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Neuroimmunomodulation with enkephalins: Effects on thymus and spleen weights in mice
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
32, 52-56 (1984)
Neuroimmunomodulation with Enkephaiins: Effects Thymus and Spleen Weights in Mice NICHOLAS P, PLOTNIKOFF, ANTHONY Departments
of Pharmacology University,
J. MURGO,
on
ANDROBERTEFAITH
and Medicine and Biomedical Research Center, School of Medicine, T&a, OkIahoma 74171
Oral
Roberts
The subcutaneous injection of either methionine-enkephalin or leucine-enkephalin in BDF, mice resulted in a significant increase in thymus weight and a significant decrease in spleen weight. This study further supports earlier findings that the enkephalins are neuroimmunomodulators.
INTRODUCTION
Two pentapeptides, methionine-enkephalin and leucine-enkephalin, were originally identified to be the endogenous ligands in the brain binding to morphine receptors (l-3). In addition to their analgesic activities, one of us (4) found antidepressant, antianxiety, and anticonvulsant activities for the enkephalins. It became apparent that there were several different receptors for morphine-type compounds as well as peptide ligands (5). A nonopiate receptor on normal human blood T lymphocytes for methionineenkephalin was originally reported by Wybran et al. (6, 7). At the same time, Hazum et al. (8) reported that cultured human lymphocytes possessed specific receptors for P-endorphins. In addition, Lopker et al. (9) reported on opiate receptors in human phagocytic leukocytes. The above studies led us to study the enkephalins as neuroimmunomodulators. We discovered that the enkephalins prolonged survival time of BDF, mice inoculated with attentuated L1210 and also caused an increased blastogenic response to phytohemagglutinin stimulation in murine splenic lymphocytes (10-12). Finally, our group observed increased active T-cell rosette-forming cells in in vitro studies of blood from normal human volunteers as well as lymphoma patients (13, 14). The present study of neuroimmunomodulation is an extension of the above studies as they relate to target lymphoid organs in the immune system. MATERIALS
AND METHODS
Mice. Four-week-old BDF, female mice were purchased from Jackson Laboratories, Bar Harbor, Maine. Animals were allowed to recover from shipping stress for 1 week before being placed on study. Mice were housed five per cage in polycarbonate shoe box cages and given food and water ad libitum. Enkephalin treatment. Mice were randomly placed in groups of ten. Groups of mice were given either methionine-enkephalin or leucine-enkephalin (10 or 30 mg/ kg SC) daily for 7 total days in a 9-day sequence. The first day (Day 0) the mice received 0.2 ml of RPM1 1640 ip to parallel previous L1210 tumor cell studies 52 0090-1229184 $1.50 Copyright
0 1984 by Academic _
Press. Inc. ~
,
NEUROIMMUNOMODULATION
WITH ENKEPHALINS
53
(10, 1I, 12). Subsequent injections of enkephalins were given SCon the mornings of Days 1, 2, 3, 4, 6, 7, and 8. Control mice received the same schedule of injections with the vehicle exclusive of enkephalin. The mice were sacrificed on the afternoon of Day 8 by cervical dislocation, body weights were taken, and the thymus, spleen, and adrenals were removed and weighed. Histology. Tissues selected for histological examination were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 pm, and routinely stained with hematoxylin and eosin. Statistical analyses. Statistical analyses were carried out with the Student t test. RESULTS
Thymus. Significant increases in relative thymus weight (percentage of body weight) were observed in mice administered either methionine-enkephalin (Fig. 1) or leucine-enkephalin (Fig. 2). In the case of methionine-enkephalin, the lower dose, 10 mg/kg, resulted in a significant increase in the weight of thymus. The higher dose of 30 mg/kg also increased the thymus weight but did not reach statistical significance. Histologically there appeared to be a significant increase in the size of the thymic cortex in the enkephalin-treated animals (Fig. 3). Absolute weights of the thymus are shown in Table 1. Spleen. In contrast to the thymus, the spleens of mice treated with either enkephalin or Leu-enkephalin were significantly decreased in size at both 10 and 30 mg/kg doses (Figs. 1 and 2). The absolute weights are shown in Table 1. Histological examination revealed that the apparent major loss of splenic tissue occurred in the red pulp. Adrenals. No significant effects on the size of the adrenals were seen with either Met-enkephalin or Leu-enkephalin at doses of 10 or 30 mgikg (Table 1). Body weights. No significant alterations of body weights of mice compared to controls were seen in mice treated with either Met-enkephalin or Leu-enkephalin at IO or 30 mgikg (Table 1). DISCUSSION
The present study supports our earlier findings that the enkephalins the immune system, as measured by increases in splenic lymphocyte THYMUS
stimulate blastoge-
SPLEEN 0.50 0.40 0.30 0.20 0.10
Control
iom_s 309 kg kg
Control
lOa
309 kg
kg
I. Effect of methionine-enkephalin on the size of thymus and spleen (percentage of body weight). Vertical bars represent the mean values of 10 mice, and vertical lines represent standard errors of the mean. *‘P < 0.05. FIG.
