IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOG'!,
13(1&2),
1-10 (1991)
STUDIES ON CHEMILUMINESCENCE A N D PROTEIN KINASE-C ACTIVITY OF CISPLATIN TREATED MOUSE PERITONEAL EXUDATE CELLS.
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
B. GEETHA, AJIT SODHI'*
a n d SUKH MAHENDRA SINGH,
School o f Biotechnologyt, D e p a r t m e n t of Z o o l o g y , Banaras Hindu U n i v e r s i t y , V a r a n a s i - 2 2 1 005, U.P., INDlA.
Key w o r d s :
C i s p l a t i n , P e r i t o n e a l exudate c e l l s , Chemiluminescence. ATP, P r o t e i n k i n a s e - - C .
SUMMARY A single i.p.
i n j e c t i o n of c i s p l a l - i n (10 mg/kg body w e i g h t ) i n t o mice
r e s u l t s i n a s i g n i f i c a n t i n c r e a s e i n c h e m i l u m i n e s c e n c e a n d ATP c o n t e n t s o f t h e p e r i t o n e a l e x u d a t e c e l l s (PEC) t h a n t h a t o f PEC from u n t r e a t e d mice.
It
is a l s o o b s e r v e d t h a t i n v i t r o t r e a t m e n t of m a c r o p h a g e s w i t h c i s p l a t i n , r I F N - t
a n d LPS show i n c r e a s e d a c t i v i t y o f t h e p r o t e i n k i n a s e - C (PK-C).
The a c t i v a t i o n
of PK-C c o u l d r e s u l t i n s t i m u l a t i o n o f NADPH-oxidase r e s u l t i n g i n i n c r e a s e d l e v e l s of chemiluminescence.
I n c r e a s e d c o n t e n t s of ATP i n PEC a f t e r c i s p l u L i i i
t r e d L n i e n t a l s o s u g g e s t s t h a t t h i s a c t i v a t i o n is e n e r g y d e p e n d e n t . INTRODUCTION 'The g e n e r a t i o n of c h e m i c a l l y react-ive niuleculrs s u c h a s s u p e r o x i d e
a n i o n s (0-2),h y d r o g e n p e r o x i d e ( H 2 0 2 ) , s i n g l e t o x y g e n a n d h y d r o x y l r a d i c a l s a s a result of r e s p i r a t o r y b u r s t a c t i v a t i o n i n m o n o n u c l e a r p h a g o c y t e s is a n essential s t e p i n t h e h o s t s d e f e n s e a g a i n s t m i c r o o r g a n i s m s (1). of L h e s e o x i d a t i v e m e t a b o l i t e s c a n b e n i e a s u r e d as l i g h t
inescence (2).
The production
m i s s i o n or chemilum-
A l t h o u g h t h e u s e of c h e m i l u m i n e s c e n c e (CL) h a s become i n c r e a s -
i n g l y p o p u l a r i n d e t e r m i n i n g f u n c t i o n a l . c h a r a c t e r i s t i c s of m o n o n u c l e a r p h a g o c y t e s ( 3 I% 4 ) . t h e m e c h a n i s m s b e h i n d t h e r e a c t i o n is f a r f r o m b e i n g
*
To whoiri a l l c o r r e s p o n d a n c e s h o u l d b e a d d r e s s e d 1
Copyright@ 1991 by Marcel Dekker, Inc.
2
GEETHA, S O D H I , AND S I N G H
understood. It is well known that the addition of luminol or lucigenin to a chemiluminescent system can amplify the response by acting as a bystander substrate for the oxidative metabolites generated during the process of activation ( 5 ) . It is known that PMA and zymosan enhance the CL of polyinorphonuclear leukocytes (6 & 7). Cisplatin (CP) is a potent anti-tumor compound which has been used successfully against number of tumors in humans ( 8 ) and animals ( 9 & 10). There are several reports suggesting the role of cisplatin as an immuno-
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
modulator (11 & 1 2 ) . It has been shown recently from our laboratory that CP activates murine macrophages to the tumoricidal state and enhanced release of H202, 0-2 and IL-1 both in vitro and in vivo (13 - 15).
