Research in
Res Exp Med (1992) 192 : 145-150
Experimental Medicine 9 Springer-Verlag 1992
Regulation of NK cell activity and the level of the intracellular cAMP in human peripheral blood lymphocytes by Met-enkephalin I. Martin-Kleiner, M. Osmak, and J. Gabrilovac Department of Experimental Biologyand Medicine, Ruder BogkovidInstitute, Bijeni~ka54, P.O. Box 1016, 41001 Zagreb, Croatia Received July 29, 1991 / accepted December 13, 1991
Summary. The effect of Met-enkephalin on natural killer (NK) activity and on intracellular cyclic adenosine monophosphate (cAMP) level in human peripheral blood lymphocytes was measured 1/4, 1, 2, and 24 h after incubation. Concentrations of Met-enkephalin ranged from 10 -12 to 10-8M. Cyclic changes in NK activity and in the intracellular cAMP level after treatment with Met-enkephalin were observed. Kinetics of changes caused by high (10 9, 10-8 M) and low concentrations (10 -i2, 10-li, 10-10 M) of Met-enkephalin differed from each other. Early, nearly threefold increase in the level of intracellular cAMP was found 15 min after treating peripheral blood lymphocytes with 10-12 M Met-enkephalin. By contrast, a nearly 75% decrease of intracellular cAMP level was found 2 h after treatment with 10 9M Met-enkephalin. Generally, early intensive changes in the cAMP level in peripheral blood lymphocytes, induced by Met-enkephalin, were followed by delayed, subtle changes in NK cell activity in the opposite direction. Key words: Met-enkephalin - NK cell activity - cAMP - Peripheral blood lymphocytes
Introduction Enkephalins, endogenous opioid pentapeptides, are modulators of the immune functions of T and B lymphocytes, granulocytes, monocytes, macrophages, and NK cells [16]. Met-enkephalin (MENK) was originally described as an in vitro [5] and in vivo [6] stimulator of NK cell activity. Recently, bimodal effects of MENK (stimulation and decrease of NK activity) have been observed [14]. Modulation is achieved by binding MENK to &class opioid receptors on cells of Offprint requests' to." I. Martin-Kleiner
146 the i m m u n e system in vivo [10]. T h e r e is evidence for expressing 6-class opioid receptors o n h u m a n a n d m u r i n e T a n d B l y m p h o c y t e s a n d m a c r o p h a g e s [3]. T h e r e is also indirect evidence for the existence of opioid receptors on purified h u m a n N K cells [12]. 8-class opioid receptors were r e p o r t e d to be negatively c o r r e l a t e d to the a d e n y l a t e cyclase system a n d to the g e n e r a t i o n of i n t r a c e l l u l a r c A M P as the second m e s s e n g e r in signal t r a n s d u c t i o n in m u r i n e leukocytes [4]. N K activity was shown to be inversely c o r r e l a t e d to the i n t r a c e l l u l a r c A M P level [15]. T h e aim of our study was to ascertain the c o r r e l a t i o n b e t w e e n the o b s e r v e d b i m o d a l effects of M E N K o n N K activity a n d the changes in i n t r a c e l l u l a r c A M P level in h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s i n d u c e d by M E N K .
Materials and methods
Preparation of the effector cell population Peripheral blood was obtained, using heparin as the anticoagulant, from healthy male donors aged 18-30 years. Mononuclear cells were separated by centrifugating blood samples on gradient of Lymphoprep (Nycomed. Oslo. Norway) [2]. Monocvtes were removed by adherence on plastic Petri dishes (Greiner. Nfirtingen. Germany), for 1 t~ at 37~ C. Thus obtained non-adherent lymphocytes served as the effector cells in the experiments (in further text designated as peripheral blood lymphocytes or PBL).
Treatment of PBL with M E N K Suspensions of peripheral blood lymphocytes, in RPMI medium supplemented with 10% fetal calf serum (FCS), were adjusted to 2 • 10 7 cell/ml. To obtain final concentrations (10 -12, 10-11, 10-~~ 10-9, and i0 SM), 0.3ml of PBL suspensions, containing 6 • 106 cells, were admixed with 0.3 ml of MENK (Sigma, St. Louis, USA) dissolved in RPMI medium with 10% FCS. Incubation with MENK lasted 15 rain or 1. 2. 3, or 24 hours at 37~ A control sample without MENK in the medium was included in each trial.
NK cell cytotoxic assay After treatment with MENK. PBL were washed, readjusted to 107/ml, and used as the effectors in a NK cytotoxic assay. A standard 4-h chromium-release assay with K-562 cells as the targets was used [8]. All samples were run in triplicate. Titration curves were based on four different effector-to-target ratios (50, 16.6, 5.5, and 1.8). NK activity was expressed as lytic units (LU) per 107 cells. One LU denotes the number of effector cells required to produce 30% of the target-cell lysis. Changes differing by more than 2 SD from control NK activity in at least two points of the titration curve were considered to be significant.