54
PLOTNIKOFF,
MURGO,
AND FAITH SPLEEN
control
10mg ii5
30mg Ri
FIG. 2. Effect of leucine-enkephalin on the size of thymus and spleen (percentage of body weight). Vertical bars represent the mean values of 10 mice, and vertical lines represent standard errors of the mean. * P < 0.05.
nesis, active T-cell rosette-forming cells in normal and lymphoma patients (14). More recently, Gilman et al. (15) found similar effects with P-endorphin which enhanced proliferative responses of spleen cells to the T-cell mitogens concanavalin A and phytohemagglutinin. Furthermore, Mathews et al. (16) just reported that Met-enkephalin as well as P-endorphin enhanced NK (natural killer) cell activity in humans. Our group has found similar effects of Met- and Leu-enkephalin on enhancing NK cell activity in humans (Faith et al., 1983, unpublished data). Therefore, the immune system can be stimulated by the enkephalins directly
FIG. 3. Effect of enkephalins on thymic histology. (A) Thymus of untreated animal (Cor = thymic cortex; Med = thymic medulla). (B) Thymus of enkephalin-treated animal showing increase in thickness of thymic cortex (labeling as for A).
NEUROIMMUNOMODULATION TABLE EFFECT
Control 10 mgikg 30 mg/kg
Control 10 mgikg 30 mgikg
OF ENKEPHALINS
ON BODY,
THYMUS,
WITH ENKEPHALINS
55
1 SPLEEN,
AND ADRENAL
Body weight (g z SEM)
Thymus (mg ? SEM)
19.6 -c 0.3 19.2 f 0.2 (N.S.)” 19.3 i 0.3 (N.S.)” (N.S.Jb
Methionine-enkephalin 68.6 or 3.6 101.8 i 3.1 78.1 t 2.9 68.3 + 2.2 (P = 0.05)~ (P < 0.001)” 79.1 * 4.5 69.8 i 1.7 (P < 0.1)” (P < 0.001) (N.S.)’ (N.S.)b
19.6 2 0.4 19.4 f 0.3 (N.S.) 19.2 2 0.2 (N.S.)” (N.S.)b
56.1 49.3 (P < 63.0 (P < (P <
WEIGHTS
Spleen (mg -r- SEM)
Leucine-enkephalin i 0.9 115.2 2 6.1 i 2.8 98.0 i 4.0 0.05) (P < 0.05y + 1.7 71.3 ct- 1.9 0.005)~ (P < o.ool)” O.OOl)b (P < O.OOl)b
Adrenals (mg + SEM) 8.6 + 1.3 10.3 f 0.8 (N..!Qa 10.1 2 0.6 (N.S.)a (N.S.)b 6.3 2 0.7 5.7 r 0.8 (N.S.) 4.5 -?I 0.8 (P = 0.1)” (N.SJb
u Statistical difference from control; N.S. = no significant difference (P > 0.1). b Statistical difference between enkephalin groups.