In vivo administration of CP at a dose of 10 mg/kg body weight to mice was optimal for acLivaLion of PEC.
It is known that rIFN-Y enhances the PK-C activity
in murine peritoneal macrophages (16) and LPS stimulates the arachidonic acid cascade and also enhances the release of reactive oxygen intermediates in response to subsequent stimulation with variety of triggers (17).
Since
it has been observed that cisplatin treatment of macrophages in vivo also
enhances the acitivity of NADP, NADPH and myeloperoxidase (unpublished observation), we studied the effect of CP on PK-C activity.
In addition the CI, studies oil PEC obtained from CP treated mice, we also studied the effect of CP on the ATP content i l l pIT. to
MATERIALS AND METHODS Inbred strain of C3H/He mice, 6 to 8 weeks old were used f o r all the experiments. Most of the chemicals and tissue culture medium RPMI 1640 used were obtained from Sigma Chemical Company, U.S.A..
All the reagents and solutions were prepared in triple glass distilled water.
Chemiluminescence Assay: Cisplatin in PBS (10 mg/kg body weight) was administered i.p. 24 and 48 h before harvesting PEC. Control mice were given injection of PBS. PEC were harvested by the method described previously (18). After centrifugation f o r 10 min at 1000 rpm the pellet was resuspended in phenol red free HBSS at a concentration of 1 x lo6 cells/ml. CL assays were performed using a luminometer (LKB 1250) at 37OC in presence or absence
of PMA (500 ng/ml) or opsonized zymosan (100 pg/ml). A curve was drawn to record the emitted CL expressed in mV, as a function of incubation time. The results presented in the study are the mean of values obtained.
ATP Assay: ATP was assayed using the LKB ATP assay kit. 2% Trichloroacetic acid (TCA) containing 2 nM EDTA was prepared. Xylenol blue was added to the TCA-EDTA solution to a final concentration of 0.02~. 100 p1 of PEC (1 x 106 cells) from in vivo CP treated or normal mice were added to TCA-xylenol mixture. This suspension was mixed and than incubated for 10 min at room
CHEMILUMINESCENCE AND PROTEIN KINASE-C ACTIVITY
3
temperature. After 10 min the extract was diluted to a final concentration of 0.1% TCA and 0.002% xylenol blue. The final 1 ml assay mixture contained 200 ul ATP monitoring reagent, 700 p l of tris-acetate buffer and 100 ~1 of test sample. The rate of light emission was measured on luminometer. ATP contents are expressed in mV. Protein Kinase-C Assay: PK-C activity was determined spectrophotometrically according to the method described by Roskoski (1983). Macrophage monolayers
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
were prepared as described earlier (15) in 24 well tissue culture plates (NUNC, Denmark). 2.5 x 106 cells per well were treated with cisplatin (10pg/ml), rIFN-Y (10 U/ml), MDP ( 5 ug/ml) and LPS (10pg/ml) for 24 h in a C02 incubator at 37OC. After the treatment monolayers were lysed with 1 ml of 0.5% Triton-X 100. This cell lysate was used to assay the PK-C activity. The final 1 ml incubation mixture contained the following components:
100 nM Morpholinopropane sulphonic acid (MOPS), 15 U Lactate dehydrogenase (LDH), 7 U Pyruvate kinase, 10 mM Potassium chloride (KCL), 1 mM Phosphoenol pyruvate (PEP), 10 mM Magnesium chloride (MgC12), 1 mM Adenosine triphosphate
200 pM Nicotinamide (ATP), lOOuM Ser-Peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), adenine dinucleotide phosphate reduced (NADPH). After recording the minor ATPase activity of the components, the reaction was initiated by adding 2 g1 of PK-C o r 2 ul of untreated or treated cell lysate incubated at 3OoC for 5 min.
