Neuroleptic and anti-depressant drug treatment abolishes conditioned immunosuppression in mice

BRAIN,

BEHAVIOR,

AND

3, 3 12-319 (1989)

IMMUNITY

Neuroleptic and Anti-depressant Drug Treatment Abolishes Conditioned lmmunosuppression in Mice’ REGINALD Departments

of

M. GORCZYNSKI

Surgery and Immunology, Room 853, 600 University

AND WENDY

Uni\jersity of Toronto, Ave., Toronto, MSGIXS

HOLMES

c/o Mt. Sinai Research Ontario, Canada

Institute,

Mice previously exposed to cyclophosphamide in the presence of saccharin-flavored water will show a decreased antibody response to challenge with sheep erythrocytes if simultaneously they are again given saccharin to drink. These mice also show conditioned taste aversion. Treatment of conditioned animals with chlorpromazine or amitriptyline after challenge with erythrocytes in the presence of saccharin reduced the degree of immunosuppression and, though to a lesser degree, the conditioned taste aversion. 0 1989 Academic Press. Inc.

INTRODUCTION

A number of independent investigators have established convincingly that a variety of immune responses can be subjected to (apparently) nonimmunological control, using the techniques of associative learning (Ader & Cohen, 1975, 1985; Gorczynski & Kennedy, 1984; Bovbjerg, Ader, & Cohen, 1982; Gorczynski, Kennedy, & Ciampi, 1985). In perhaps the most well-developed model system for such analysis, mice or rats previously exposed to saccharin-flavored drinking water (Sacc) in the context of intraperitoneal injection with cyclophosphamide (Cy: a known immunosuppressive drug) subsequently have a decreased antibody response on challenge with sheep erythrocytes (SE) if they are also reexposed to saccharin-flavored water (Ader & Cohen, 1975; Gorczynski, 1987a). Like other classical conditioning phenomena these responses can be extinguished by repeated nonreinforced exposure to conditioning cues alone (Gorczynski & Kennedy, 1984; Gorczynski, 1987a). The mechanism by which these conditioned immunological changes occur is not well understood. Lymphoid tissue is innervated by /3-adrenergic and cholinergic neurones (Felten, Overhage, Felten, & Schemdtje, 1981; Bulloch & Pomerantz, 1984). Lymphocytes have been shown to possess receptors for neurotransmitter or neuroendocrine substances, which in turn can modify immune responses in vivo and in vitro (Kusnecov, Husband, King, Pang, & Smith, 1987; Johnson, Torres, Smith, Dion, & Blalock, 1984). Changes in neurotransmitters within the CNS have also been documented after peripheral immunization (Besodovsky, de1 Rey, & Sorkin, 1983; Carlson, Felten, Livnat, & Felten, 1987). The immunological effects seen following CNS lesioning (Cross, Markesberry, Brooks, & Roszman, 1980), along with measured changes in neuronal firing following immuniza-

’ Supported by the MRC of Canada (Grant MA-5440) 312 0889.1591/89 $3.00 Copyright All rights

0 1989 by Academic Press. Inc. of reproduction in any form reserved.

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tion (Besedovsky, Felix, & Haas, 1977), further imply that important functional CNS-immune system interactions can occur in a bidirectional manner. In previous studies of classical conditioning of immune responses reciprocal adoptive transfer of cells from normal or conditioned mice into normal or conditioned irradiated hosts was used to document an important role for both cells and host environment in the development of conditioned immunity (Gorczynski, 1987a, b). In this report we examined the ability of psychoactive drugs to modify the expression of conditioned responses as another approach to an analysis of the the mechanism(s) behind conditioned immunoregulation. MATERIALS

