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Effects of amphetamine on influenza virus infection in mice
Life Sciences, Vol. Printed in the USA
52, pp.
PL 73-78
Pergamon
PHARMACOLOGY Accelerated
Press
LETTERS
Communication
EFFECTS OF AMPHETAMINE ON INFLUENZA VIRUS INFECTION IN MICE
MJ. N~6ez, J.C. Fern~ndez-Rial, J. Couceiro, J.A. Su~rez, D.E. G6mez-Fern~ndez, M. Rey-M~ndez and M. Freire-Garabal. Neuroinmunologfa-Universidadde Santiago (NIMUS), University of Santiago de Compostela. 15705-Santiago de Compostela. Spain. (Submitted September I0, 1992; accepted October 6, 1992; received in final form December 18, 1992)
Abstract Several experiments were conducted to evaluate the effects of chronic amphetamine on the infuenza A (PR-8/34) virus specific immune injury in CD-1 mice. Treatment with amphetamine resulted in a significant increase of lung virus titers and pulmonar vascular permeability. Amphetamine also increased the lethality of infected mice.
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 reduction of the number and functional capacities of T-cells (1,2), as well as in a suppression of the activity of phagocytosis (3). It is well known that deficiencies in natural and specific immune responses predispose the host to more severe virus infection (4,5). In this regard, in a previous report we observed that amphetamine decreased the resistance of mice against (MTV)induced tumors (6). To further elucidate this latter interaction, in the present paper we report the effects of amphetamine on the pathogenicity of the mouse-adapted strain of influenza virus PR-8/34 in mice. Methods Mice
Male 3-week-old, pathogen-free, CD-1 mice (Interfauna Ib~rica S.A., Barcelona, Spain) were used. They were housed, 7 days before experiments, four per cage in an aseptic and sound-proof chamber kept between 21°C and 22°C and maintained on an alternating 12-hr light/dark cycle. Sterilyzed food (Panlab Diet A.03) and water were given ad libitum. Procedure
Mice were randomly dealed in three groups according to the treatment they were submitted to: GROUP A. controls, GROUP B. saline, GROUP C. amphetamine. Virus
The mouse-adapted strain of influenza virus PR-8/34 was grown in the allantoic fluid of embryonated eggs after serial passage in mice to increase virulence. Influenza virus infectivity was assayed according to the procedures described by Lennett and Schmidt (7). Log dilutions of lung homogenates were added in a volume of 0.1 ml to the aliantoic sac of 10 day-old embryonated chicken eggs. The eggs were maintained at 35°C. After 48 h the allantoic fluid was harvested and tested at each dilution for the hemagglutination activity. The eggs were scored positive for infectivity at a given virus dilution when the allantoic fluid demonstrated positive hemagglutination of chicken erythrocytes (8). The microtiter procedure was used to determine the hemagglutination of influenza virus. The dose required to infect 50 % of the embryonated eggs (EID50) was determined by the method of Reed and Muench (9). Virus was inoculated intranasally 24 h after the beginning of drugs administration. Corresponding Author: ManuelFreire-Garabal. c/Hu~ffanas19.15703-Santiagode Compo~ela. SPAIN. 0024-3205/93 $6.00 + .00 Copyright
© 1993 Pergamon
Press Ltd
All rights
reserved.
PL-74
Amphetamine
on I n f l u e n z a
Virus
Infection
Vol.
52, No.
i0,
1993
Controls
100
Saine Amphetamine ILl L~
80
Z
Ll.I 0n," LU Q. -J
60
[]
l
© 40
N 20
1 HAU
0
0
"
l
J
t
I
I
I
I
I
2
4
6
8
I0
12
14
TIME
AFTER
INFECTION
A v
v
.__
J
16
(DAYS)
100
tu
8O
z
w
O tr LU O,. ,_1
60
w
D
¢'r m
©
211
5 HAU
~ e
I
I
I
I
I
I
2
4
6
8
10
12
I
14
16
FIGURE 1
Effect of amphetamine on the survival of PR8/34-infected mice. Twenty-four hours after the beginning of experiments, mice were inoculated i.n. with PR8 virus (1 HAU/mouse, A; 5 HAU/mouse, B). Survival curves represent the values of two experiments, p < 0,05 for the mortality ratios between saline and amphetamine.