The difference between the background (ATPase activity) and the rate
obtained in the presence of PK-C were calculated. The rates were monitored at 340 nm in a spectrophotometer against blank prepared by the same batch of reaction mixture without any enzyme. Statistical analysis:
All experiments were done at least three times in
triplicate. The difference between various groups was checked by Student's t-test, RESULTS First we investigated the role of in vivo administration of CP on activation of PEC using CL assay. As shown in Fig.1 lucigenin dependent CL of PEC obtained from CP treated mice was significantly higher than that from untreated mice. PEC collected from CP treated mice after 24 h showed a maximum CL of 15 mV after 1 min, whereas PEC obtained from control mice showed only a maximum CL of 7 mV. Addition of PMA to PEC from CP treated mice showed a maximum CL of 18 mV. The CP treated PEC (without PMA) showed CL of 15 mV. However, when CL of CP treated PEC attained a steady state (10 mV) after 5 min. treatment of PEC with PMA showed a immediate sharp increase in CL to 23 mV (Fig.2). I n another set of experiment simultaneous addition of PMA to CP treated PEC for 48 h (PEC removed after 48 h of CP treatment) showed a maximum CL of 21 mV after 1-2 min (Fig.4). We
GEETHA, S O D H I , AND S I N G H
4 ol?tfQM A W bntr.1 Rca
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
A Cdrol
Fig.1.
Time course of chemiluminescence emission i n PEC ( 1 x 106 ) obtained from normal o r CP t r e a t e d mice ( 2 4 h) i n response t o
PMA (500 ng/ml). dent experiments.
The values a r e r e p r e s e n t a t i v e of t h r e e indepenSIbwere c o n s i s t e n t l y < 1 0 % of mean.
a l s o compared t h e e f f e c t of Zymosan and PMA on CI. a s described i n M a t e r i a l s and Methods.
A s i g n i f i c a n t d i f f e r e n c e i n CL was observed a f t e r a d d i t i o n of
Zymosan or PMA t o untreated and CP t r e a t e d ( 48 h) PEC (Fig.3 & 4 ) .
I t was
observed that a c t i v a t i o n of PEC w i t h PMA i s more e f f e c t i v e and quick (within
1-2 min) than w i t h Zymosan (within 7-15 min). The r e s u l t s of ATP assay a r e shown i n Fig.5.
A significant difference
i n ATP contents of PEC from untreated and i n vivo CP t r e a t e d mice a f t e r 2 4 h
o f treatment was observed.
CP t r e a t e d PEC showed CL of 86 m V , whereas
untreated PEC showed only 15 mV.
T h e ATP c o n t e n t s of PEC from 48 h CP t r e a t e d
mice showed a maximum CL of 17.3 mV.
Normal PEC showed a CL of 14 mV.
I t i s c l e a r from table-1 t h a t treatment of macrophage monolayers w i t h C P , rIFN-Y and LPS r e s u l t s i n increased PK-C a c t i v i t y a s compared t o untreated M a x i m u m PK-C a c t i v i t y was ohserved i n marrophages t r e a t e d w i t h
macrophages.
CP and rIFN-'I.
PK-C a c t i v i t y .
LPS t r e a t e d macrophages a l s o showed s i g n i f i c a n t l y increased However, MOP t r e a t e d macrophages showed decreased PK-C a c t i v i t y .
C H E M I L U M I N E S C E N C E AND P R O T E I N K I N A S E - C A C T I V I T Y
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
25
F i g . 2 . Chemiluminescence of PEC (1 x lo6) from mice treated with CP (10 mg/kg) in response to PMA.
PMA (500 ng/ml) was added either
at the begining of the assay (open circles) o r at the time indicated with arrow (closed circles).
independent experiments.
