Physiology & Behavior,Vol. 51, pp. 349-352. ©Pergamon Press plc, 1992. Printed in the U.S.A.
0031-9384/92 $5.00 + .00
Prostaglandins Do Not Mediate Interferon-oL Effects on Mouse Behavior L I N D A S. C R N I C 1 A N D M A R K A. S E G A L L
Departments o f Pediatrics and Psychiatry, University o f Colorado School o f Medicine, Denver, CO 80262 R e c e i v e d 8 April 1991 CRNIC, L. S. AND M. A. SEGALL. Prostaglandins do not mediate interferon-or effects on mouse behavior. PHYSIOL BEHAV 51(2) 349-352, 1992.--Interferons play an important role in cell function as well as host defense against virus infection. Interferon-ct stimulates cyclooxygenase-catalyzed prostaglandin synthesis. Therefore, a cyclooxygenase inhibitor was used to determine whether the behavioral effects of mouse interferon-c~ in mice are mediated by increased prostaglandin synthesis. Male DBA/2J mice were exposed to 1600 U/g mouse interferon-a, the buffer in which the interferon was diluted, 5 mg/kg of a cyclooxygenase inhibitor, indomethacin, or 5 mg/kg indomethacin plus 1600 U/g mouse interferon-et. As in previous studies, interferon decreased activity and food pellets obtained in the first 4 hours after administration. Indomethacin had no effect on this effect of interferon; therefore, we conclude that the behavioral effects of interferon are not mediated by the fever or anoretic effects of prostaglandins. Interferon alpha
Prostaglandin
DBA mouse
Indomethacin
INTERFERON-as are soluble proteins produced by all cells in response to virus infection and other stimuli (16). While named for their antiviral effects, interferon-as have a variety of effects upon cell differentiation and function (16). When used for treatment of cancer, the dose-limiting effects of interferons are often the neuropsychological effects [e.g., (1)]. Using an animal model, decreases in activity, food acquisition and water intake were noted in the first several hours after administration of mouse interferon-et to DBA/2J mice (17). The decrease in food acquisition and water intake did not seem to be due to nausea produced by interferon-a, as interferon-a could not be used to support a conditioned taste aversion (18). Only small amounts of interferon enter the brain when injected peripherally [e.g., (2)]. While the small amounts of interferon that enter the brain may be active there, it is important to determine whether substances released due to interferon treatment might account for its behavioral effects. Interferons stimulate prostaglandin formation (7). Prostaglandins induced by interferon have been shown to be responsible for the fever induced by interferon (6) and are well known to produce anorexia [e.g., (14)]. Thus, the possibility that prostaglandins are responsible for the behavioral effects of interferon administration was tested using the cyclooxygenase inhibitor indomethacin to abolish prostaglandin synthesis.
Activity
Food intake
extreme in response to interferon. Mice were housed individually with ad lib access to food and water. The light schedule was 12 hours on, 12 hours off, with lights off at 1900 hours.
Interferon Mouse interferon-a (Cat. 22061, lots 87077 and 85092) was obtained from Lee BioMolecular (San Diego, CA) and diluted in a glycine buffer, pH 3.5. A dose of 1600 U/g was used as it had been shown in previous studies to significantly suppress activity and food and water acquisition (17). This dose is in the range of tissue levels of interferon found in virus-infected mice (11).
Indomethacin Indomethacin (Sigma, St. Louis) was diluted in sterile normal saline. Pilot trials demonstrated that 1 and 5 mg/kg indomethacin had no effect upon the variables to be measured, while 10 mg/kg suppressed food intake. As 5 mg/kg has been shown to produce reliable behavioral effects in this and other strains of mice (4,8) and did not affect food intake in the pilot trials, this dose was used. This dose of indomethacin is in the range of those shown to increase NK activity in mice (23) and higher than that needed to antagonize IL-1 effects (3 mg/kg in rats) (22). Most relevant, 2.5 mg/kg in rats attenuate the effects of endotoxin on anorexia (13). Indomethacin has been shown to be effective by 15 minutes after injection (4,22). In the present study, it was administered at the same time as interferon-or. This was done because the behavioral effects of interferon take 1-2 hours to become evident and to avoid multiple disruptions of the mice for injections. All drugs were administered IP in a volume of 0.2 ml.