Vol.
52, NO.
I0,
1993
Amphetamine
on I n f l u e n z a
Virus
Infection
PL-75
Lethality assay. Mice were inoculated i.n. with 1 HAU/mouse (20 mice/group) and 5 HAU/mouse (20 mice/group) PR-8/34 virus. Survival percentages were recorded daily during 16 days (10).
Virus titers The pathogenesis of influenza virus infection in mice was determined by quantitation of virus titers (8). Mice were inoculated intranasally with 0.1 LD50 of influenza A virus. After 12 and 24 h and 3, 6 and 9 days of infection, 6 mice from each group were sacrificed by cervical dislocation and immediately necropsied. Their lungs were removed under aseptic conditions, minced with scissors, weighed and stored at -70°C. A 20 % (w:vol) lung suspension was prepared by homogenizing lung tissue in Hank's balanced salt solution. This suspension was clarified by centrifugation at 500 x g for 10 min at 4°C. The infectious virus was assayed in embryonated eggs as previously described (8).
Calculation of alveolar capillary leak Permeability index (PI), that indicates increased pulmonary vascular permeability, was calculated to determine tissue injury (8). After 3, 6, 9, 12 and 15 days of inoculation with 0.1 LD50 influenza A virus, 1251labeled bovine serum albumin (100 I = 140,000 cpm) was injected into the tail vein of six mice from each experimental group. After 2 h, the mice were anesthetized with ketamine (150 mg/kg), 100 I of caval blood was withdrawn and the heart and lungs removed in block (8). The pulmonary vasculature was perfused with 2 ml saline via the right ventricle to remove the blood-associated radioactivity in the pulmonary system. Lung tissue and blood were counted in a gamma counter (LKB Wallac 1275 Gamma Counter). The ratio of radioactivity present in the lungs to that present in the animal's blood was expressed as the Pi.
Treatment with drugs 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 ml/kg 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 ml/kg of 0.9% saline solution. Drugs were daily administered at 09.30 a.m.
Statistical analysis Mortality ratios and mean times to death were analyzed with Fisher's exact test and the Mann-Whitney U test, respectively. Analysis of mean differences in virus titers and PI was performed according to the Student's t test. Significance was achieved at p < 0.05. Results Figure 1 shows the effect of amphetamine on the survival of mice infected with 1 HAU and 5 HAU of PR-8/34 virus. Amphetamine significantly increased the lethality to virus infection. This effect was very evident at 5 HAU/mouse. Calculation of mean time to death showed that significantly earlier times of death occurred among mice of the GROUP C (5.1 +- 0.2 days after infection) in comparison with mice of the GROUP B (6.9 _+0.5 days after infection). Differences berween gruops A and B were not significant. Virus titers in the lungs of mice sacrificed 0.5, 1 and 3 days after infection were greater in GROUP C in comparison with GROUP A (Figure 2). Differences between groups A and B were only significant on the 3rd day assay. As shown in figure 3 the PI was significantly greater in GROUP C mice in comparison with GROUP B mice. Differences between groups A and B were only significant on the 9th day assay. Discussion Our results show that amphetamine exacerbates influenza virus infection. Calculation of mean time to death indicated that significantly earlier times of death occurred amongst mice mice injected with amphetamine compared with mice injected with saline. Pathologic findings in this report show that influenza A virus titers in the lungs of mice were greater for mice injected with amphetamine in comparison with mice injected with saline. Furthermore, alveolar-capillary leakage was greater in this group. Treatment with saline resulted in higher virus titers and PI in comparison
PL-76
Amphetamine
on I n f l u e n z a
lO, O
Controls
10 e
Amp
Virus
Infection
Vol.
