c t i o n of _4. b r a z i l i e n s i s
n.s.
2
(1979)
THE EFFECT OF PERITONEAL NACROPlIAGES FROM M I C E INJECTED WITH LEISHMANIA BRAZILIENSIS ON THE I N VITRO GROWTH I F TUMOR CELLS Fernando l l e r i n o and -1uan L u i s Departainento d e M e d i c i n a E x p e r i m e n t i l , I n s t i t u t o Venezolano d e I n v e s t i g a c i o n e s C i e n t i f i c i s , A p a r t a d o 1 8 2 7 , Caracas, Venezuela
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
"Abstract" Leishmania b r a z i l i e n s i s r e p o r t e d l y i s c a p a b l e of p r o d u c i n g a r e d u c t i o n i n t h e g r o w t h of s o l i d and a s c i t e s m u r i n e l e u k e m i a a d lymphomas. The p o s s i b i l i t y t h a t t h e i n h i b i t o r y e f f e c t on t h e a s c i t e s tumor was produced by t h e a c t i v a t i o n of t h e macrophage p e r i t o n e a l c e l l p o p u l a t i o n was e x p l o r e d . I t w a s o b s e r v e d t h a t a d h e r e n t c e l l s from t h e p e r i t o n e a l c a v i t y of b r a z i l i e n s i s i n j e c t e d m i c e c a u s e d a marked i n h i b i t o r v e f € e c t on t h e i n v i t r o growth of t h e 6C3HED lymphoma and EL-4 l e u k e m i a c e l l s . T h i s e f f e c t w a s d e p e n d e n t on t h e d e g r e e of t h e macrophage a c t i v a t i o n , w a s n o t produced by s u p e r n a t a n t s from c u l t u r e s of a c t i v a t e d macrophages, and i t seems t h a t conIn a d d i t i o n t o t a c t c e l l between t a r g e t and e f f e c t o r c e l l s i s n e c e s s a r y . I,. b r a z i l i e n s i s : b e i n g c y t o s t a t i c , t h i s e f f e c t was a l s o c y t o t - o x i c . T h e a c t i v a t e d macrophages w e r e a l s o c a p a b l e of s u p p r e s s i n g t h e m u l t i p l i c a t i o n of normal c e l l s i n d u c e d by m i t o g e n , b u t t h i s w a s n o t o b s e r v e d i f t h e c e l l s were a l r e a d y u n d e r g o i n g m u l t i p l i c a t i o n . A s i m i l a r c y t o s t a t i c e f f e c t cm t h e tumor c e l l s was o b s e r v e d t o be produced by t h e p e r i t o n e a l non-adherent c e l l p o p u l a t i o n of t h e L. b r a z i l i e n s i s i n j e c t e d mice.
I t h a s been w e l l e s t a b l i s h e d t h a t p e r i t o n e a l macrophages o b t a i n e d . from a n i m a l s i n j e c t e d w i t h b a c i l l u s Calmette-Guei-in
(BCC:)
,
chemicals, o r tliose
from a n i m a l s c h r o n i c a l l y i n f e c t e d by p a r a s i i - e s i n h i b i t , o r g r e a t l y r e d u c e ,
-
i n v i v o a n d i n v i t r o tumor g r o w t h ( 1 , 2 , 3 ) .
._
P r e v i o u s s t u d i e s have shown t h a t t h e a d m i n i s t r a t i o n of a n o n - p a t h o g e n i c s t r a i n o f Leishmania b r a z i l i e n s i s i n f l u e n c e d t h e g r o w t h of a s c i t i c and s o l i d lymphoma and l e u k e m i a ( 4 ) .
I t was a l s o d e m o n s t r a t e d t h a t a c t - i v a t e d macro-
p h a g e s which c a u s e d a marked i n h i b i t o r y e f f e c t on t.he i n v i t r o growth of s u c h tumors (5) w e r e p r e s e n t i n the p e r i t o n e a l c a v j t y of t h e mice p r e v i o u s l y in.jected w i t h t h e p a r a s i t e .