as outlined above. The enkephalins may naturally influence immune function during stress by release from the adrenals (17, 18). This function together with the catecholamines and the steroids from the adrenals may represent an integrated modulation system(s) between the central nervous system and the pituitary adrenal axis and the immune system (I 1). This integration and modulation among the central nervous system, endocrine, and immune systems represent reactivity to stress. In the present study, this may include the release of growth hormone and/or prolactin (reported in rats with the enkephalins) with consequent increase in thymic weight (19). However, it is possible that the enkephalins have direct effects on the thymus and spleen accounting for the weight differences reported. Indeed histological examination revealed an apparent increase in size of the thymic cortex and reduction of splenic red pulp. The change in thymus could result from either an increase in the number of immature cells migrating to the thymus or a slowing in the rate of cellular migration through the thymus. It is felt that the splenic change is due to a contraction of the splenic capsule resulting in expu.lsion of red pulp cells into circulation. Additionally the enkephalins may influence these tissues by their effects on lymphocyte differentiation indicated in previous studies (13, 14). These studies may provide a basis for identifying the modulatory effects between the central nervous system and the immune system. REFERENCES 1. Hughes, I., Smith, T. W., Kosterlitz, Nature
258, 577, 1975.
H. W., Fothergill, L. A., Morgan, B. A., and Morris, H. T..
56
PLOTNIKOFF,
MURGO,
AND FAITH
2. Simantov, R., and Snyder, S., Proc. Natl. Acad. Sci. USA 73, 2515, 1976. 3. Kosterlitz, H. W.,“Opiates and Endogenous Opioid Peptides,” ElsevieriNorth-Holland Biomedical Press, Amsterdam, 1976. 4. Plotnikoff, N. P., Kastin, A. J., Coy, D. H., Christensen, C. W., Schally, A. V., and Spirtes, M. A., Life Sci. 19, 1283, 1976. 5. Bloom, F. E., Annu. Rev. Pkarmacol. Toxicol. 23, 151, 1983. 6. Wybran, J., Appelboom, T., Famaey, J-P., and Govaerts, A., J Immunol. 123(3), 1068, 1979. 7. Wybran, J., Appelboom, T., Famaey, J-P., and Govaerts, A., In “International Symposium on New Trends in Human Immunology and Cancer Immunotherapy” (B. Serrou and C. Rosenfeld, Eds.), pp. 48-55, Doin Press, Paris, 1980. 8. Hazum, E., Chang, K. J., and Cuatrecasas, P., Science 205, 1033, 1979. 9. Lopker, A., Abood, L. G., Hoss, W., and Lionetti, F. J., Biockem. Pkarmacol. 29, 1361, 1980. 10. Plotnikoff, N. P., Miller, G. C., and Murgo, A. J., ht. J. Zmmunopkarmaco/. 4, 366, 1982. 11. Plotnikoff, N. P., Psyckopkarmacol. Bull. U(4), 148, 1982. 12. Plotnikoff, N. P., and Miller, G. C., Int. J. Immunopkarmacol. 5, 437, 1983. 13. Miller, G. C., Murgo, A. J., and Plotnikoff, N. P., ht. .I. Immunopkarmacol. 4, 366, 1982. 14. Miller, G. C., Murgo, A. J., and Plotnikoff, N. P.. Clin. Zmmunol. Immunopatkol. 26, 446, 1983. 15. Gilman, S. C., Schwartz, J. M., Milner, R. J., Bloom, F. E., and Feldman, J. D., Proc. Nut/. Acad.
Sci.
16. Mathews,
USA
79, 4226,
1982.
P. M., Froelich, C. J., Sibbitt, W. L., Jr., and Bankhurst, A. D., J. Immunol.
130,
1658, 1983.