The values are representative of three
SDs were consistently
13(1&2),
1-10 (1991)
STUDIES ON CHEMILUMINESCENCE A N D PROTEIN KINASE-C ACTIVITY OF CISPLATIN TREATED MOUSE PERITONEAL EXUDATE CELLS.
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
B. GEETHA, AJIT SODHI'*
a n d SUKH MAHENDRA SINGH,
School o f Biotechnologyt, D e p a r t m e n t of Z o o l o g y , Banaras Hindu U n i v e r s i t y , V a r a n a s i - 2 2 1 005, U.P., INDlA.
Key w o r d s :
C i s p l a t i n , P e r i t o n e a l exudate c e l l s , Chemiluminescence. ATP, P r o t e i n k i n a s e - - C .
SUMMARY A single i.p.
i n j e c t i o n of c i s p l a l - i n (10 mg/kg body w e i g h t ) i n t o mice
r e s u l t s i n a s i g n i f i c a n t i n c r e a s e i n c h e m i l u m i n e s c e n c e a n d ATP c o n t e n t s o f t h e p e r i t o n e a l e x u d a t e c e l l s (PEC) t h a n t h a t o f PEC from u n t r e a t e d mice.
It
is a l s o o b s e r v e d t h a t i n v i t r o t r e a t m e n t of m a c r o p h a g e s w i t h c i s p l a t i n , r I F N - t
a n d LPS show i n c r e a s e d a c t i v i t y o f t h e p r o t e i n k i n a s e - C (PK-C).
The a c t i v a t i o n
of PK-C c o u l d r e s u l t i n s t i m u l a t i o n o f NADPH-oxidase r e s u l t i n g i n i n c r e a s e d l e v e l s of chemiluminescence.
I n c r e a s e d c o n t e n t s of ATP i n PEC a f t e r c i s p l u L i i i
t r e d L n i e n t a l s o s u g g e s t s t h a t t h i s a c t i v a t i o n is e n e r g y d e p e n d e n t . INTRODUCTION 'The g e n e r a t i o n of c h e m i c a l l y react-ive niuleculrs s u c h a s s u p e r o x i d e
a n i o n s (0-2),h y d r o g e n p e r o x i d e ( H 2 0 2 ) , s i n g l e t o x y g e n a n d h y d r o x y l r a d i c a l s a s a result of r e s p i r a t o r y b u r s t a c t i v a t i o n i n m o n o n u c l e a r p h a g o c y t e s is a n essential s t e p i n t h e h o s t s d e f e n s e a g a i n s t m i c r o o r g a n i s m s (1). of L h e s e o x i d a t i v e m e t a b o l i t e s c a n b e n i e a s u r e d as l i g h t
inescence (2).
The production
m i s s i o n or chemilum-
A l t h o u g h t h e u s e of c h e m i l u m i n e s c e n c e (CL) h a s become i n c r e a s -
i n g l y p o p u l a r i n d e t e r m i n i n g f u n c t i o n a l . c h a r a c t e r i s t i c s of m o n o n u c l e a r p h a g o c y t e s ( 3 I% 4 ) . t h e m e c h a n i s m s b e h i n d t h e r e a c t i o n is f a r f r o m b e i n g
*
To whoiri a l l c o r r e s p o n d a n c e s h o u l d b e a d d r e s s e d 1
Copyright@ 1991 by Marcel Dekker, Inc.
2
GEETHA, S O D H I , AND S I N G H
understood. It is well known that the addition of luminol or lucigenin to a chemiluminescent system can amplify the response by acting as a bystander substrate for the oxidative metabolites generated during the process of activation ( 5 ) . It is known that PMA and zymosan enhance the CL of polyinorphonuclear leukocytes (6 & 7). Cisplatin (CP) is a potent anti-tumor compound which has been used successfully against number of tumors in humans ( 8 ) and animals ( 9 & 10). There are several reports suggesting the role of cisplatin as an immuno-
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
modulator (11 & 1 2 ) . It has been shown recently from our laboratory that CP activates murine macrophages to the tumoricidal state and enhanced release of H202, 0-2 and IL-1 both in vitro and in vivo (13 - 15).