457 Copyright 0 1979 by Marcel Dekker, Inc. All Rights Reserved Neither this w o r k n o r dny p d r t may he reproduced o r transmitted in any f o r m o r by a n y means. electronic or mechanical, mcludmy phoiocopylng. microfilming, a n d recording, or by any information storage and retrieval s y s t e m , without permission in writing f r o m t h e publisher.
MERINO AND LUIS
458
T h i s r e p o r t d e s c r i b e s t h e e f f e c t of c e l l s from t h e p e r i t o n e a l c a v i t y of mice i n j e c t e d w i t h
&.
b r a z i l i e n s i s on t h e growth of tumor c e l l s a s w e l l
a s s u g g e s t i o n s about t h e p o s s i b l e mechanisms.
M a t e r i a l s and Methods
Mice of t h e s t r a i n s C3H/HeJ and C57BL/6J w e r e o b t a i n e d from The Jackson Laboratory (Bar Harbor, Maine, USA) and maintained i n o u r animal colony. Food and water were a d m i n i s t e r e d ad l i b i t u m .
The a s c i t e s tumors used were
t h e 6C3HEU lymphoma, and t h e lymphoblastic leukemia EL-4.
Both tumors were
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
o b t a i n e d from D r . F. Milgrom ( S t a t e U n i v e r s i t y of New York, Buffalo) and maintained i n syngenic mice by weekly t r a n s f e r s . The s t r a i n of
L.
b r a z i l i e n s i s used was o r i g i n a l l y i s o l a t e d from a
human c a s e of l e i s h m a n i a s i s and has been maintained by t h e Department of C e l l Biology, School of S c i e n c e s , Universidad C e n t r a l de Venezuela i n v i t r o i n Davis' medium f o r more than 500 passages. I n our experiments, t h i s s t r a i n was maintained i n NCTC 135 medium ( G I B C O , USA) supplemented w i t h 5% f e t a l c a l f serum.
Leishmania growth h a s
been maintained f o r over one y e a r i n t h i s medium and no pathogenic e f f e c t was seen when t h e s e p a r a s i t e s were i n j e c t e d i n mice ( 4 ) . In _ v i t_ ro _
s t u d i e s on t h e e f f e c t of p e r i t o n e a l macrophages on t h e growth
of a s c i t e s tumor c e l l s were performed as d e s c r i b e d by D r o l l e r and Remington I n b r i e f , p e r i t o n e a l exudate c e l l s from mice i n j e c t e d w i t h I._.
(6).
bra-
z i l i e n s i s and u n i n o c u l a t e d c o n t r o l mice were h a r v e s t e d i n h e p a r i n i z e d medium 199.
Cells ( 2 ~ 1 0 ~ were ) p l a t e d i n g l a s s t e s t t u b e s and incubated
a t 37°C i n an atmosphere of 5% COz.
A f t e r two hours, t h e non-adherent
c e l l s were removed by washing t h e t u b e s t h r e e times w i t h medium 199, and
lo5
a s c i t e s tumor c e l l s were then added t o t h e t u b e s c o n t a i n i n g t h e adherent
cells.
T h i s p r o p o r t i o n of c e l l s was always used u n l e s s o t h e r w i s e s t a t e d .
Before being added, t h e tumor c e l l s were p l a t e d i n P e t r i d i s h e s f o r two hours.
The non-adherent
c e l l s were t h e n removed, and used f o r t h e t e s t .
Tumor c e l l growth was e v a l u a t e d by determining t h e i n c o r p o r a t i o n of t r i t i a t e d
459
,SE.lSHMANIA BRAZILIENSIS AND TUMOR CELL GROWTH t.hymidine (3€I-TdR,
s p e c i f i c a c t i v i t y 1 . 9 Ci/mfl, N e w Iln;%landN u c l e a r , U S A ) .
One p C i of t h e i s o t o p e w a s added t o e a c h t u h e and t h e t u b e s were i n c u b a t e d f o r 24 h o u r s .