17. Kilpatrick, D. L., Lewis, R. V., Stein, S., and Udenfriend, S., Proc. Natl. Acad. Sci. USA 77, 7743, 1980. 18. Holaday, J. W., Annu. Rev. Pkarmacol. Toxico/. 23, 541, 1983. 19. Labrie, F., DuPont, L. C., Lissitzky, J. C., Lepine, J., Raymond, V., and Coy, D. H., In “Endorphins in Mental Health Research” (E. Usdin and W. E. Bunney, Jr., Eds.), Oxford Univ. Press, New York, 1979. Received November 29, 1983; accepted with revisions February 17, 1984.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
32, 52-56 (1984)
Neuroimmunomodulation with Enkephaiins: Effects Thymus and Spleen Weights in Mice NICHOLAS P, PLOTNIKOFF, ANTHONY Departments
of Pharmacology University,
J. MURGO,
on
ANDROBERTEFAITH
and Medicine and Biomedical Research Center, School of Medicine, T&a, OkIahoma 74171
Oral
Roberts
The subcutaneous injection of either methionine-enkephalin or leucine-enkephalin in BDF, mice resulted in a significant increase in thymus weight and a significant decrease in spleen weight. This study further supports earlier findings that the enkephalins are neuroimmunomodulators.
INTRODUCTION
Two pentapeptides, methionine-enkephalin and leucine-enkephalin, were originally identified to be the endogenous ligands in the brain binding to morphine receptors (l-3). In addition to their analgesic activities, one of us (4) found antidepressant, antianxiety, and anticonvulsant activities for the enkephalins. It became apparent that there were several different receptors for morphine-type compounds as well as peptide ligands (5). A nonopiate receptor on normal human blood T lymphocytes for methionineenkephalin was originally reported by Wybran et al. (6, 7). At the same time, Hazum et al. (8) reported that cultured human lymphocytes possessed specific receptors for P-endorphins. In addition, Lopker et al. (9) reported on opiate receptors in human phagocytic leukocytes. The above studies led us to study the enkephalins as neuroimmunomodulators. We discovered that the enkephalins prolonged survival time of BDF, mice inoculated with attentuated L1210 and also caused an increased blastogenic response to phytohemagglutinin stimulation in murine splenic lymphocytes (10-12). Finally, our group observed increased active T-cell rosette-forming cells in in vitro studies of blood from normal human volunteers as well as lymphoma patients (13, 14). The present study of neuroimmunomodulation is an extension of the above studies as they relate to target lymphoid organs in the immune system. MATERIALS
AND METHODS
Mice. Four-week-old BDF, female mice were purchased from Jackson Laboratories, Bar Harbor, Maine. Animals were allowed to recover from shipping stress for 1 week before being placed on study. Mice were housed five per cage in polycarbonate shoe box cages and given food and water ad libitum. Enkephalin treatment. Mice were randomly placed in groups of ten. Groups of mice were given either methionine-enkephalin or leucine-enkephalin (10 or 30 mg/ kg SC) daily for 7 total days in a 9-day sequence. The first day (Day 0) the mice received 0.2 ml of RPM1 1640 ip to parallel previous L1210 tumor cell studies 52 0090-1229184 $1.50 Copyright
0 1984 by Academic _
Press. Inc. ~
,
NEUROIMMUNOMODULATION
WITH ENKEPHALINS
53
(10, 1I, 12). Subsequent injections of enkephalins were given SCon the mornings of Days 1, 2, 3, 4, 6, 7, and 8. Control mice received the same schedule of injections with the vehicle exclusive of enkephalin. The mice were sacrificed on the afternoon of Day 8 by cervical dislocation, body weights were taken, and the thymus, spleen, and adrenals were removed and weighed. Histology. Tissues selected for histological examination were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 pm, and routinely stained with hematoxylin and eosin. Statistical analyses. Statistical analyses were carried out with the Student t test. RESULTS
Thymus. Significant increases in relative thymus weight (percentage of body weight) were observed in mice administered either methionine-enkephalin (Fig. 1) or leucine-enkephalin (Fig. 2). In the case of methionine-enkephalin, the lower dose, 10 mg/kg, resulted in a significant increase in the weight of thymus. The higher dose of 30 mg/kg also increased the thymus weight but did not reach statistical significance. Histologically there appeared to be a significant increase in the size of the thymic cortex in the enkephalin-treated animals (Fig. 3). Absolute weights of the thymus are shown in Table 1. Spleen. In contrast to the thymus, the spleens of mice treated with either enkephalin or Leu-enkephalin were significantly decreased in size at both 10 and 30 mg/kg doses (Figs. 1 and 2). The absolute weights are shown in Table 1. Histological examination revealed that the apparent major loss of splenic tissue occurred in the red pulp. Adrenals. No significant effects on the size of the adrenals were seen with either Met-enkephalin or Leu-enkephalin at doses of 10 or 30 mgikg (Table 1). Body weights. No significant alterations of body weights of mice compared to controls were seen in mice treated with either Met-enkephalin or Leu-enkephalin at IO or 30 mgikg (Table 1). DISCUSSION
The present study supports our earlier findings that the enkephalins the immune system, as measured by increases in splenic lymphocyte THYMUS
stimulate blastoge-
SPLEEN 0.50 0.40 0.30 0.20 0.10
Control
iom_s 309 kg kg
Control
lOa
309 kg
kg
I. Effect of methionine-enkephalin on the size of thymus and spleen (percentage of body weight). Vertical bars represent the mean values of 10 mice, and vertical lines represent standard errors of the mean. *‘P < 0.05. FIG.