METHOD
Subjects Fifty DBA/2J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were 3-5 months old at the time of testing. This strain of mouse was chosen because it is intermediate in the production of interferon (19) and, thus, not likely to be
~Requests for reprints should be addressed to Linda S. Crnic, Ph.D., Box C233, 4200 E. 9th Ave., Denver, CO 80262.
349
350
CRNIC ANI) 5EG&I.i
Procedure
7~00
During the experiment, mice were continually housed in chambers in which horizontal activity, food pellet acquisition and water intake were measured 24 hours a day as previously described (17). An activity monitor (Omnitech, Columbus, OH) was used to measure activity, and a photobeam system was used to deliver food pellets (20 mg; Noyes, Lancaster, NH) 15 seconds after a head poke into a food chamber. On the first day of testing, mice were weighed, injected IP with a glycine buffer and placed into the chamber at 1500 hours. At the same time on the next day, they were removed, weighed and injected with either: glycine buffer (n = 15); 1600 U/g mouse interferon-c~ (n = 14); 5 mg/kg indomethacin ( n = 8); 5 mg/kg indomethacin plus 1600 U/g mouse interferon-a (n = 13). They were returned to the chamber and behavior recorded for the next 23 hours. This testing procedure differs from our previous study (16) in that mice were allowed only one day rather than one week to accommodate to the testing chamber before drug exposure. We have found that this decreases the sensitivity of the measures to effects of interferon but still allows detection of reliable effects. Dependent variables recorded every half hour, 23 hours per day, were graphed and showed that the pattern of effects was similar to previous studies, namely, the maximal effects of interferon occurred in the first several hours post injection (Fig. 1). Therefore, data were summed over the first 4 hours postinjection and analyzed by two-way analysis of variance with planned contrasts.
................................................................................................................ i:
:>- 6 0 0 0
,
t /' ~ " //~
\XXN
o I
2-
\\ \\ \\ N \ i
4000.
\\\\i
uffer
Indometh
1
Interferon
o
Interferon
Buffer
&
Indometh
Indometh
Interferon Interferon & Indometh
FIG. 3. Total water intake during the 23 hour test period. 80.
p=.08
p=.91
indomethacin to potentiate rather than alleviate the effects of interferon-or in the present study. The decreases in activity and food-related behavior after interferon-or administration must be mediated by prostaglandin-independent effects of interferon. Potential candidates for alternate pathways of interferon-or effects on behavior, in addition to other metabolites of arachidonic acid, include opiate systems [e.g., (5,12)1. The data in this paper provide evidence that the anoretic effects of interferon differ from those of another cytokine, interleukin-1 (IL-1), and endotoxin which appear to be mediated by prostaglandins, and are ameliorated by cyclooxygenase inhibitors (10,13). Interferon also differs from IL-1 in that the latter can support a conditioned taste aversion (20) while IFN does not (18).
p-.43 u~
60.
+
"5 40. E
D Z
20
0
Buffer
Indometh
Interferon Interferon
&
Indometh
FIG. 2. Mean and standard deviations of horizontal activity and food pellets delivered summed over the four hours immediately following interferon administration. Indometh = indomethacin.
arachidonic acid from the cyclooxygenase to the epoxygenase pathway (15). This might account for the trend observed for the
ACKNOWLEDGEMENTS This work was supported by grants MH44453 and MH00621 to L. Cmic and MH09718 to M. Segall from the National Institute of Mental Health and by the Developmental Psychobiology Endowment Fund of the Department of Psychiatry of the University of Colorado School of Medicine.
