Immunobiol., vol. 183, pp. 55-68 (1991)
1 VA
Medical Center and Tulane University School of Medicine, New Orleans, Louisiana, Miller Medical Group, Nashville, Tennessee, and 3 East Tennessee State University, Johnson City, Tennessee, USA 2
Failure of Met-Enkephalin to Enhance Natural Killer Cell Activity 1 2 ABBAJ. KASTIN , JANET SELIGSON! ,JOHN H. STRIMAS ,
and DAVID S. CHI 3
Received January 14, 1991 . Accepted in Revised Form April 16, 1991
Abstract Several papers have reported the enhancing effects of opiate pep tides, like Met-enkephalin, on natural killer (NK) cell activity. We examined the actions of Met-enkephalin on NK activity in blood obtained from 18 different donors, of different ages, many of them on several occasions, at several ratios of effector to target cells, several concentrations of peptide, in several types of flasks, with the purity and identity of the pentapeptide verified by chromatography, in a system responsive to interferon, and with results calculated in different ways. No significant increase was found for the peptide for any ratio of cells, any concentration of peptide, or any single subject, even when the subjects with the lowest baseline NK cell activity were used or when the subjects were more than 60 years old. Instead of an increase, the combined results for all subjects at all ratios at all concentrations of Met-enkephalin showed an overall decrease of 31.3 % specific cytotoxicity. These results fail to support the reports of an enhancing effect of Met-enkephalin on NK cell activity.
Introduction In 1975, methionine [MetJ-enkephalin was described as an endogenous opiate peptide (1). In 1979, we provided evidence for another peptide that could block the analgesic effect of some opiates like enkephalin, and we proposed the existence of a class of naturally occurring opiate antagonists (2, 3). The many reports of the involvement of endogenous opiate peptides in the modulation of the immune system have been ably summarized (4). It was logical for us to examine the influence of an anti opiate peptide on these opiate effects, which we did with several systems, including E-rosette formation (5, 6). We were disappointed, however, in the great variability of the effects of the opiate and antiopiates in both active and total E-rosette formation and, therefore, sought other tests in which endogenous opiate peptides like Met-enkephalin had been reported to be active. Abbreviations: NK = natural killer; Met phocytes; ANOVA = analysis of variance
=
methionine; PBL
=
peripheral blood lym-
56 . A. J. KASTIN, J. SELIGSON, J. H. STRIMAS, and D. S. CHI
The largest number of papers dealing with the effects of opiate pep tides on the immune system used natural killer (NK) cell activity of human lymphocytes (7-18). 'Upon testing this system, we were surprised by our inability to find the reported enhancing effect of Met-enkephalin on NK activity, despite consideration of factors like age and sex that other investigators emphasized to explain discrepancies among their studies. We describe our failure in this paper.
Materials and Methods Subject Blood was obtained from 18 healthy subjects, age 23 to 79, 10 men and 8 women. Written informed consent was obtained in each case, as specifically approved by the Institutional Review Board for human use.
Peptides Met-enkephalin was obtained from Sigma Co., St. Louis, MO, USA, and compared with the same peptide obtained from Bachem, Philadelphia, P A, USA. ~-endorphin was obtained from Bachem. Met-enkephalin was tested in samples from all subjects; ~-endorphin was tested in the 7 subjects over 60 years of age and in 1 younger subject. These pep tides were tested at a concentration of 1O-8M in blood from each subject and at additional concentrations in most subjects. In samples from the 11 younger subjects, the concentrations tested were usually 10-7, 10-8 , and 1O-9M. In samples from the 7 subjects over 60 years old, the concentrations ranged from 10-3M to 1O-15M.
Procedure The method described by MATHEWS et al. (7) was followed. This method consists of a 4 h assay measuring the release of 51 Cr from K562 tumor cells as targets. It involves the isolation of peripheral blood lymphocytes (PBL) by density centrifugation with Ficoll-Hypaque. The PBL were then washed three times in PBS (pH 7.2) and re-suspended in RPMI 1640 with HEPES, 10% fetal calf serum, bacitracin (10-6M), penicillin (1 %), and streptomycin (1 %). PBL adherent to plastic were removed by incubation at 37°C, 5 % CO 2 for 1 h in polystyrene culture flasks. Non-adherent cells were then washed with PBS, counted, and tested for viability with 0.4 % Trypan blue stain. Viability was usually greater than 98 %. PBL were preincubated 1 h with peptide, washed with PBS, and resuspended in media with fresh peptide. The assay was performed in triplicate in round-bottom microtiter plates (Linbro, Hamden, CT, USA) in a total volume of 0.2 ml. Most tests with Met-enkephalin were performed at effector to target (E:T) cell ratios of 6.25:1,12.5:1,25:1, and 50:1; in 5 samples, a ratio of 100:1 was also used. In the 8 subjects over 60 years old, the E:T ratios were 10:1 and 20:1. After the incubation for 4h at 37°C with 51Cr labeled K562 cells, the plate was centrifuged (140 g) and 0.1 ml supernatant was harvested from each well. Radioactivity of each sample was determined with a gamma counter (Beckman Instruments, Inc, Irvine, CA, USA). The percentage of lysed target cells was calculated as follows: 'f' . . Percent speCl IC CytotoXICity
=
cpmexp - cpmsr 100 x -=----'------''------''''-cpmmr - cpmsr
where cpmexp was equal to the mean of the observed triplicate assay, cpm" was the spontaneous release of 51Cr by the target cells, and cpmmr was the maximum release of 51Cr obtained by treating the target cells with 1 % Triton X-100.