In vivo administration of CP at a dose of 10 mg/kg body weight to mice was optimal for acLivaLion of PEC.
It is known that rIFN-Y enhances the PK-C activity
in murine peritoneal macrophages (16) and LPS stimulates the arachidonic acid cascade and also enhances the release of reactive oxygen intermediates in response to subsequent stimulation with variety of triggers (17).
Since
it has been observed that cisplatin treatment of macrophages in vivo also
enhances the acitivity of NADP, NADPH and myeloperoxidase (unpublished observation), we studied the effect of CP on PK-C activity.
In addition the CI, studies oil PEC obtained from CP treated mice, we also studied the effect of CP on the ATP content i l l pIT. to
MATERIALS AND METHODS Inbred strain of C3H/He mice, 6 to 8 weeks old were used f o r all the experiments. Most of the chemicals and tissue culture medium RPMI 1640 used were obtained from Sigma Chemical Company, U.S.A..
All the reagents and solutions were prepared in triple glass distilled water.
Chemiluminescence Assay: Cisplatin in PBS (10 mg/kg body weight) was administered i.p. 24 and 48 h before harvesting PEC. Control mice were given injection of PBS. PEC were harvested by the method described previously (18). After centrifugation f o r 10 min at 1000 rpm the pellet was resuspended in phenol red free HBSS at a concentration of 1 x lo6 cells/ml. CL assays were performed using a luminometer (LKB 1250) at 37OC in presence or absence
of PMA (500 ng/ml) or opsonized zymosan (100 pg/ml). A curve was drawn to record the emitted CL expressed in mV, as a function of incubation time. The results presented in the study are the mean of values obtained.
ATP Assay: ATP was assayed using the LKB ATP assay kit. 2% Trichloroacetic acid (TCA) containing 2 nM EDTA was prepared. Xylenol blue was added to the TCA-EDTA solution to a final concentration of 0.02~. 100 p1 of PEC (1 x 106 cells) from in vivo CP treated or normal mice were added to TCA-xylenol mixture. This suspension was mixed and than incubated for 10 min at room
CHEMILUMINESCENCE AND PROTEIN KINASE-C ACTIVITY
3
temperature. After 10 min the extract was diluted to a final concentration of 0.1% TCA and 0.002% xylenol blue. The final 1 ml assay mixture contained 200 ul ATP monitoring reagent, 700 p l of tris-acetate buffer and 100 ~1 of test sample. The rate of light emission was measured on luminometer. ATP contents are expressed in mV. Protein Kinase-C Assay: PK-C activity was determined spectrophotometrically according to the method described by Roskoski (1983). Macrophage monolayers
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
were prepared as described earlier (15) in 24 well tissue culture plates (NUNC, Denmark). 2.5 x 106 cells per well were treated with cisplatin (10pg/ml), rIFN-Y (10 U/ml), MDP ( 5 ug/ml) and LPS (10pg/ml) for 24 h in a C02 incubator at 37OC. After the treatment monolayers were lysed with 1 ml of 0.5% Triton-X 100. This cell lysate was used to assay the PK-C activity. The final 1 ml incubation mixture contained the following components:
100 nM Morpholinopropane sulphonic acid (MOPS), 15 U Lactate dehydrogenase (LDH), 7 U Pyruvate kinase, 10 mM Potassium chloride (KCL), 1 mM Phosphoenol pyruvate (PEP), 10 mM Magnesium chloride (MgC12), 1 mM Adenosine triphosphate
200 pM Nicotinamide (ATP), lOOuM Ser-Peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), adenine dinucleotide phosphate reduced (NADPH). After recording the minor ATPase activity of the components, the reaction was initiated by adding 2 g1 of PK-C o r 2 ul of untreated or treated cell lysate incubated at 3OoC for 5 min.