After t h i s pulse period, t h e culture
moving t h e non-adherent
c e l l s and t h e m o u n t of 3Ii-TdK
c e l l s w a s determined i n t r i c h l o r o a c e t i c
t e r m i n a t e d by re-
1.135
i n c o r p o r a t e d by t h e s e
acicl p r e c i p i t geed m a t e r i a l by
c o u n t i n g i n a s c i n t i l l a t i o n l i q u i d f l u i d composed of t - o l u e n e i n a P a c k a r d "ode1 3330 s p e c t r o m e t e r .
L'IIP,
dm-POPOP and
Triplicate cultures were
a l w a y s performed i n e a c h e x p e r i m e n t . M i t o g e n i c r e s p o n s e of s p l e e n l y m p h o c y t e s w a s s t d i e d by c u l t u r i n g
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
;!.5 x l o 5 mononuclear s p l e e n c e l l s (obtainecl by p r e s s i n g s p l e e n s t h r o u g h a s t a i n l e s s s t e e l g a u z e ) i n 0 . 2 m l KPM 1640 medium ( G I ~ B I : : ~ ,USA) s u p p l e m e n t e d
w i t 1 1 p e n i c i l l i n and s t r e p t o n y c i n and 5% f r e s h human s8iirum i n r , i i c r o t i t e r p l a t e s ( F a l c o n 3 0 4 0 ) a t 3 7 O C i n a 5% CO2 a t m o s p h e r e .
Twenty u 1 phytohemag-
g l u t i n i n M ( D i f c o , USA) w e r e added t o e a c h c u l t u r e an(l t h e r e s p o n s e e v a l u a t e d by t h e i n c o r p o r a t i o n o f 3H-TdR
o f . 24 h o u r s .
on d a y 2 of t h e cultun:, by a l l o w i n g a p u l s e
C u l t u r e s w e r e t e r m i n a t e d by c a l l a s p i r a t ion on g l a s s f i k r
f ' i l t e r s (Reeve Angel) i n a MASH I1 h a r v e s t e r (Microbi.':)logical A s s o c i a t e s ) . A f t e r d r y i n g , t h e p l a t e s w e r e c o u n t e d i n s c j n t i l l a t i o n f l u i d composed of PPO, dm-POPOP and t o l u e n e i n a P a c k a r d model 3330 s p e r t r o m e t e r a t 5 7 % efficiency. A l l experiments w e r e performed i n t r i p l . i c a t e .
S t a t i s t i c a l comparisons
w e r e p e r f o r r i e d by S t u d e n t ' s t - t e s t .
Results
k.
The e f f e c t of t h e i n t r a p e r i t o n e a l a d m i n i s t r a t i o n of
b r a z i l i e n s i s on
--
t h e i n v i t r o tumor g r o w t h w a s d e t e r m i n e d a t d i f f e r e n t t i n e i n t e r v a l s a E t e r
t h e a d m i n i s t r a t i o n of l e i s h m a n i a .
A s c a n be s e e n i n T a b l e I , t h e macro-
phages of t h e s e animals e x e r t e d a s t r o n g i n h i b i t o r y e f f e c t on t h e i n v i t r o growth of t h e 6C3HED lymphoma when t h e y were o b t a i n e d cine t o t h r e e d a y s a f t e r t h e i n j e c t i o n , and d e c r e a s e d i n t h e f o l l o w i n g d a y s . was o b t a i n e d when u s i n g EL-4
l e u k e m i a c e l l s as a t a r g e t
A s i m i l a r result
( T a b l e 11).
Since
MWINO AND LUIS
460 TABLE I
t. b r a z i l i e n s i s
E f f e c t of p e r i t o n e a l macrophages of mice i n j e c t e d with on t h e i n v i t r o growth of lymphoma 6C3HED
3H-TdB i n c o r p o r a t i o n (mean 5 S.D.) by 6C3HED c e l l s Tumor c e l l s c u l t u r e d i n t h e presence o f :
P
CPM 60245
t 12645
1
17427
3
21682
5
49134
5 5001 5 7720 2 9124
7
55613
14
58184
Normal macrophages
Immunol Invest Downloaded from informahealthcare.com by McMaster University on 11/04/14 For personal use only.
Macrophages taken a t day a f t e r i n j e c t i o n of _4. b r a z i l i e n s i s
n.s.
2