REFERENCES
1. Adams, F.; Quesada, J. R.; Gutterman, J. U. Neuropsychiatilc manifestations of human leukocyte interferon therapy in patients with cancer. JAMA 252:938-941; 1984. 2. Billiau, A.; Heremans, H.; Ververken, D.; Van Damme, J.; Carton, H.; De Somer, P. Tissue distribution of human interferons after exogenous administration in rabbits, monkeys, and mice. Arch. Virol. 68:19-25, 1981. 3. Capdevila, J. H.; Falck, J. R. Cytochrome P-450 and the bioactivation of arachidonic acid. Blood Vessels 26:54-57; 1989. 4. Collins, A. C.; Gilliam, D. M.; Miner, L. L. Indomethacin pretreatment blocks the effects of high concentrations of ethanol. Alcohol.: Clin. Exp. Res. 9:371-376; 1985. 5. Dafny, N.; Lee, J. R.; Dougherty, P. M. Immune response products alter CNS activity: Interferon modulates central opioid functions. J. Neurosci. Res. 19:130-139; 1988. 6. Dinarello, C. A.; Bernheim, H. A.; Duff, G. W.; Le, H. V.; Nagabhushan, T. L.; Hamilton; N. C.; Coceani, F. Mechanisms of fever induced by recombinant human interferon. J. Clin. Invest. 74: 906-913; 1984. 7. Fitzpatrick, F. A.; Stringfellow, D. A. Virus and interferon effects on cellular prostaglandin biosynthesis. J. Immunol. 125:431-437; 1980. 8. George, F. R.; Collins, A. C. Prostaglandin synthetase inhibitors antagonize the depressant effects of ethanol. Pharmacol. Biochem.
Behav. 10:865-869; 1979. 9. Hannigan, G. E.; Williams, B. R. G. Signal transduction by interferon-o~ through arachidonic acid metabolism. Science 251:204-207; 1991. 10. Hellerstein, M. K.; Meydani, S. N.; Meydani, M.; Wu, K.; Dinarello, C. A. Interleukin-l-induced anorexia in the rat. J. Clin. Invest. 84: 228-235; 1989. 11. Heremans, H.; Billiau, A.; De Somer, P. Interferon in experimental viral infections in mice: Tissue interferon levels resulting from the virus infection and from exogenous interferon therapy. Infect. Immun. 30:513-522; 1980. 12. Kuriyama, K.; Hoil, T.; Moil, T.; Nakashima, T. Actions of interferon-a and interlenkin-lB on the glucose-responsive neurons in the ventromedial hypothalamus. Brain Res. Bull. 24:803-810; 1990. 13. Langhans, W.; Harlacher, R.; Scharrer, E. Verapamil and indomethacin attenuate endotoxin-induced anorexia. Physiol. Behav. 46: 535-539; 1989. 14. Levine, A. S.; Morley, J. E. The effect of prostaglandins (PGE2 and PGF2a) on food intake in rats. Pharmacol. Biochem. Behav. 15:735-738; 1981. 15. McGiff, J. C. Cytochrome P-450 metabolism of arachidonic acid. Annu. Rev. Pharmacol. 31:339-369; 1991. 16. Mannering, G. J.; Deloria, L. B. The pharmacology and toxicology of the interferons: An overview. Annu. Rev. Pharmacol. Toxicol.
352
26:455-515; 1986. 17. Segall, M. A.; Crnic, L. S. An animal model for the behavioral effects of interferon. Behav. Neurosci. 104:612--618; 1990. 18. Segall, M. A.; Crnic, L. S. A test of conditioned taste aversion with mouse interferon-a. Brain Behav. Immun. 4:223-231: 1990. 19. Shirahata, T.; Mori, A.; Ishikawa, H.; Goto, H. Strain differences of interferon-generating capacity and resistance in toxoplasma-infected mice. Microbiol. Immunol. 30:1307-1316; 1986. 20. Tazi, A.; Dantzer, R.; Crestiani, F.; Le Moal, M. Interleukin-1 induces conditioned taste aversion in rats: A possible explanation for its pituitary-adrenal stimulating activity. Brain Res. 473:369-
C R N I C A N [ ) S E G A I 1.
371; t988. 21. Tovey, M. G.; Gresser, l ; Rochette-Egly, C ; Begon-[ oup,-Gu.~marho, J.; Bandu, M.-T.: Man U, C. lndomethacin and aspirin do not inhibit the antiviral or antiproliferative actions of interfero~ ! Gen. Virol. 63:505-508; 1982. 22. Uehara, A.; Okumura. T.: Kumei, Y.: Takasugi, ~.: Namiki, M. Indomethacin reverses interleukin-I hyperinsulinemia in conscious and freely moving rats. Eur. J. Pharmacol. 192:185-187; lt)cJ!. 23. Voth, R.; Chmielarczyk, W.; Storch, E.; Kirchner, H. Induction of natural killer celt activity in mice by injection of indomethacin. Nat. Immun. Cell Growth Regul. 5:317-324: 1986.