Measurement of the intracellular cAMP level The level of cAMP was measured by a RIA kit (cAMP ~25IDual Assay System, RPA 509, Amersham, UK). After treatment with MENK, as described, the cell suspensions were deproteinized with 6% trichloroacetic acid, which was followed by liquid phase extraction. For cAMP measurement, non-acetylation radio-immunoassay protocol was used according to the detailed instruction of the manufacturer, cAMP concentration was expressed as fmol/106 cells. All samples were run in duplicate. As the difference between parallel samples never exceeded 8%, results differing by more than 2 SD from the controls were considered significant.
147 Results
O n e h o u r a f t e r t r e a t m e n t with low c o n c e n t r a t i o n s o f M E N K (10 -12, 10 -11, 10-1~ s t i m u l a t i o n of N K activity was n o t e d . It was f o l l o w e d b y a t r a n s i e n t fall at 2 h a n d a r e n e w e d s t i m u l a t i o n at 3 h a n d 2 4 h (Fig. l a ) . T r e a t m e n t with high c o n c e n t r a t i o n s o f M E N K (10 -9, 10 8M) r e s u l t e d in a d e c r e a s e o f N K activity in the 2nd a n d 3rd h, f o l l o w e d at 24 h by a s t i m u l a t i o n s t r o n g e r t h a n it was a f t e r t r e a t m e n t with low c o n c e n t r a t i o n s of M E N K (Fig. l b ) .
MENK concentrations ._J O 1-
o 10-12 M lOmM A 10-10M
18o
v 10-~9M 7
/,i,,~
~4o I.,.
.~f00 > tO 5~
z
60 1/4 1
VA 2
Time
3
24
~/4 1 with
incubation
of
2 3 24 MENK (h)
Fig. la, b. Cyclic changes in NK activity induced by a low and b high concentrations of MENK. Endogenous NK activity was 38LU. Full symbols represent significant results. The results are expressed as the percent of control calculated as a ratio of NK activity of PBL treated with MENK vs untreated PBL
NENK
concentrations
r
1500
[3 10-11M
o 'LO-12M
?
. No MENK
A 10-10M
o 10-8M
1500
10-9 M
O3
9 o 1000
I000
t-
f
o
E 13..
Res Exp Med (1992) 192 : 145-150
Experimental Medicine 9 Springer-Verlag 1992
Regulation of NK cell activity and the level of the intracellular cAMP in human peripheral blood lymphocytes by Met-enkephalin I. Martin-Kleiner, M. Osmak, and J. Gabrilovac Department of Experimental Biologyand Medicine, Ruder BogkovidInstitute, Bijeni~ka54, P.O. Box 1016, 41001 Zagreb, Croatia Received July 29, 1991 / accepted December 13, 1991
Summary. The effect of Met-enkephalin on natural killer (NK) activity and on intracellular cyclic adenosine monophosphate (cAMP) level in human peripheral blood lymphocytes was measured 1/4, 1, 2, and 24 h after incubation. Concentrations of Met-enkephalin ranged from 10 -12 to 10-8M. Cyclic changes in NK activity and in the intracellular cAMP level after treatment with Met-enkephalin were observed. Kinetics of changes caused by high (10 9, 10-8 M) and low concentrations (10 -i2, 10-li, 10-10 M) of Met-enkephalin differed from each other. Early, nearly threefold increase in the level of intracellular cAMP was found 15 min after treating peripheral blood lymphocytes with 10-12 M Met-enkephalin. By contrast, a nearly 75% decrease of intracellular cAMP level was found 2 h after treatment with 10 9M Met-enkephalin. Generally, early intensive changes in the cAMP level in peripheral blood lymphocytes, induced by Met-enkephalin, were followed by delayed, subtle changes in NK cell activity in the opposite direction. Key words: Met-enkephalin - NK cell activity - cAMP - Peripheral blood lymphocytes
Introduction Enkephalins, endogenous opioid pentapeptides, are modulators of the immune functions of T and B lymphocytes, granulocytes, monocytes, macrophages, and NK cells [16]. Met-enkephalin (MENK) was originally described as an in vitro [5] and in vivo [6] stimulator of NK cell activity. Recently, bimodal effects of MENK (stimulation and decrease of NK activity) have been observed [14]. Modulation is achieved by binding MENK to &class opioid receptors on cells of Offprint requests' to." I. Martin-Kleiner
146 the i m m u n e system in vivo [10]. T h e r e is evidence for expressing 6-class opioid receptors o n h u m a n a n d m u r i n e T a n d B l y m p h o c y t e s a n d m a c r o p h a g e s [3]. T h e r e is also indirect evidence for the existence of opioid receptors on purified h u m a n N K cells [12]. 8-class opioid receptors were r e p o r t e d to be negatively c o r r e l a t e d to the a d e n y l a t e cyclase system a n d to the g e n e r a t i o n of i n t r a c e l l u l a r c A M P as the second m e s s e n g e r in signal t r a n s d u c t i o n in m u r i n e leukocytes [4]. N K activity was shown to be inversely c o r r e l a t e d to the i n t r a c e l l u l a r c A M P level [15]. T h e aim of our study was to ascertain the c o r r e l a t i o n b e t w e e n the o b s e r v e d b i m o d a l effects of M E N K o n N K activity a n d the changes in i n t r a c e l l u l a r c A M P level in h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s i n d u c e d by M E N K .