54
PLOTNIKOFF,
MURGO,
AND FAITH SPLEEN
control
10mg ii5
30mg Ri
FIG. 2. Effect of leucine-enkephalin on the size of thymus and spleen (percentage of body weight). Vertical bars represent the mean values of 10 mice, and vertical lines represent standard errors of the mean. * P < 0.05.
nesis, active T-cell rosette-forming cells in normal and lymphoma patients (14). More recently, Gilman et al. (15) found similar effects with P-endorphin which enhanced proliferative responses of spleen cells to the T-cell mitogens concanavalin A and phytohemagglutinin. Furthermore, Mathews et al. (16) just reported that Met-enkephalin as well as P-endorphin enhanced NK (natural killer) cell activity in humans. Our group has found similar effects of Met- and Leu-enkephalin on enhancing NK cell activity in humans (Faith et al., 1983, unpublished data). Therefore, the immune system can be stimulated by the enkephalins directly
FIG. 3. Effect of enkephalins on thymic histology. (A) Thymus of untreated animal (Cor = thymic cortex; Med = thymic medulla). (B) Thymus of enkephalin-treated animal showing increase in thickness of thymic cortex (labeling as for A).
NEUROIMMUNOMODULATION TABLE EFFECT
Control 10 mgikg 30 mg/kg
Control 10 mgikg 30 mgikg
OF ENKEPHALINS
ON BODY,
THYMUS,
WITH ENKEPHALINS
55
1 SPLEEN,
AND ADRENAL
Body weight (g z SEM)
Thymus (mg ? SEM)
19.6 -c 0.3 19.2 f 0.2 (N.S.)” 19.3 i 0.3 (N.S.)” (N.S.Jb
Methionine-enkephalin 68.6 or 3.6 101.8 i 3.1 78.1 t 2.9 68.3 + 2.2 (P = 0.05)~ (P < 0.001)” 79.1 * 4.5 69.8 i 1.7 (P < 0.1)” (P < 0.001) (N.S.)’ (N.S.)b
19.6 2 0.4 19.4 f 0.3 (N.S.) 19.2 2 0.2 (N.S.)” (N.S.)b
56.1 49.3 (P < 63.0 (P < (P <
WEIGHTS
Spleen (mg -r- SEM)
Leucine-enkephalin i 0.9 115.2 2 6.1 i 2.8 98.0 i 4.0 0.05) (P < 0.05y + 1.7 71.3 ct- 1.9 0.005)~ (P < o.ool)” O.OOl)b (P < O.OOl)b
Adrenals (mg + SEM) 8.6 + 1.3 10.3 f 0.8 (N..!Qa 10.1 2 0.6 (N.S.)a (N.S.)b 6.3 2 0.7 5.7 r 0.8 (N.S.) 4.5 -?I 0.8 (P = 0.1)” (N.SJb
u Statistical difference from control; N.S. = no significant difference (P > 0.1). b Statistical difference between enkephalin groups.