0031-9384/92 $5.00 + .00
Prostaglandins Do Not Mediate Interferon-oL Effects on Mouse Behavior L I N D A S. C R N I C 1 A N D M A R K A. S E G A L L
Departments o f Pediatrics and Psychiatry, University o f Colorado School o f Medicine, Denver, CO 80262 R e c e i v e d 8 April 1991 CRNIC, L. S. AND M. A. SEGALL. Prostaglandins do not mediate interferon-or effects on mouse behavior. PHYSIOL BEHAV 51(2) 349-352, 1992.--Interferons play an important role in cell function as well as host defense against virus infection. Interferon-ct stimulates cyclooxygenase-catalyzed prostaglandin synthesis. Therefore, a cyclooxygenase inhibitor was used to determine whether the behavioral effects of mouse interferon-c~ in mice are mediated by increased prostaglandin synthesis. Male DBA/2J mice were exposed to 1600 U/g mouse interferon-a, the buffer in which the interferon was diluted, 5 mg/kg of a cyclooxygenase inhibitor, indomethacin, or 5 mg/kg indomethacin plus 1600 U/g mouse interferon-et. As in previous studies, interferon decreased activity and food pellets obtained in the first 4 hours after administration. Indomethacin had no effect on this effect of interferon; therefore, we conclude that the behavioral effects of interferon are not mediated by the fever or anoretic effects of prostaglandins. Interferon alpha
Prostaglandin
DBA mouse
Indomethacin
INTERFERON-as are soluble proteins produced by all cells in response to virus infection and other stimuli (16). While named for their antiviral effects, interferon-as have a variety of effects upon cell differentiation and function (16). When used for treatment of cancer, the dose-limiting effects of interferons are often the neuropsychological effects [e.g., (1)]. Using an animal model, decreases in activity, food acquisition and water intake were noted in the first several hours after administration of mouse interferon-et to DBA/2J mice (17). The decrease in food acquisition and water intake did not seem to be due to nausea produced by interferon-a, as interferon-a could not be used to support a conditioned taste aversion (18). Only small amounts of interferon enter the brain when injected peripherally [e.g., (2)]. While the small amounts of interferon that enter the brain may be active there, it is important to determine whether substances released due to interferon treatment might account for its behavioral effects. Interferons stimulate prostaglandin formation (7). Prostaglandins induced by interferon have been shown to be responsible for the fever induced by interferon (6) and are well known to produce anorexia [e.g., (14)]. Thus, the possibility that prostaglandins are responsible for the behavioral effects of interferon administration was tested using the cyclooxygenase inhibitor indomethacin to abolish prostaglandin synthesis.
Activity
Food intake
extreme in response to interferon. Mice were housed individually with ad lib access to food and water. The light schedule was 12 hours on, 12 hours off, with lights off at 1900 hours.
Interferon Mouse interferon-a (Cat. 22061, lots 87077 and 85092) was obtained from Lee BioMolecular (San Diego, CA) and diluted in a glycine buffer, pH 3.5. A dose of 1600 U/g was used as it had been shown in previous studies to significantly suppress activity and food and water acquisition (17). This dose is in the range of tissue levels of interferon found in virus-infected mice (11).
Indomethacin Indomethacin (Sigma, St. Louis) was diluted in sterile normal saline. Pilot trials demonstrated that 1 and 5 mg/kg indomethacin had no effect upon the variables to be measured, while 10 mg/kg suppressed food intake. As 5 mg/kg has been shown to produce reliable behavioral effects in this and other strains of mice (4,8) and did not affect food intake in the pilot trials, this dose was used. This dose of indomethacin is in the range of those shown to increase NK activity in mice (23) and higher than that needed to antagonize IL-1 effects (3 mg/kg in rats) (22). Most relevant, 2.5 mg/kg in rats attenuate the effects of endotoxin on anorexia (13). Indomethacin has been shown to be effective by 15 minutes after injection (4,22). In the present study, it was administered at the same time as interferon-or. This was done because the behavioral effects of interferon take 1-2 hours to become evident and to avoid multiple disruptions of the mice for injections. All drugs were administered IP in a volume of 0.2 ml.