Enkephalin and NK Activity . 57 Interferon Human interferon was obtained from Sigma and used according to established methods (19, 20) in the same way as were the pep tides (7). Blood from 4 subjects was tested at concentrations of 1 x 103 and 1 x 104 IU/m!. Chromatography
Thin layer chromatography of Met-enkephalin was run in two systems. The first was butanol:acetic acid:water in a ratio of 4:2:2. The second was ethyl acetate:pyridine:acetic acid:water in a ratio of 60:20:6: 11. These were visualized with ninhydrin under UV light. Statistics
Unless otherwise indicated, the results were expressed in the usual way as % specific cytotoxicity. They were analyzed by analysis of variance (ANOV A) followed by Duncan's multiple range test.
Results
The Met-enkephalin used in this study showed biological actlVlty m reducing electrically stimulated contractions of the vas deferens. Chromatographic analysis of the Met-enkephalin obtained from both Sigma and Bachem revealed a single spot in both systems used. Each spot showed the same Rf reported by Bachem in their accompanying analytical data sheet. Separate ANOV A's performed with the data from each of the 18 subjects failed to show statistically significant differences in NK activity in the blood
80
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Figure 2. NK cell activity (± SEM) from all 18 subjects combined from assays with effector:target cell ratios of 100:1, 50:1,25:1,20:1, 12.5:1, 10:1, and 6.25:1. The number of samples measured in triplicate containing Met-enkephalin was 244; the number of media controls was 117." P < 0.05 vs. media.
from any subject. Duncan's multiple range test failed to show significant differences for any single subject for either peptide at any of the different concentrations tested. The results for each subject are shown in Figure 1.
30
>E-< U >< 0 E-
1 VA
Medical Center and Tulane University School of Medicine, New Orleans, Louisiana, Miller Medical Group, Nashville, Tennessee, and 3 East Tennessee State University, Johnson City, Tennessee, USA 2
Failure of Met-Enkephalin to Enhance Natural Killer Cell Activity 1 2 ABBAJ. KASTIN , JANET SELIGSON! ,JOHN H. STRIMAS ,
and DAVID S. CHI 3
Received January 14, 1991 . Accepted in Revised Form April 16, 1991
Abstract Several papers have reported the enhancing effects of opiate pep tides, like Met-enkephalin, on natural killer (NK) cell activity. We examined the actions of Met-enkephalin on NK activity in blood obtained from 18 different donors, of different ages, many of them on several occasions, at several ratios of effector to target cells, several concentrations of peptide, in several types of flasks, with the purity and identity of the pentapeptide verified by chromatography, in a system responsive to interferon, and with results calculated in different ways. No significant increase was found for the peptide for any ratio of cells, any concentration of peptide, or any single subject, even when the subjects with the lowest baseline NK cell activity were used or when the subjects were more than 60 years old. Instead of an increase, the combined results for all subjects at all ratios at all concentrations of Met-enkephalin showed an overall decrease of 31.3 % specific cytotoxicity. These results fail to support the reports of an enhancing effect of Met-enkephalin on NK cell activity.
Introduction In 1975, methionine [MetJ-enkephalin was described as an endogenous opiate peptide (1). In 1979, we provided evidence for another peptide that could block the analgesic effect of some opiates like enkephalin, and we proposed the existence of a class of naturally occurring opiate antagonists (2, 3). The many reports of the involvement of endogenous opiate peptides in the modulation of the immune system have been ably summarized (4). It was logical for us to examine the influence of an anti opiate peptide on these opiate effects, which we did with several systems, including E-rosette formation (5, 6). We were disappointed, however, in the great variability of the effects of the opiate and antiopiates in both active and total E-rosette formation and, therefore, sought other tests in which endogenous opiate peptides like Met-enkephalin had been reported to be active. Abbreviations: NK = natural killer; Met phocytes; ANOVA = analysis of variance
=
methionine; PBL
=
peripheral blood lym-
56 . A. J. KASTIN, J. SELIGSON, J. H. STRIMAS, and D. S. CHI
The largest number of papers dealing with the effects of opiate pep tides on the immune system used natural killer (NK) cell activity of human lymphocytes (7-18). 'Upon testing this system, we were surprised by our inability to find the reported enhancing effect of Met-enkephalin on NK activity, despite consideration of factors like age and sex that other investigators emphasized to explain discrepancies among their studies. We describe our failure in this paper.
