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Effects of amphetamine on the activity of phagocytosis in mice
Life Sciences, Vol. Printed in the USA
51, pp. PL 145-148
Pergamon
Press
PHARMACOLOGY LETTERS Accelerated Communication
EFFECTS OF AMPHETAMINE ON THE ACTIVITY OF PHAGOCYTOSIS IN MICE
Manuel Freire-Garabal, Mar(a J. Nt~6ez,Jos~ L Balboa, Jos~ C. Fern~mdez-Rial and Angel Belmonte. Department of Pharmacology. School of Medicine. University of Santiago de Compostela. 15705-Santiago de Compostela. Spain. (Submitted May 21, 1992; accepted May 27, 1992; received in final form July 30, 1992)
Abstract Experiments were conducted to evaluate the influences of chronic treatment with amphetamine (0.4 mg/kg/day) on the activity of phagocytosis in mice. Results show a decrease of the in vitro and in vivo phagocytosis measured by using the zymosan-particle uptake method and the carbon clearance test, respectively.
Introduction In previous investigations we observed adverse effects of amphetamine on the immune system of mice. Daily injection with 0.4 mg/kg of amphetamine resulted in a suppression of T-cell populations and functional capacities of T-cells (1). Amphetamine also markedly reduced the development and passive transfer of immunity to Listeria monocytogenes and decreased the latency of MTV-induced tumors in mice. These effects were associated with increases in plasma ACTH and corticosterone levels. In the present paper, we report the effect of amphetamine on phagocytosis measured in vitro and in vivo in mice. Methods Mice Female mice (7 to 9 weeks old) of the BALB/c strain (Interfauna Ib~rica S.A., Barcelona, Spain) were used. They were housed four per cage in well ventilated rooms that were kept between 21°C and 22°C and maintained on an alternating 12-hr light/dark cycle. Food (Panlab Diet A.03) and water were given ad libitum. Procedure Mice were divided into two groups according to the treatment they were submitted to (placebo or amphetamine). After 1,2,3,4,8,12,16 and 20 days of drug treatment, two lots of six mice from each group were used for in vitro and in vitro assays, respectively. Experiments were always performed at (04.00 p.m.).
In vitro ohaoocvtosis of macrophaqes Macrophages were collected by washing the peritoneal cavity with ice-cold PBS (0.1 M, pH 7.3). The peritoneal cells were washed twice with RPMI culture medium (GIBCO Laboratories, Grand Island, N.Y.) by centrifugation (1,000 rpm, 5 min, 0°C) and finally resuspended in an adequate volume of RPMI medium supplemented with 10% heat-inactivated fetal calf serum (FCS, GIBCO) to give a concentration of 1 x 106 nucleated cells per ml. Aliquots (1 ml) of this suspension were seeded in 35 mm petri dishes and incubated at 37°C in a humidified 5% CO2 and 95% air incubator. Three hours later, non-adherent cells were washedoff, and 1 ml fresh medium containing 10% (FCS) was added. Zymosan was added to give 5 x 106 particles/ml and incubation was continued. Thirty minutes later, particle uptake was measured by light microscopy. Cells containing three or more zymosan particles were counted as phagocytic (2). Corresponding Author: Manuel Freire-Garabal. c/Hu~rfanas 19. 15703-Santiago de Compostela. SPAIN.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
PL-146
Amphetamine
Effects
on P h a g o c y t o s i s
Vol.
51, No.
15, 1992
In vivo phaoocvtosis of macroDhaqes Phagocytic activity was estimated by using the carbon clearance test (2). Carbon suspension (Pelikan cl 1/1432a) was centrifuged at 5,000 r.p.m, for 15 min, and the suparnatant was diluted 3-fold with sterile 1.5% gelatin saline to bring the carbon concentration to about 30 mg/ml. Diluted carbon suspension was injected at 0,1 ml/10 g body weight into the tail vein of mice. After 0.5 and 10 min of injection, a blood sample (0.05 ml) was collected by puncturing the retro-orbital venous plexus. Blood was hemolyzed by the addition of 1 ml 0.1% Na2CO 3 solution, followed by measurement of its optical density at 600 nm. Phagocytic index, K, was derived from the following equation (2): 1
K= -
tlo-to.5
log -
CO.5 C10
where C0. 5 and C 10 express the carbon concentration at time to. 5 and t I 0, respectively. Treatment with druqs Racemic amphetamine sulphate (Sigma Chemical Co, St Louis, Mo.) was subcutaneously injected at a dosage of 0.4 mg/kg, in a volume of 1 m l / k g of 0.9 saline solution. The basis for employing this low dose of amphetamine is based on previous dose-response assays that showed to affect the immune system. Placebo mice were subcutaneously injected with 1 m i / k g of 0.9% saline solution. Drugs were daily administered at 09.30 a.m.