Materials and methods
Preparation of the effector cell population Peripheral blood was obtained, using heparin as the anticoagulant, from healthy male donors aged 18-30 years. Mononuclear cells were separated by centrifugating blood samples on gradient of Lymphoprep (Nycomed. Oslo. Norway) [2]. Monocvtes were removed by adherence on plastic Petri dishes (Greiner. Nfirtingen. Germany), for 1 t~ at 37~ C. Thus obtained non-adherent lymphocytes served as the effector cells in the experiments (in further text designated as peripheral blood lymphocytes or PBL).
Treatment of PBL with M E N K Suspensions of peripheral blood lymphocytes, in RPMI medium supplemented with 10% fetal calf serum (FCS), were adjusted to 2 • 10 7 cell/ml. To obtain final concentrations (10 -12, 10-11, 10-~~ 10-9, and i0 SM), 0.3ml of PBL suspensions, containing 6 • 106 cells, were admixed with 0.3 ml of MENK (Sigma, St. Louis, USA) dissolved in RPMI medium with 10% FCS. Incubation with MENK lasted 15 rain or 1. 2. 3, or 24 hours at 37~ A control sample without MENK in the medium was included in each trial.
NK cell cytotoxic assay After treatment with MENK. PBL were washed, readjusted to 107/ml, and used as the effectors in a NK cytotoxic assay. A standard 4-h chromium-release assay with K-562 cells as the targets was used [8]. All samples were run in triplicate. Titration curves were based on four different effector-to-target ratios (50, 16.6, 5.5, and 1.8). NK activity was expressed as lytic units (LU) per 107 cells. One LU denotes the number of effector cells required to produce 30% of the target-cell lysis. Changes differing by more than 2 SD from control NK activity in at least two points of the titration curve were considered to be significant.
Measurement of the intracellular cAMP level The level of cAMP was measured by a RIA kit (cAMP ~25IDual Assay System, RPA 509, Amersham, UK). After treatment with MENK, as described, the cell suspensions were deproteinized with 6% trichloroacetic acid, which was followed by liquid phase extraction. For cAMP measurement, non-acetylation radio-immunoassay protocol was used according to the detailed instruction of the manufacturer, cAMP concentration was expressed as fmol/106 cells. All samples were run in duplicate. As the difference between parallel samples never exceeded 8%, results differing by more than 2 SD from the controls were considered significant.
147 Results
O n e h o u r a f t e r t r e a t m e n t with low c o n c e n t r a t i o n s o f M E N K (10 -12, 10 -11, 10-1~ s t i m u l a t i o n of N K activity was n o t e d . It was f o l l o w e d b y a t r a n s i e n t fall at 2 h a n d a r e n e w e d s t i m u l a t i o n at 3 h a n d 2 4 h (Fig. l a ) . T r e a t m e n t with high c o n c e n t r a t i o n s o f M E N K (10 -9, 10 8M) r e s u l t e d in a d e c r e a s e o f N K activity in the 2nd a n d 3rd h, f o l l o w e d at 24 h by a s t i m u l a t i o n s t r o n g e r t h a n it was a f t e r t r e a t m e n t with low c o n c e n t r a t i o n s of M E N K (Fig. l b ) .
MENK concentrations ._J O 1-
o 10-12 M lOmM A 10-10M
18o
v 10-~9M 7
/,i,,~
~4o I.,.
.~f00 > tO 5~
z
60 1/4 1
VA 2
Time
3
24
~/4 1 with
incubation
of
2 3 24 MENK (h)
Fig. la, b. Cyclic changes in NK activity induced by a low and b high concentrations of MENK. Endogenous NK activity was 38LU. Full symbols represent significant results. The results are expressed as the percent of control calculated as a ratio of NK activity of PBL treated with MENK vs untreated PBL
NENK
concentrations
r
1500
[3 10-11M
o 'LO-12M
?
. No MENK
A 10-10M
o 10-8M
1500
10-9 M
O3
9 o 1000
I000
t-
f
o
E 13..