Materials and Methods Subject Blood was obtained from 18 healthy subjects, age 23 to 79, 10 men and 8 women. Written informed consent was obtained in each case, as specifically approved by the Institutional Review Board for human use.
Peptides Met-enkephalin was obtained from Sigma Co., St. Louis, MO, USA, and compared with the same peptide obtained from Bachem, Philadelphia, P A, USA. ~-endorphin was obtained from Bachem. Met-enkephalin was tested in samples from all subjects; ~-endorphin was tested in the 7 subjects over 60 years of age and in 1 younger subject. These pep tides were tested at a concentration of 1O-8M in blood from each subject and at additional concentrations in most subjects. In samples from the 11 younger subjects, the concentrations tested were usually 10-7, 10-8 , and 1O-9M. In samples from the 7 subjects over 60 years old, the concentrations ranged from 10-3M to 1O-15M.
Procedure The method described by MATHEWS et al. (7) was followed. This method consists of a 4 h assay measuring the release of 51 Cr from K562 tumor cells as targets. It involves the isolation of peripheral blood lymphocytes (PBL) by density centrifugation with Ficoll-Hypaque. The PBL were then washed three times in PBS (pH 7.2) and re-suspended in RPMI 1640 with HEPES, 10% fetal calf serum, bacitracin (10-6M), penicillin (1 %), and streptomycin (1 %). PBL adherent to plastic were removed by incubation at 37°C, 5 % CO 2 for 1 h in polystyrene culture flasks. Non-adherent cells were then washed with PBS, counted, and tested for viability with 0.4 % Trypan blue stain. Viability was usually greater than 98 %. PBL were preincubated 1 h with peptide, washed with PBS, and resuspended in media with fresh peptide. The assay was performed in triplicate in round-bottom microtiter plates (Linbro, Hamden, CT, USA) in a total volume of 0.2 ml. Most tests with Met-enkephalin were performed at effector to target (E:T) cell ratios of 6.25:1,12.5:1,25:1, and 50:1; in 5 samples, a ratio of 100:1 was also used. In the 8 subjects over 60 years old, the E:T ratios were 10:1 and 20:1. After the incubation for 4h at 37°C with 51Cr labeled K562 cells, the plate was centrifuged (140 g) and 0.1 ml supernatant was harvested from each well. Radioactivity of each sample was determined with a gamma counter (Beckman Instruments, Inc, Irvine, CA, USA). The percentage of lysed target cells was calculated as follows: 'f' . . Percent speCl IC CytotoXICity
=
cpmexp - cpmsr 100 x -=----'------''------''''-cpmmr - cpmsr
where cpmexp was equal to the mean of the observed triplicate assay, cpm" was the spontaneous release of 51Cr by the target cells, and cpmmr was the maximum release of 51Cr obtained by treating the target cells with 1 % Triton X-100.
Enkephalin and NK Activity . 57 Interferon Human interferon was obtained from Sigma and used according to established methods (19, 20) in the same way as were the pep tides (7). Blood from 4 subjects was tested at concentrations of 1 x 103 and 1 x 104 IU/m!. Chromatography
Thin layer chromatography of Met-enkephalin was run in two systems. The first was butanol:acetic acid:water in a ratio of 4:2:2. The second was ethyl acetate:pyridine:acetic acid:water in a ratio of 60:20:6: 11. These were visualized with ninhydrin under UV light. Statistics
Unless otherwise indicated, the results were expressed in the usual way as % specific cytotoxicity. They were analyzed by analysis of variance (ANOV A) followed by Duncan's multiple range test.
Results
The Met-enkephalin used in this study showed biological actlVlty m reducing electrically stimulated contractions of the vas deferens. Chromatographic analysis of the Met-enkephalin obtained from both Sigma and Bachem revealed a single spot in both systems used. Each spot showed the same Rf reported by Bachem in their accompanying analytical data sheet. Separate ANOV A's performed with the data from each of the 18 subjects failed to show statistically significant differences in NK activity in the blood
80
>< b
70
U X
60
b
50
0
0
_
MEDIA
t!QQSl
MET- ENKEPHALlN
~ /I-ENDORPHIN
b
>
t u >< 0 E-< 0 E-
u u
-u ~
r.J
c.. CI'J
35 30 25
•
20 15 10
~
5 0
Figure 2. NK cell activity (± SEM) from all 18 subjects combined from assays with effector:target cell ratios of 100:1, 50:1,25:1,20:1, 12.5:1, 10:1, and 6.25:1. The number of samples measured in triplicate containing Met-enkephalin was 244; the number of media controls was 117." P < 0.05 vs. media.
from any subject. Duncan's multiple range test failed to show significant differences for any single subject for either peptide at any of the different concentrations tested. The results for each subject are shown in Figure 1.
30
>E-< U >< 0 E-