Statistical analvsis Statistical analysis was performed using the Student's t-test analysis. Differences were considered to be significant when the probability (P) value was < 0.05. Results The results of in vitro phagocytic activity of peritoneal macrophages are shown in Table I. Despite the same number of macrophages being collected in all experimental groups, the number of macrophages containing zymosan particles were reduced to 32 % of the control by 8 days in mice injected with amphetamine. Then values began to return to around a 55 % of the control by 20 days. In vivo studies also showed a decrease of the activity of phagocytosis in amphetamine-treated mice. After 8 days of treatment with amphetamine, the carbon clearance rate was significantly decreased at 25 % of the control. Then values began to return to around 50 % of the control by 20 days of treatment (Figure 1). TABLE I Effects of amphetamine on in vitro phagocytosis by peritoneal macrophages % particle (zymosan) uptake of 100 ceils Untreated controls
56.4 ± 8.17
Days of treatment
Placebo
Amphetamine
1 2 3 4 8 12 16 20
57"3 + 8"2 54'7 + 5"5 55"9 + 1 "9 53"1 + 3 " 0 55"3 + 5"3 57.6+4.5 56-4 + 7"3 58"7 + 4"6
42"8 + 8"1 * 35"5 + 2"6 28"2 + 7'4 23"2+4"5 18.4 + 5"3 20.1 + 4 " 0 27"0 + 6"1 31 "5 + 9'7
BALB/c mice were daily injected with saline or amphetamine. Macrophageswere collected by washing the peritoneal cavity of these mice and cultured in RPMI-1640medium containing 10% FCS with 5 x 106 zymosan particles for 30 rain. Particle uptake was measured as described under Materials and Methods. The results represent the mean -+ S.D. of 6 animals. Differences between placebo and amphetamine were significant at p < 0.01 and * p < 0.05. Differences between controls and mice injected with placebo were not significant.
Vol. 51, No. 15, 1992
Amphetamine Effects on Phagocytosis
O
Control
•
PllGebo
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AmphetAmine
0,02
PL-147
0,015 X
"o t,D .2
0,01
o o co.
0,005
0
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1
2
3
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I
I
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I
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8
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12
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16
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I
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20
Days of treatment
FIGURE 1
Effect of amphetamine on the carbon clearanceactivity in mice. The carbon suspension (30 mg/ml) was injected at 0.1 ml/10 g body weight into the tail vein of mica. After 0.5 and 10 min of injection, a blood sample (0.05 ml) was collected by puncturing the retro-orbital venous plexus. Phagocytic index (K) was calculated as described under Materiel end Methods. The results represent the mean -+ S.D. of 6 animals. Differences between placebo end amphetamine were significant at p < 0.01 and * p < 0.05. Differences between controls and mice injected with placebo were not significant. Discussion Our data show that chronic treatment with amphetamine powerfully inhibited the activity of phagocytosis in mice. In vitro studies showed a sharp fall in the zymosan uptake by peritoneal macrophages after 8 days of treatment with amphetamine. Furthermore, the carbon clearance test showed a similar pattern of decrease in the in vivo phagocytosis. In both experiments, values began to return towards those of the controls to the end of experiments, perhaps as expression of tolerance to the effects of the drug. The mechanism of action of amphetamine might be either direct (at target cell) or indirect (affecting neuroendocrine pathways). Although direct effects of amphetamine should not be excluded, one can hypothesize that the inhibition of phagocytosis may be secondary to a mediator involved in expressing the drug's effect. Amphetamine has shown numerous effects on neuronal and endocrine systems. Molecular products of cells of the nervous and immune systems provide a means of communication between the two systems (3-6). Many of the effects of amphetamine involve the drug modulation of the adrenergic system and mimic stresslike states (7-11). Cellular immune activity is partially regulated by the adrenergic nervous system (12). Macrophages have been shown to possess 8-adrenergic receptors and their activation was found to affect their functional capacities (13,14). A second point to be considered concerns the neuroendocrinological effects of amphetamine (6,15). The stimulatory effect of amphetamine on adrenocorticotropic hormone (ACTH) and adrenocorticoids should be
PL-148
Amphetamine
Effects
on Phagocytosis
Vol.