52, No.
i0,
1993
Saline
¢D
.= ¢n
10 e 10 7
. tm-
>
o a m LU
lO e 10 6
t/
lO 4 O
lO 3 Time after infection (Days)
u
10 2
0.5
1
3
6
9
FIGURE 2 Virus titers in lung tissue of mice infected with 0.1 LD50 influenza A virus and sampled at given times after inoculation. The results represent the mean -+ S.D. of 6 animals. * Differences between saline and amphetamine were significant at p < 0.05. • Differences between controls and mice injected with saline significant at p < 0.05.
0,8 Controls
----o-Amphetamine
O.
0,6
\\\\\\\
X e.-
~
0,4
e~
E ~. 0,2 a
om
Time after infection (Days)
0 3
l
I
[
6
9
12
•
15
FIGURE 3 PI for mice infected with 0.1 LD50 influenza A virus and sampled at given times after inoculation. The results represent the mean +_ S.D. of 6 animals. * Differences between amphetamine and saline significant at p < 0.05. • Differences between controls and mice injected with saline significant at p < 0.05.
Vol.
52, No.
I0,
1993
Amphetamine
on I n f l u e n z a
Virus
Infection
PL-77
with untreated controls in one interval per assay. This latter effect may be due to stress induced by drug administration. The results presented here are in good agreement with previous reports on the adverse effects of CNS stimulant on virus infection. Amphetamine has been found to increase the incidence and decrease the latency of MTV-tumors in mice. There was also appreciated a correlation with the lethality of mice. Di Francesco et al. also found that both acute and subachronic treatment with cocaine significantly decreased the resistance to PR-8 infection (10). Research has proved the role of thymus-dependent immune response in influenza A virus infected mice. The development of T-cells with cytotoxic activity specific for influenza virus infected cells appears to be the critical factor in limiting the viral infection to the respiratory tract and in recovery (10). Wells wt al (11) found that transfer of a population of lymphocytes containing cytotoxic T-cell activity from BALB/c mice infected with influenza A virus 8 days previously results in an earlier and greater reduction in pulmonary virus titers in T-cell deficient recipient mice. Compared to immunocompetent controls, nude mice also showed a higher mortality following influenza A inoculation, and those mice which survived tended to have persistent viral presence in their lungs. By contrast, the onset of death following inoculation was somewhat delayed in the nude mice group. By contrast, experiments with cyclosporine A-treated mice are not fully in the line with our findings (12). This drug acts early during the process of T-cell activation by blocking the release of lymphokines, thereby preventing the formation of effector T-cells. Cyclosporine A, when administered to BALB/c mice with a moderate size inoculation of influenza A virus, substantially alters the immune response to the infection. Nevertheless, although infected mice treated for 21 days with cyclosporine had higher virus titers in their lungs than infected controls not exposed to the immunosuppressive agent, cyclosporine-induced suppressive effects of certain populations of T-cells seemed to be beneficial in reducing the pathogenesis of influenza A-virus pulmonary infection, as the hystopathology seen in the lungs was less severe. In our previous studies we observed adverse effects of amphetamine administration on the number and functional capacities of T-cells. Amphetamine also suppressed the activity of phagocytosis under in vivo and in vitro conditions. Since the mouse-adapted strain of influenza virus PR-8/34 affects several immune functions (10), we postulate that changes in the pathogenicity should be attributed to the amphetamineinduced immunosuppression. The mechanism of action of amphetamine on the immune system may be either direct or indirect. Although direct effects of amphetamine (at target cell) should not be excluded, one can hypothesize that the inhibition of the immune response 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 (13). Many of the effects of amphetamine involve the drug modulation of the adrenergic system and mimic stresslike states (14-18). Cellular immune activity is partially regulated by the adrenergic nervous system (19). A second point to be considered concerns the neuroendocrinological effects of amphetamine. The stimulatory effect of amphetamine on adrenocorticotropic hormone (ACTH) and adrenocorticoids should be involved. First, ACTH from the pituitary gland and even ir-ACTH from lymphocyte origin, has a direct inhibitory effect on functional capacities of immune cells. Second, the rise in plasma corticosterone concentrations, via ACTH secretion enhancement, suppresses various aspects of immune function (20). Our previous investigations show a stimulatory effect of chronic amphetamine on ACTH secretion, proportional to the decrease in the functional activities of spleen cells and 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. In conclussion, our data at present show that amphetamine, through known and unknown neuroendocrine pathways should injury the elements of the immunological apparatus, which may leave the subject vulnerable to the action of viruses.