AND METHODS

Mice. BALBK mice were purchased from the Jackson Laboratories, Bar Harbor, Maine. Mice were housed five per cage and allowed food and water ad libitum (except when on a restricted water schedule-see text). Cell preparution. Spleen cells were prepared aseptically in phosphate-buffered saline (PBS) containing 0.3% bovine serum albumin (BSA) as described elsewhere (Gorczynski & Cunningham, 1978). Sheep erythrocytes (SE) were obtained every 2 weeks from Woodland Farms, Guelph, and washed three times in PBS prior to use. When cells were cultured in Marbrook vessels for in vitro antibody formation 10 x IO6 spleen cells were cultured with 20 x lo6 SE in 1 ml in the inner vessel of a Marbrook chamber with 10 ml of feeder medium in the outer chamber. The medium used was a-Minimal Essential Medium supplemented with 10% heatactivated fetal calf serum (cr-FlO). Antibody-forming-cells (AFC) were enumerated using modified Cunningham chambers (Gorczynski & Cunningham, 1978) at Day 5 of culture. Where drugs were added to in vitro cultures the drug was included in the concentrations shown (Table 2) in both inner and outer vessels of the Marbrook chamber. Conditioning of mice. Mice were maintained on a daily watering schedule in which water was available for only a 30-min period (OS:OO-OS:30 AM). After 10 days animals were exposed once to 0.1% Sacc (conditioned stimulus, CS) in their drinking water and immediately thereafter received an injection of Cy (100 mg/kg) in 0.5 ml PBS given intraperitoneally (unconditioned stimulus, US). Reexposure to Cy and Sacc (CS + US) was repeated on two further occasions at 21-day intervals. Between such exposures all animals were given water only: mean daily consumption was 2-3 ml/mouse. Animals were challenged with SE (5 x 10s i.p.) 21 days after the last exposure to CS + US and were sacrificed for measurement of IgG-AFC 7 days later. In some experiments some groups of mice also began to receive different psychoactive drugs in their water supply 7 days after the last CS + US treatment. The drug concentration (diazepam, 2 kg/ml; amitriptyline, 20 kg/ml; chlorpromazine, 70 kg/ml) was chosen to represent a known therapeutic daily dosage for man (15 mg, 150 mg, and 500 mg, respectively) assuming a daily fluid consumption of 2 ml per mouse. The range of drug exposure for mice in these groups was (human equivalents) 10-23 mg, 100-230 mg, and 350-750 mg, respectively. Control studies indicated that no groups (conditioned or nonconditioned) showed any avoidance behavior or preference for the drug solution relative to water.

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When taste aversion (for Sacc-flavored water) was assessed, a preference test was used. Animals were offered plain water or plain Sacc (control groups) or water with drug and Sacc or Sacc with drug at the same time (in separate bottles) and preference (for Sacc) calculated as: (Sacc-flavored

water consumed - water consumed) (total fluid intake)

This ranges from + 1 (total Sacc preference) to - 1 (total taste aversion). Statistical analyses. Differences in antibody responses between groups were initially assessed by one-way analysis of variance (ANOVA). Thereafter, pairwise comparison of groups using a nonparametric test (Wilcoxon Rank Sum) was used (individual AFC responses within any group follow a log-normal distribution). RESULTS

Effect of psychoactive drugs on conditioned immunosappression of antibody responses in vivo. As reported elsewhere, mice receiving treatment trials of Sacc-

flavored water in association with the immunosuppressive drug cyclophosphamide, show a diminished antibody response after challenge with SE if they are also reexposed to Sacc (Ader & Cohen, 1975; Gorczynski, 1987a). Apparently Sacc elicits some of the immunosuppressive properties of Cy. Control studies show that these effects are not seen if Sacc and Cy are not given (during the conditioning trials) in associative form, and that the conditioned immunosuppression can be extinguished by repeated reexposure to Sacc only (nonreinforced cues; Gorczynski, 1987a). Typical data for groups of nonconditioned (Cy only) and conditioned mice challenged with SE and given either Sacc or water to drink are shown in rows 1, 2 and 9, 10 of Table 1 (one of two studies of this type). It is clear that the conditioned mice show a marked taste aversion to Sacc-flavored water (-0.37 + 0.24) as well as reduced IgG-AFC responses (7430 vs 16,900 [conditioned not exposed to Sacc]; 7430 vs 20,240 [nonconditioned exposed to Sacc]) . If conditioned mice were treated for the last 21 days before sacrifice with a variety of psychoactive drugs, quite different results were obtained. As noted under Materials and Methods, the concentration of drug used in the drinking supply was that calculated to represent a therapeutic dose for man and had no detectable effect on fluid intake. Taste aversion was reduced in all drug-treatment groups (compare rows 9, 10 with 11, 12; 13, 14; 15, 16; p < .05). However, preference for Sacc was still not as pronounced as in the NCS controls (compare rows 3-8 with rows 11-16; .02 < p < .05). We are unaware of any published data which has addressed this issue of the effect of such drugs on the expression of taste-aversion behavior in mice. Conditioned mice started on diazepam prior to exposure to SE (with/without Sacc) still demonstrated conditioned immunosuppression of antibody responses. In contrast, treatment with amitriptyline or chlorpromazine abolished conditioned immunosuppression in experimental mice (rows 9, 10 vs rows 13-16). Noncondi-