51, No. 15, 1992
involved. First, ACTH from the pituitary gland and even ir-ACTH from lymphocyte origin, has a direct inhibitory effect on functional capacities of macrophages. About 47 % of unstimulated peritoneal macrophages express ACTH receptors (16). Second, the rise in plasma corticosterone concentrations, via ACTH secretion enhancement, suppresses various aspects of immune function, including phagocytic activity (2,17). Our previous investigations show a stimulatory effect of chronic amphetamine on ACTH secretion, proportional to the decrease in the activity of phagocytosis. Nevertheless, we observed that adrenalectomized mice showed less but statistically significant immunosuppression in response to amphetamine administration. So, this led us to believe that other neuropeptides and neurotransmitters could be involved in the immunological response to amphetamine. The large number of interactions at molecular, cellular and functional levels between the nervous system and the immune system characterizing the operational compositions and expressions of the neuroimmune network make complex the isolation of the pathways in which amphetamine may be involved in the regulation of the immune responses. So, many questions are still to be addressed in order to understand more fully the immunosuppressive characteristics of amphetamine. Moreover, biological significance and health relatedness of this immunological effect should be assessed. Acknowledqements We wish to express our gratitude to E. Cancio and J.A. Veira for their technical support. References 1. M. FREIRE-GARABAL, J.L. BALBOA, M.J. NUI~IEZ, MT. CASTAI~IO, J.B. LLOVO, J.C. FERNANDEZ-RIAL and A. BELMONTE, Life Sci. 49(16) 107-112 (1991). 2. T. OKIMURA, M. OGAWA and T. YAMAUCHI, Jpn. J. Pharmacol. 4__11,229-235 (1986). 3. J.E. BLALOCK, Physiol. Rev. 6_.991-32 (1989). 4. R. ADER, D. FELTEN and N. COHEN, Annu. Rev. Pharmacol. Toxicol. 30 561-602 (1990). 5. J.E. BLALOCK, Int. J. Neurosci. 51 363-364 (1990). 6. D.A. WEIGENT, D.J. CARR and J.E. BLALOCK, Ann. N. Y. Acad. Sci. 57__.-917-27 (1990). 7. I. GELLER and J. SEIFTER, Psychopharmacoiogy 1 482-492 (1960). 8. S.M. ANTELMAN, A.J. EICHLER, C.A. BLACK and D. KOCAN, Science 20_._.77329-331 (1980). 9. R.E. SU'I-FON, G.F. KOOB, M. LE MOAL, J. RIVIER and W. VALE, Nature 297 331-333 (1982). 10. K.T. 8RITI'ON, J. MORGAN, J. RIVIER, W. VALE and G.F. KOOB, Psychopharmacology 8__66170-174 (1985). 11. K.T. BRI'I-I'ON, G. LEE and G.F. KOOB, Psychopharmacology 94 306-311 (1988). 12. N. BELLUARDO, G. MUDO, V CARDILE, G. MIGLIORATI, C. RICCARDI, S. CELLA and M. BINDONI, Cell Growth Regul _926-35 (1990). 13. C.K. ABRASS, W. O'CONNER, P.J. SCARPACE and I.B. ABRASS, J. Immunol. 13__.551338-1341 (1985). 14. N.H. BISHOPRIC, H.J. COHEN and R.J. LEFKOWlTZ, J. Allergy Clin. Immunol. 6,5 29-33 (1980). 15. R.W. FULLER and H.D. SNODDY, Endocrinology 109 1026-1028 (1981). 16. E.M. SMITH, D.V. HARBOUR, T.K. HUGHES, T. KENT, M.J. EBAUGH, A. JAZAYERI and W.J. MEYER, Stress and Immunity. N. Plotnikoff, A. Murgo, R. Faith and J. Wybran (eds), 453-479, CRC Press, Boca Raton, Florida (1991). 17. R.M. SCHULTZ, M.A. CHIRIGOS, J.N. STOYCHKOV and N.A. PAVLIDIS, J. Reticuloendoth. Soc. 26 8392(1979).