PL-78
Amphetamine
on I n f l u e n z a V i r u s
Infection
Vol.
52, No.
i0,
1993
Acknowledgements We wish to express our gratitude to Ignacio Fern&ndez-Rial, Jos&A. Veira and Esperanza Cancio for their technical support and to Gelines Costoya and Pilar Fernandez-Vila for their bibliographic assistance. References 1. M. FREIRE-GARABAL, J.L. BALBOA, MJ. NUNEZ, M.T. CASTAI~IO, J.B. LLOVO, J.C. FERNANDEZ-RIAL and A. BELMONTE, Life Sci. 49(16) 107-112 (1991). 2. M. FREIRE-GARABAL, M.J. NUNEZ, J.L BALBOA, J.C. FERNANDEZ-RIAL and A. BELMONTE (in press). 3. M. FREIRE-GARABAL and M.J. NUIgEZ (in press). 4. G.L. ADA and P.D. JONES, Current topics in microbiology and immunology. G.L. Ada and P.D. Jones (eds), vol 128, 1-54, Springer, Heidelberg, Berlin (1986). 5. E. GARACI, A. MASTINO and C. FAVALLI, Bull. N.Y. Acad. Med. 6,5 111-119 (1989). 6. M. FREIRE-GARABAL, M.J. NU~IEZ, J.L. BALBOA, J.A. SUAREZ, A. GALLEGO and A. BELMONTE (in press). 7. E.H. LENNETT and N.J. SCHMIDT, Diagnostic Procedure for Viral, Rickettsial and Chamydial Infections, vol 5, American Public Health Association, Washington (1979). 8. A.M. PENNA, K.J. JOHNSON, J. CAMILLERI and P.R. KNIGHT, Intervirology 3_! 188-196 (1990). 9. 46. L.M. REED and H.A. Muench, Am. J. Hyg. 27 439-497 (1938). 10. P. DI FRANCESCO, F. PICA, C. CROCE, C. FAVALLI, E. TUBARO and E. GARACI, Nat. Immun. Cell Growth Regul. 9 397-405 (1990). 11. M.A. WELLS, F.A. ENNIS and P. ALBRETCH J. Immunot. 126 1042-1046 (1981). 12. E. SCHILTKNECHT and G.L. ADA, Cell Immunol. 9_~1227-239 (1985). 13. J.E. BLALOCK, Physiol. Rev. 6.._991-32 (1989). 14. I. GELLER and J. SEIFTER, Psychopharmacology 1 482-492 (1960). 15. S,M. ANTELMAN, A.J. EICHLER, C.A. BLACK and D. KOCAN, Science 207 329-331 (1980). 16. R.E. SU]-ION, G.F. KOOB, M LE MOAL, J. RIVlER and W. VALE, Nature 29__Z331-333 (1982). 17. K.T. BRITTON, J. MORGAN, J. RIVIER, W. VALE and G.F. KOOB, Psychopharmacology 8..66170-174 (1985). 18. K.T. BRITTON, G. LEE and G.F. KOOB, Psychopharmacology 94 306-311 (1988). 19. N. 8ELLUARDO, G. MUDO, V CARDILE, G. MIGLIORATI, C. RICCARDI, S. CELLA and M. 81NDONI, Cell Growth Regul 9 26-35 (1990). 20. V. RILEY, Science. 212 1100-1109 (1981).
52, pp.