as outlined above. The enkephalins may naturally influence immune function during stress by release from the adrenals (17, 18). This function together with the catecholamines and the steroids from the adrenals may represent an integrated modulation system(s) between the central nervous system and the pituitary adrenal axis and the immune system (I 1). This integration and modulation among the central nervous system, endocrine, and immune systems represent reactivity to stress. In the present study, this may include the release of growth hormone and/or prolactin (reported in rats with the enkephalins) with consequent increase in thymic weight (19). However, it is possible that the enkephalins have direct effects on the thymus and spleen accounting for the weight differences reported. Indeed histological examination revealed an apparent increase in size of the thymic cortex and reduction of splenic red pulp. The change in thymus could result from either an increase in the number of immature cells migrating to the thymus or a slowing in the rate of cellular migration through the thymus. It is felt that the splenic change is due to a contraction of the splenic capsule resulting in expu.lsion of red pulp cells into circulation. Additionally the enkephalins may influence these tissues by their effects on lymphocyte differentiation indicated in previous studies (13, 14). These studies may provide a basis for identifying the modulatory effects between the central nervous system and the immune system. REFERENCES 1. Hughes, I., Smith, T. W., Kosterlitz, Nature
258, 577, 1975.
H. W., Fothergill, L. A., Morgan, B. A., and Morris, H. T..
56
PLOTNIKOFF,
MURGO,
AND FAITH
2. Simantov, R., and Snyder, S., Proc. Natl. Acad. Sci. USA 73, 2515, 1976. 3. Kosterlitz, H. W.,“Opiates and Endogenous Opioid Peptides,” ElsevieriNorth-Holland Biomedical Press, Amsterdam, 1976. 4. Plotnikoff, N. P., Kastin, A. J., Coy, D. H., Christensen, C. W., Schally, A. V., and Spirtes, M. A., Life Sci. 19, 1283, 1976. 5. Bloom, F. E., Annu. Rev. Pkarmacol. Toxicol. 23, 151, 1983. 6. Wybran, J., Appelboom, T., Famaey, J-P., and Govaerts, A., J Immunol. 123(3), 1068, 1979. 7. Wybran, J., Appelboom, T., Famaey, J-P., and Govaerts, A., In “International Symposium on New Trends in Human Immunology and Cancer Immunotherapy” (B. Serrou and C. Rosenfeld, Eds.), pp. 48-55, Doin Press, Paris, 1980. 8. Hazum, E., Chang, K. J., and Cuatrecasas, P., Science 205, 1033, 1979. 9. Lopker, A., Abood, L. G., Hoss, W., and Lionetti, F. J., Biockem. Pkarmacol. 29, 1361, 1980. 10. Plotnikoff, N. P., Miller, G. C., and Murgo, A. J., ht. J. Zmmunopkarmaco/. 4, 366, 1982. 11. Plotnikoff, N. P., Psyckopkarmacol. Bull. U(4), 148, 1982. 12. Plotnikoff, N. P., and Miller, G. C., Int. J. Immunopkarmacol. 5, 437, 1983. 13. Miller, G. C., Murgo, A. J., and Plotnikoff, N. P., ht. .I. Immunopkarmacol. 4, 366, 1982. 14. Miller, G. C., Murgo, A. J., and Plotnikoff, N. P.. Clin. Zmmunol. Immunopatkol. 26, 446, 1983. 15. Gilman, S. C., Schwartz, J. M., Milner, R. J., Bloom, F. E., and Feldman, J. D., Proc. Nut/. Acad.
Sci.
16. Mathews,
USA
79, 4226,
1982.
P. M., Froelich, C. J., Sibbitt, W. L., Jr., and Bankhurst, A. D., J. Immunol.
130,
1658, 1983.
17. Kilpatrick, D. L., Lewis, R. V., Stein, S., and Udenfriend, S., Proc. Natl. Acad. Sci. USA 77, 7743, 1980. 18. Holaday, J. W., Annu. Rev. Pkarmacol. Toxico/. 23, 541, 1983. 19. Labrie, F., DuPont, L. C., Lissitzky, J. C., Lepine, J., Raymond, V., and Coy, D. H., In “Endorphins in Mental Health Research” (E. Usdin and W. E. Bunney, Jr., Eds.), Oxford Univ. Press, New York, 1979. Received November 29, 1983; accepted with revisions February 17, 1984.