METHOD
Subjects Fifty DBA/2J mice were obtained from Jackson Laboratories (Bar Harbor, ME) and were 3-5 months old at the time of testing. This strain of mouse was chosen because it is intermediate in the production of interferon (19) and, thus, not likely to be
~Requests for reprints should be addressed to Linda S. Crnic, Ph.D., Box C233, 4200 E. 9th Ave., Denver, CO 80262.
349
350
CRNIC ANI) 5EG&I.i
Procedure
7~00
During the experiment, mice were continually housed in chambers in which horizontal activity, food pellet acquisition and water intake were measured 24 hours a day as previously described (17). An activity monitor (Omnitech, Columbus, OH) was used to measure activity, and a photobeam system was used to deliver food pellets (20 mg; Noyes, Lancaster, NH) 15 seconds after a head poke into a food chamber. On the first day of testing, mice were weighed, injected IP with a glycine buffer and placed into the chamber at 1500 hours. At the same time on the next day, they were removed, weighed and injected with either: glycine buffer (n = 15); 1600 U/g mouse interferon-c~ (n = 14); 5 mg/kg indomethacin ( n = 8); 5 mg/kg indomethacin plus 1600 U/g mouse interferon-a (n = 13). They were returned to the chamber and behavior recorded for the next 23 hours. This testing procedure differs from our previous study (16) in that mice were allowed only one day rather than one week to accommodate to the testing chamber before drug exposure. We have found that this decreases the sensitivity of the measures to effects of interferon but still allows detection of reliable effects. Dependent variables recorded every half hour, 23 hours per day, were graphed and showed that the pattern of effects was similar to previous studies, namely, the maximal effects of interferon occurred in the first several hours post injection (Fig. 1). Therefore, data were summed over the first 4 hours postinjection and analyzed by two-way analysis of variance with planned contrasts.
................................................................................................................ i:
:>- 6 0 0 0
,
t /' ~ " //~
\XXN
o I
2-
\\ \\ \\ N \ i
4000.
\\\\i
uffer
Indometh
1
Interferon
o
Interferon
Buffer
&
Indometh
Indometh
Interferon Interferon & Indometh
FIG. 3. Total water intake during the 23 hour test period. 80.
p=.08
p=.91
indomethacin to potentiate rather than alleviate the effects of interferon-or in the present study. The decreases in activity and food-related behavior after interferon-or administration must be mediated by prostaglandin-independent effects of interferon. Potential candidates for alternate pathways of interferon-or effects on behavior, in addition to other metabolites of arachidonic acid, include opiate systems [e.g., (5,12)1. The data in this paper provide evidence that the anoretic effects of interferon differ from those of another cytokine, interleukin-1 (IL-1), and endotoxin which appear to be mediated by prostaglandins, and are ameliorated by cyclooxygenase inhibitors (10,13). Interferon also differs from IL-1 in that the latter can support a conditioned taste aversion (20) while IFN does not (18).
p-.43 u~
60.
+
"5 40. E
D Z
20
0
Buffer
Indometh
Interferon Interferon
&
Indometh
FIG. 2. Mean and standard deviations of horizontal activity and food pellets delivered summed over the four hours immediately following interferon administration. Indometh = indomethacin.
arachidonic acid from the cyclooxygenase to the epoxygenase pathway (15). This might account for the trend observed for the
ACKNOWLEDGEMENTS This work was supported by grants MH44453 and MH00621 to L. Cmic and MH09718 to M. Segall from the National Institute of Mental Health and by the Developmental Psychobiology Endowment Fund of the Department of Psychiatry of the University of Colorado School of Medicine.