PL 73-78
Pergamon
PHARMACOLOGY Accelerated
Press
LETTERS
Communication
EFFECTS OF AMPHETAMINE ON INFLUENZA VIRUS INFECTION IN MICE
MJ. N~6ez, J.C. Fern~ndez-Rial, J. Couceiro, J.A. Su~rez, D.E. G6mez-Fern~ndez, M. Rey-M~ndez and M. Freire-Garabal. Neuroinmunologfa-Universidadde Santiago (NIMUS), University of Santiago de Compostela. 15705-Santiago de Compostela. Spain. (Submitted September I0, 1992; accepted October 6, 1992; received in final form December 18, 1992)
Abstract Several experiments were conducted to evaluate the effects of chronic amphetamine on the infuenza A (PR-8/34) virus specific immune injury in CD-1 mice. Treatment with amphetamine resulted in a significant increase of lung virus titers and pulmonar vascular permeability. Amphetamine also increased the lethality of infected mice.
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 reduction of the number and functional capacities of T-cells (1,2), as well as in a suppression of the activity of phagocytosis (3). It is well known that deficiencies in natural and specific immune responses predispose the host to more severe virus infection (4,5). In this regard, in a previous report we observed that amphetamine decreased the resistance of mice against (MTV)induced tumors (6). To further elucidate this latter interaction, in the present paper we report the effects of amphetamine on the pathogenicity of the mouse-adapted strain of influenza virus PR-8/34 in mice. Methods Mice
Male 3-week-old, pathogen-free, CD-1 mice (Interfauna Ib~rica S.A., Barcelona, Spain) were used. They were housed, 7 days before experiments, four per cage in an aseptic and sound-proof chamber kept between 21°C and 22°C and maintained on an alternating 12-hr light/dark cycle. Sterilyzed food (Panlab Diet A.03) and water were given ad libitum. Procedure
Mice were randomly dealed in three groups according to the treatment they were submitted to: GROUP A. controls, GROUP B. saline, GROUP C. amphetamine. Virus
The mouse-adapted strain of influenza virus PR-8/34 was grown in the allantoic fluid of embryonated eggs after serial passage in mice to increase virulence. Influenza virus infectivity was assayed according to the procedures described by Lennett and Schmidt (7). Log dilutions of lung homogenates were added in a volume of 0.1 ml to the aliantoic sac of 10 day-old embryonated chicken eggs. The eggs were maintained at 35°C. After 48 h the allantoic fluid was harvested and tested at each dilution for the hemagglutination activity. The eggs were scored positive for infectivity at a given virus dilution when the allantoic fluid demonstrated positive hemagglutination of chicken erythrocytes (8). The microtiter procedure was used to determine the hemagglutination of influenza virus. The dose required to infect 50 % of the embryonated eggs (EID50) was determined by the method of Reed and Muench (9). Virus was inoculated intranasally 24 h after the beginning of drugs administration. Corresponding Author: ManuelFreire-Garabal. c/Hu~ffanas19.15703-Santiagode Compo~ela. SPAIN. 0024-3205/93 $6.00 + .00 Copyright
© 1993 Pergamon
Press Ltd
All rights
reserved.
PL-74
Amphetamine
on I n f l u e n z a
Virus
Infection
Vol.
52, No.
i0,
1993
Controls
100
Saine Amphetamine ILl L~
80
Z
Ll.I 0n," LU Q. -J
60
[]
l
© 40
N 20
1 HAU
0
0
"
l
J
t
I
I
I
I
I
2
4
6
8
I0
12
14
TIME
AFTER
INFECTION
A v
v
.__
J
16
(DAYS)
100
tu
8O
z
w
O tr LU O,. ,_1
60
w
D
¢'r m
©
211
5 HAU
~ e
I
I
I
I
I
I
2
4
6
8
10
12
I
14
16
FIGURE 1
Effect of amphetamine on the survival of PR8/34-infected mice. Twenty-four hours after the beginning of experiments, mice were inoculated i.n. with PR8 virus (1 HAU/mouse, A; 5 HAU/mouse, B). Survival curves represent the values of two experiments, p < 0,05 for the mortality ratios between saline and amphetamine.
Vol.
52, NO.
I0,
1993
Amphetamine
on I n f l u e n z a
Virus
Infection
PL-75
Lethality assay. Mice were inoculated i.n. with 1 HAU/mouse (20 mice/group) and 5 HAU/mouse (20 mice/group) PR-8/34 virus. Survival percentages were recorded daily during 16 days (10).