51, pp. PL 145-148
Pergamon
Press
PHARMACOLOGY LETTERS Accelerated Communication
EFFECTS OF AMPHETAMINE ON THE ACTIVITY OF PHAGOCYTOSIS IN MICE
Manuel Freire-Garabal, Mar(a J. Nt~6ez,Jos~ L Balboa, Jos~ C. Fern~mdez-Rial and Angel Belmonte. Department of Pharmacology. School of Medicine. University of Santiago de Compostela. 15705-Santiago de Compostela. Spain. (Submitted May 21, 1992; accepted May 27, 1992; received in final form July 30, 1992)
Abstract Experiments were conducted to evaluate the influences of chronic treatment with amphetamine (0.4 mg/kg/day) on the activity of phagocytosis in mice. Results show a decrease of the in vitro and in vivo phagocytosis measured by using the zymosan-particle uptake method and the carbon clearance test, respectively.
Introduction In previous investigations we observed adverse effects of amphetamine on the immune system of mice. Daily injection with 0.4 mg/kg of amphetamine resulted in a suppression of T-cell populations and functional capacities of T-cells (1). Amphetamine also markedly reduced the development and passive transfer of immunity to Listeria monocytogenes and decreased the latency of MTV-induced tumors in mice. These effects were associated with increases in plasma ACTH and corticosterone levels. In the present paper, we report the effect of amphetamine on phagocytosis measured in vitro and in vivo in mice. Methods Mice Female mice (7 to 9 weeks old) of the BALB/c strain (Interfauna Ib~rica S.A., Barcelona, Spain) were used. They were housed four per cage in well ventilated rooms that were kept between 21°C and 22°C and maintained on an alternating 12-hr light/dark cycle. Food (Panlab Diet A.03) and water were given ad libitum. Procedure Mice were divided into two groups according to the treatment they were submitted to (placebo or amphetamine). After 1,2,3,4,8,12,16 and 20 days of drug treatment, two lots of six mice from each group were used for in vitro and in vitro assays, respectively. Experiments were always performed at (04.00 p.m.).
In vitro ohaoocvtosis of macrophaqes Macrophages were collected by washing the peritoneal cavity with ice-cold PBS (0.1 M, pH 7.3). The peritoneal cells were washed twice with RPMI culture medium (GIBCO Laboratories, Grand Island, N.Y.) by centrifugation (1,000 rpm, 5 min, 0°C) and finally resuspended in an adequate volume of RPMI medium supplemented with 10% heat-inactivated fetal calf serum (FCS, GIBCO) to give a concentration of 1 x 106 nucleated cells per ml. Aliquots (1 ml) of this suspension were seeded in 35 mm petri dishes and incubated at 37°C in a humidified 5% CO2 and 95% air incubator. Three hours later, non-adherent cells were washedoff, and 1 ml fresh medium containing 10% (FCS) was added. Zymosan was added to give 5 x 106 particles/ml and incubation was continued. Thirty minutes later, particle uptake was measured by light microscopy. Cells containing three or more zymosan particles were counted as phagocytic (2). Corresponding Author: Manuel Freire-Garabal. c/Hu~rfanas 19. 15703-Santiago de Compostela. SPAIN.
Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights
reserved.
PL-146
Amphetamine
Effects
on P h a g o c y t o s i s
Vol.
51, No.
15, 1992
In vivo phaoocvtosis of macroDhaqes Phagocytic activity was estimated by using the carbon clearance test (2). Carbon suspension (Pelikan cl 1/1432a) was centrifuged at 5,000 r.p.m, for 15 min, and the suparnatant was diluted 3-fold with sterile 1.5% gelatin saline to bring the carbon concentration to about 30 mg/ml. Diluted carbon suspension was injected at 0,1 ml/10 g body weight into the tail vein of mice. After 0.5 and 10 min of injection, a blood sample (0.05 ml) was collected by puncturing the retro-orbital venous plexus. Blood was hemolyzed by the addition of 1 ml 0.1% Na2CO 3 solution, followed by measurement of its optical density at 600 nm. Phagocytic index, K, was derived from the following equation (2): 1
K= -
tlo-to.5
log -
CO.5 C10
where C0. 5 and C 10 express the carbon concentration at time to. 5 and t I 0, respectively. Treatment with druqs Racemic amphetamine sulphate (Sigma Chemical Co, St Louis, Mo.) was subcutaneously injected at a dosage of 0.4 mg/kg, in a volume of 1 m l / k g of 0.9 saline solution. The basis for employing this low dose of amphetamine is based on previous dose-response assays that showed to affect the immune system. Placebo mice were subcutaneously injected with 1 m i / k g of 0.9% saline solution. Drugs were daily administered at 09.30 a.m.