REFERENCES
1. Adams, F.; Quesada, J. R.; Gutterman, J. U. Neuropsychiatilc manifestations of human leukocyte interferon therapy in patients with cancer. JAMA 252:938-941; 1984. 2. Billiau, A.; Heremans, H.; Ververken, D.; Van Damme, J.; Carton, H.; De Somer, P. Tissue distribution of human interferons after exogenous administration in rabbits, monkeys, and mice. Arch. Virol. 68:19-25, 1981. 3. Capdevila, J. H.; Falck, J. R. Cytochrome P-450 and the bioactivation of arachidonic acid. Blood Vessels 26:54-57; 1989. 4. Collins, A. C.; Gilliam, D. M.; Miner, L. L. Indomethacin pretreatment blocks the effects of high concentrations of ethanol. Alcohol.: Clin. Exp. Res. 9:371-376; 1985. 5. Dafny, N.; Lee, J. R.; Dougherty, P. M. Immune response products alter CNS activity: Interferon modulates central opioid functions. J. Neurosci. Res. 19:130-139; 1988. 6. Dinarello, C. A.; Bernheim, H. A.; Duff, G. W.; Le, H. V.; Nagabhushan, T. L.; Hamilton; N. C.; Coceani, F. Mechanisms of fever induced by recombinant human interferon. J. Clin. Invest. 74: 906-913; 1984. 7. Fitzpatrick, F. A.; Stringfellow, D. A. Virus and interferon effects on cellular prostaglandin biosynthesis. J. Immunol. 125:431-437; 1980. 8. George, F. R.; Collins, A. C. Prostaglandin synthetase inhibitors antagonize the depressant effects of ethanol. Pharmacol. Biochem.
Behav. 10:865-869; 1979. 9. Hannigan, G. E.; Williams, B. R. G. Signal transduction by interferon-o~ through arachidonic acid metabolism. Science 251:204-207; 1991. 10. Hellerstein, M. K.; Meydani, S. N.; Meydani, M.; Wu, K.; Dinarello, C. A. Interleukin-l-induced anorexia in the rat. J. Clin. Invest. 84: 228-235; 1989. 11. Heremans, H.; Billiau, A.; De Somer, P. Interferon in experimental viral infections in mice: Tissue interferon levels resulting from the virus infection and from exogenous interferon therapy. Infect. Immun. 30:513-522; 1980. 12. Kuriyama, K.; Hoil, T.; Moil, T.; Nakashima, T. Actions of interferon-a and interlenkin-lB on the glucose-responsive neurons in the ventromedial hypothalamus. Brain Res. Bull. 24:803-810; 1990. 13. Langhans, W.; Harlacher, R.; Scharrer, E. Verapamil and indomethacin attenuate endotoxin-induced anorexia. Physiol. Behav. 46: 535-539; 1989. 14. Levine, A. S.; Morley, J. E. The effect of prostaglandins (PGE2 and PGF2a) on food intake in rats. Pharmacol. Biochem. Behav. 15:735-738; 1981. 15. McGiff, J. C. Cytochrome P-450 metabolism of arachidonic acid. Annu. Rev. Pharmacol. 31:339-369; 1991. 16. Mannering, G. J.; Deloria, L. B. The pharmacology and toxicology of the interferons: An overview. Annu. Rev. Pharmacol. Toxicol.
352
26:455-515; 1986. 17. Segall, M. A.; Crnic, L. S. An animal model for the behavioral effects of interferon. Behav. Neurosci. 104:612--618; 1990. 18. Segall, M. A.; Crnic, L. S. A test of conditioned taste aversion with mouse interferon-a. Brain Behav. Immun. 4:223-231: 1990. 19. Shirahata, T.; Mori, A.; Ishikawa, H.; Goto, H. Strain differences of interferon-generating capacity and resistance in toxoplasma-infected mice. Microbiol. Immunol. 30:1307-1316; 1986. 20. Tazi, A.; Dantzer, R.; Crestiani, F.; Le Moal, M. Interleukin-1 induces conditioned taste aversion in rats: A possible explanation for its pituitary-adrenal stimulating activity. Brain Res. 473:369-
C R N I C A N [ ) S E G A I 1.
371; t988. 21. Tovey, M. G.; Gresser, l ; Rochette-Egly, C ; Begon-[ oup,-Gu.~marho, J.; Bandu, M.-T.: Man U, C. lndomethacin and aspirin do not inhibit the antiviral or antiproliferative actions of interfero~ ! Gen. Virol. 63:505-508; 1982. 22. Uehara, A.; Okumura. T.: Kumei, Y.: Takasugi, ~.: Namiki, M. Indomethacin reverses interleukin-I hyperinsulinemia in conscious and freely moving rats. Eur. J. Pharmacol. 192:185-187; lt)cJ!. 23. Voth, R.; Chmielarczyk, W.; Storch, E.; Kirchner, H. Induction of natural killer celt activity in mice by injection of indomethacin. Nat. Immun. Cell Growth Regul. 5:317-324: 1986.