Virus titers The pathogenesis of influenza virus infection in mice was determined by quantitation of virus titers (8). Mice were inoculated intranasally with 0.1 LD50 of influenza A virus. After 12 and 24 h and 3, 6 and 9 days of infection, 6 mice from each group were sacrificed by cervical dislocation and immediately necropsied. Their lungs were removed under aseptic conditions, minced with scissors, weighed and stored at -70°C. A 20 % (w:vol) lung suspension was prepared by homogenizing lung tissue in Hank's balanced salt solution. This suspension was clarified by centrifugation at 500 x g for 10 min at 4°C. The infectious virus was assayed in embryonated eggs as previously described (8).
Calculation of alveolar capillary leak Permeability index (PI), that indicates increased pulmonary vascular permeability, was calculated to determine tissue injury (8). After 3, 6, 9, 12 and 15 days of inoculation with 0.1 LD50 influenza A virus, 1251labeled bovine serum albumin (100 I = 140,000 cpm) was injected into the tail vein of six mice from each experimental group. After 2 h, the mice were anesthetized with ketamine (150 mg/kg), 100 I of caval blood was withdrawn and the heart and lungs removed in block (8). The pulmonary vasculature was perfused with 2 ml saline via the right ventricle to remove the blood-associated radioactivity in the pulmonary system. Lung tissue and blood were counted in a gamma counter (LKB Wallac 1275 Gamma Counter). The ratio of radioactivity present in the lungs to that present in the animal's blood was expressed as the Pi.
Treatment with drugs 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 ml/kg 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 ml/kg of 0.9% saline solution. Drugs were daily administered at 09.30 a.m.
Statistical analysis Mortality ratios and mean times to death were analyzed with Fisher's exact test and the Mann-Whitney U test, respectively. Analysis of mean differences in virus titers and PI was performed according to the Student's t test. Significance was achieved at p < 0.05. Results Figure 1 shows the effect of amphetamine on the survival of mice infected with 1 HAU and 5 HAU of PR-8/34 virus. Amphetamine significantly increased the lethality to virus infection. This effect was very evident at 5 HAU/mouse. Calculation of mean time to death showed that significantly earlier times of death occurred among mice of the GROUP C (5.1 +- 0.2 days after infection) in comparison with mice of the GROUP B (6.9 _+0.5 days after infection). Differences berween gruops A and B were not significant. Virus titers in the lungs of mice sacrificed 0.5, 1 and 3 days after infection were greater in GROUP C in comparison with GROUP A (Figure 2). Differences between groups A and B were only significant on the 3rd day assay. As shown in figure 3 the PI was significantly greater in GROUP C mice in comparison with GROUP B mice. Differences between groups A and B were only significant on the 9th day assay. Discussion Our results show that amphetamine exacerbates influenza virus infection. Calculation of mean time to death indicated that significantly earlier times of death occurred amongst mice mice injected with amphetamine compared with mice injected with saline. Pathologic findings in this report show that influenza A virus titers in the lungs of mice were greater for mice injected with amphetamine in comparison with mice injected with saline. Furthermore, alveolar-capillary leakage was greater in this group. Treatment with saline resulted in higher virus titers and PI in comparison
PL-76
Amphetamine
on I n f l u e n z a
lO, O
Controls
10 e
Amp
Virus
Infection
Vol.
52, No.
i0,
1993
Saline
¢D
.= ¢n
10 e 10 7
. tm-
>
o a m LU
lO e 10 6
t/
lO 4 O
lO 3 Time after infection (Days)
u
10 2
0.5
1
3
6
9
FIGURE 2 Virus titers in lung tissue of mice infected with 0.1 LD50 influenza A virus and sampled at given times after inoculation. The results represent the mean -+ S.D. of 6 animals. * Differences between saline and amphetamine were significant at p < 0.05. • Differences between controls and mice injected with saline significant at p < 0.05.
0,8 Controls
----o-Amphetamine
O.