Statistical analvsis Statistical analysis was performed using the Student's t-test analysis. Differences were considered to be significant when the probability (P) value was < 0.05. Results The results of in vitro phagocytic activity of peritoneal macrophages are shown in Table I. Despite the same number of macrophages being collected in all experimental groups, the number of macrophages containing zymosan particles were reduced to 32 % of the control by 8 days in mice injected with amphetamine. Then values began to return to around a 55 % of the control by 20 days. In vivo studies also showed a decrease of the activity of phagocytosis in amphetamine-treated mice. After 8 days of treatment with amphetamine, the carbon clearance rate was significantly decreased at 25 % of the control. Then values began to return to around 50 % of the control by 20 days of treatment (Figure 1). TABLE I Effects of amphetamine on in vitro phagocytosis by peritoneal macrophages % particle (zymosan) uptake of 100 ceils Untreated controls
56.4 ± 8.17
Days of treatment
Placebo
Amphetamine
1 2 3 4 8 12 16 20
57"3 + 8"2 54'7 + 5"5 55"9 + 1 "9 53"1 + 3 " 0 55"3 + 5"3 57.6+4.5 56-4 + 7"3 58"7 + 4"6
42"8 + 8"1 * 35"5 + 2"6 28"2 + 7'4 23"2+4"5 18.4 + 5"3 20.1 + 4 " 0 27"0 + 6"1 31 "5 + 9'7
BALB/c mice were daily injected with saline or amphetamine. Macrophageswere collected by washing the peritoneal cavity of these mice and cultured in RPMI-1640medium containing 10% FCS with 5 x 106 zymosan particles for 30 rain. Particle uptake was measured as described under Materials and Methods. The results represent the mean -+ S.D. of 6 animals. Differences between placebo and amphetamine were significant at p < 0.01 and * p < 0.05. Differences between controls and mice injected with placebo were not significant.
Vol. 51, No. 15, 1992
Amphetamine Effects on Phagocytosis
O
Control
•
PllGebo
0
AmphetAmine
0,02
PL-147
0,015 X
"o t,D .2
0,01
o o co.
0,005
0
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1
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8
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20
Days of treatment
FIGURE 1
Effect of amphetamine on the carbon clearanceactivity in mice. The carbon suspension (30 mg/ml) was injected at 0.1 ml/10 g body weight into the tail vein of mica. After 0.5 and 10 min of injection, a blood sample (0.05 ml) was collected by puncturing the retro-orbital venous plexus. Phagocytic index (K) was calculated as described under Materiel end Methods. The results represent the mean -+ S.D. of 6 animals. Differences between placebo end amphetamine were significant at p < 0.01 and * p < 0.05. Differences between controls and mice injected with placebo were not significant. Discussion Our data show that chronic treatment with amphetamine powerfully inhibited the activity of phagocytosis in mice. In vitro studies showed a sharp fall in the zymosan uptake by peritoneal macrophages after 8 days of treatment with amphetamine. Furthermore, the carbon clearance test showed a similar pattern of decrease in the in vivo phagocytosis. In both experiments, values began to return towards those of the controls to the end of experiments, perhaps as expression of tolerance to the effects of the drug. The mechanism of action of amphetamine might be either direct (at target cell) or indirect (affecting neuroendocrine pathways). Although direct effects of amphetamine should not be excluded, one can hypothesize that the inhibition of phagocytosis may be secondary to a mediator involved in expressing the drug's effect. Amphetamine has shown numerous effects on neuronal and endocrine systems. Molecular products of cells of the nervous and immune systems provide a means of communication between the two systems (3-6). Many of the effects of amphetamine involve the drug modulation of the adrenergic system and mimic stresslike states (7-11). Cellular immune activity is partially regulated by the adrenergic nervous system (12). Macrophages have been shown to possess 8-adrenergic receptors and their activation was found to affect their functional capacities (13,14). A second point to be considered concerns the neuroendocrinological effects of amphetamine (6,15). The stimulatory effect of amphetamine on adrenocorticotropic hormone (ACTH) and adrenocorticoids should be
PL-148
Amphetamine
Effects
on Phagocytosis
Vol.