The difference between the background (ATPase activity) and the rate
obtained in the presence of PK-C were calculated. The rates were monitored at 340 nm in a spectrophotometer against blank prepared by the same batch of reaction mixture without any enzyme. Statistical analysis:
All experiments were done at least three times in
triplicate. The difference between various groups was checked by Student's t-test, RESULTS First we investigated the role of in vivo administration of CP on activation of PEC using CL assay. As shown in Fig.1 lucigenin dependent CL of PEC obtained from CP treated mice was significantly higher than that from untreated mice. PEC collected from CP treated mice after 24 h showed a maximum CL of 15 mV after 1 min, whereas PEC obtained from control mice showed only a maximum CL of 7 mV. Addition of PMA to PEC from CP treated mice showed a maximum CL of 18 mV. The CP treated PEC (without PMA) showed CL of 15 mV. However, when CL of CP treated PEC attained a steady state (10 mV) after 5 min. treatment of PEC with PMA showed a immediate sharp increase in CL to 23 mV (Fig.2). I n another set of experiment simultaneous addition of PMA to CP treated PEC for 48 h (PEC removed after 48 h of CP treatment) showed a maximum CL of 21 mV after 1-2 min (Fig.4). We
GEETHA, S O D H I , AND S I N G H
4 ol?tfQM A W bntr.1 Rca
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
A Cdrol
Fig.1.
Time course of chemiluminescence emission i n PEC ( 1 x 106 ) obtained from normal o r CP t r e a t e d mice ( 2 4 h) i n response t o
PMA (500 ng/ml). dent experiments.
The values a r e r e p r e s e n t a t i v e of t h r e e indepenSIbwere c o n s i s t e n t l y < 1 0 % of mean.
a l s o compared t h e e f f e c t of Zymosan and PMA on CI. a s described i n M a t e r i a l s and Methods.
A s i g n i f i c a n t d i f f e r e n c e i n CL was observed a f t e r a d d i t i o n of
Zymosan or PMA t o untreated and CP t r e a t e d ( 48 h) PEC (Fig.3 & 4 ) .
I t was
observed that a c t i v a t i o n of PEC w i t h PMA i s more e f f e c t i v e and quick (within
1-2 min) than w i t h Zymosan (within 7-15 min). The r e s u l t s of ATP assay a r e shown i n Fig.5.
A significant difference
i n ATP contents of PEC from untreated and i n vivo CP t r e a t e d mice a f t e r 2 4 h
o f treatment was observed.
CP t r e a t e d PEC showed CL of 86 m V , whereas
untreated PEC showed only 15 mV.
T h e ATP c o n t e n t s of PEC from 48 h CP t r e a t e d
mice showed a maximum CL of 17.3 mV.
Normal PEC showed a CL of 14 mV.
I t i s c l e a r from table-1 t h a t treatment of macrophage monolayers w i t h C P , rIFN-Y and LPS r e s u l t s i n increased PK-C a c t i v i t y a s compared t o untreated M a x i m u m PK-C a c t i v i t y was ohserved i n marrophages t r e a t e d w i t h
macrophages.
CP and rIFN-'I.
PK-C a c t i v i t y .
LPS t r e a t e d macrophages a l s o showed s i g n i f i c a n t l y increased However, MOP t r e a t e d macrophages showed decreased PK-C a c t i v i t y .
C H E M I L U M I N E S C E N C E AND P R O T E I N K I N A S E - C A C T I V I T Y
Immunopharmacology and Immunotoxicology 1991.13:1-10. Downloaded from informahealthcare.com by McMaster University on 01/09/15. For personal use only.
25
F i g . 2 . Chemiluminescence of PEC (1 x lo6) from mice treated with CP (10 mg/kg) in response to PMA.
PMA (500 ng/ml) was added either
at the begining of the assay (open circles) o r at the time indicated with arrow (closed circles).
independent experiments.
The values are representative of three
SDs were consistently