0,6
\\\\\\\
X e.-
~
0,4
e~
E ~. 0,2 a
om
Time after infection (Days)
0 3
l
I
[
6
9
12
•
15
FIGURE 3 PI for mice infected with 0.1 LD50 influenza A virus and sampled at given times after inoculation. The results represent the mean +_ S.D. of 6 animals. * Differences between amphetamine and saline significant at p < 0.05. • Differences between controls and mice injected with saline significant at p < 0.05.
Vol.
52, No.
I0,
1993
Amphetamine
on I n f l u e n z a
Virus
Infection
PL-77
with untreated controls in one interval per assay. This latter effect may be due to stress induced by drug administration. The results presented here are in good agreement with previous reports on the adverse effects of CNS stimulant on virus infection. Amphetamine has been found to increase the incidence and decrease the latency of MTV-tumors in mice. There was also appreciated a correlation with the lethality of mice. Di Francesco et al. also found that both acute and subachronic treatment with cocaine significantly decreased the resistance to PR-8 infection (10). Research has proved the role of thymus-dependent immune response in influenza A virus infected mice. The development of T-cells with cytotoxic activity specific for influenza virus infected cells appears to be the critical factor in limiting the viral infection to the respiratory tract and in recovery (10). Wells wt al (11) found that transfer of a population of lymphocytes containing cytotoxic T-cell activity from BALB/c mice infected with influenza A virus 8 days previously results in an earlier and greater reduction in pulmonary virus titers in T-cell deficient recipient mice. Compared to immunocompetent controls, nude mice also showed a higher mortality following influenza A inoculation, and those mice which survived tended to have persistent viral presence in their lungs. By contrast, the onset of death following inoculation was somewhat delayed in the nude mice group. By contrast, experiments with cyclosporine A-treated mice are not fully in the line with our findings (12). This drug acts early during the process of T-cell activation by blocking the release of lymphokines, thereby preventing the formation of effector T-cells. Cyclosporine A, when administered to BALB/c mice with a moderate size inoculation of influenza A virus, substantially alters the immune response to the infection. Nevertheless, although infected mice treated for 21 days with cyclosporine had higher virus titers in their lungs than infected controls not exposed to the immunosuppressive agent, cyclosporine-induced suppressive effects of certain populations of T-cells seemed to be beneficial in reducing the pathogenesis of influenza A-virus pulmonary infection, as the hystopathology seen in the lungs was less severe. In our previous studies we observed adverse effects of amphetamine administration on the number and functional capacities of T-cells. Amphetamine also suppressed the activity of phagocytosis under in vivo and in vitro conditions. Since the mouse-adapted strain of influenza virus PR-8/34 affects several immune functions (10), we postulate that changes in the pathogenicity should be attributed to the amphetamineinduced immunosuppression. The mechanism of action of amphetamine on the immune system may be either direct or indirect. Although direct effects of amphetamine (at target cell) should not be excluded, one can hypothesize that the inhibition of the immune response 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 (13). Many of the effects of amphetamine involve the drug modulation of the adrenergic system and mimic stresslike states (14-18). Cellular immune activity is partially regulated by the adrenergic nervous system (19). A second point to be considered concerns the neuroendocrinological effects of amphetamine. The stimulatory effect of amphetamine on adrenocorticotropic hormone (ACTH) and adrenocorticoids should be involved. First, ACTH from the pituitary gland and even ir-ACTH from lymphocyte origin, has a direct inhibitory effect on functional capacities of immune cells. Second, the rise in plasma corticosterone concentrations, via ACTH secretion enhancement, suppresses various aspects of immune function (20). Our previous investigations show a stimulatory effect of chronic amphetamine on ACTH secretion, proportional to the decrease in the functional activities of spleen cells and 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. In conclussion, our data at present show that amphetamine, through known and unknown neuroendocrine pathways should injury the elements of the immunological apparatus, which may leave the subject vulnerable to the action of viruses.
PL-78
Amphetamine
on I n f l u e n z a V i r u s
Infection
Vol.
52, No.
i0,
1993
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