51, No. 15, 1992
involved. First, ACTH from the pituitary gland and even ir-ACTH from lymphocyte origin, has a direct inhibitory effect on functional capacities of macrophages. About 47 % of unstimulated peritoneal macrophages express ACTH receptors (16). Second, the rise in plasma corticosterone concentrations, via ACTH secretion enhancement, suppresses various aspects of immune function, including phagocytic activity (2,17). Our previous investigations show a stimulatory effect of chronic amphetamine on ACTH secretion, proportional to the decrease in the activity of phagocytosis. Nevertheless, we observed that adrenalectomized mice showed less but statistically significant immunosuppression in response to amphetamine administration. So, this led us to believe that other neuropeptides and neurotransmitters could be involved in the immunological response to amphetamine. The large number of interactions at molecular, cellular and functional levels between the nervous system and the immune system characterizing the operational compositions and expressions of the neuroimmune network make complex the isolation of the pathways in which amphetamine may be involved in the regulation of the immune responses. So, many questions are still to be addressed in order to understand more fully the immunosuppressive characteristics of amphetamine. Moreover, biological significance and health relatedness of this immunological effect should be assessed. Acknowledqements We wish to express our gratitude to E. Cancio and J.A. Veira for their technical support. References 1. M. FREIRE-GARABAL, J.L. BALBOA, M.J. NUI~IEZ, MT. CASTAI~IO, J.B. LLOVO, J.C. FERNANDEZ-RIAL and A. BELMONTE, Life Sci. 49(16) 107-112 (1991). 2. T. OKIMURA, M. OGAWA and T. YAMAUCHI, Jpn. J. Pharmacol. 4__11,229-235 (1986). 3. J.E. BLALOCK, Physiol. Rev. 6_.991-32 (1989). 4. R. ADER, D. FELTEN and N. COHEN, Annu. Rev. Pharmacol. Toxicol. 30 561-602 (1990). 5. J.E. BLALOCK, Int. J. Neurosci. 51 363-364 (1990). 6. D.A. WEIGENT, D.J. CARR and J.E. BLALOCK, Ann. N. Y. Acad. Sci. 57__.-917-27 (1990). 7. I. GELLER and J. SEIFTER, Psychopharmacoiogy 1 482-492 (1960). 8. S.M. ANTELMAN, A.J. EICHLER, C.A. BLACK and D. KOCAN, Science 20_._.77329-331 (1980). 9. R.E. SU'I-FON, G.F. KOOB, M. LE MOAL, J. RIVIER and W. VALE, Nature 297 331-333 (1982). 10. K.T. 8RITI'ON, J. MORGAN, J. RIVIER, W. VALE and G.F. KOOB, Psychopharmacology 8__66170-174 (1985). 11. K.T. BRI'I-I'ON, G. LEE and G.F. KOOB, Psychopharmacology 94 306-311 (1988). 12. N. BELLUARDO, G. MUDO, V CARDILE, G. MIGLIORATI, C. RICCARDI, S. CELLA and M. BINDONI, Cell Growth Regul _926-35 (1990). 13. C.K. ABRASS, W. O'CONNER, P.J. SCARPACE and I.B. ABRASS, J. Immunol. 13__.551338-1341 (1985). 14. N.H. BISHOPRIC, H.J. COHEN and R.J. LEFKOWlTZ, J. Allergy Clin. Immunol. 6,5 29-33 (1980). 15. R.W. FULLER and H.D. SNODDY, Endocrinology 109 1026-1028 (1981). 16. E.M. SMITH, D.V. HARBOUR, T.K. HUGHES, T. KENT, M.J. EBAUGH, A. JAZAYERI and W.J. MEYER, Stress and Immunity. N. Plotnikoff, A. Murgo, R. Faith and J. Wybran (eds), 453-479, CRC Press, Boca Raton, Florida (1991). 17. R.M. SCHULTZ, M.A. CHIRIGOS, J.N. STOYCHKOV and N.A. PAVLIDIS, J. Reticuloendoth. Soc. 26 8392(1979).