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TABLE 1 in Mice Given Psychoactive Drugs Post-Conditioning .~-

Drug treatment Non-conditioned None Diazepam (2 t&ml) Amitriptyline (20w&4 Chlorpromazine (70 dml) Conditioned Mice None

Cues”

Taste aversionb (Sacc preference)

IgG-AFCispleen (range)

_._-

Row

Mice H,O SACC W SACC H,O SACC W’ SACC

0.63 2 0.26 0.71 ? 0.31 0.69 i 0.28 0.75 k 0.22

18360 20240 18350 19620 21200 19650 20100 19210

( 13670-24830) ( 14760-28000) (12090-28000) ( 13770-27720) (14760-30330) ( 13630-28670) (14760-27450) (13360-30330)

I 2 3 4 5 6 7 8

16900 (12460-23160) 9 W SACC -0.37 t 0.24 7430 (44%12330)** 10 Diazepam H,O 18130 (13230-25080) 11 0.09 k 0.17 8750 (4960-15520)** SACC 12 (2 dml) Amitriptyline Hz0 19290 (12960-28850) 13 SACC 0.02 + 0.18 16210 (8350-30330) (20 w/ml) 14 Chlorpromazine Hz0 18210 (1284&25850) I5 0.32 + 0.19 (70 &ml) SACC 15680 (9800-25080) 16 __~-__ --. __’ Groups of lo-week-old BALBK mice (16 mice per group) on a restricted daily water intake were given three trials (21-day intervals) of cyclophosphamide (Cy) alone, groups 1-8, or Cy + Sacc (CS + US), conditioned groups 9-16. Cy was injected at a dose of 100 mg/kg. Seven days after the last Cy treatment animals in some groups also began receiving the drugs shown in their daily drinks. Twentyone days after the last Cy treatment subgroups of eight mice/group were exposed to water alone (= NCS) or SACC (= CS) and water in a SACC preference test. Consumption of each was assessed independently. Thirty minutes after fluid intake all mice were injected i.p. with 5 x IO8 SE in 0.5 ml PBS. Where SACC reexposure was used mice were again given SACC on Days 2, 4, and 6 post-SE. ’ Taste aversion in groups offered SACC and water (see Materials and Methods for more details). ’ Geometric mean IgG-AFClgroup at 7 days. Mean cell recovery was the same in all groups (130 & 30 X IOh cells/spleen). ** p i .05, Wilcoxon rank Stm Test.

tioned mice (rows 1-8) showed no significant differences in their response profiles that could be attributed to either Sacc or drug exposure. Effect of in vitro drug exposure on antibody responses. While the failure of nonconditioned mice to show any significant changes in immune response in the presence of the drugs used argues against a direct peripheral effect of the drugs on immune cells per se as an explanation for the data of Table 1, it was of interest to test this more directly by adding drugs to isolated suspension of lymphocytes in culture. Mouse spleen cells were pooled from six normal BALB/C mice and cultured in Marbrook vessels with SE for 5 days (see Materials and Methods). Drugs at the concentrations shown in Table 2 were included in the medium of both the inner and outer chambers of the Marbrook vessels. These concentrations were chosen to overlap those known to represent therapeutic serum levels in vivo in man (i.e., amitriptyline, 100 rig/ml; chlorpromazine, 250 rig/ml). In addition Metenkaphalin, a neurotransmitter reported to modulate immune responses in vitro

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AND HOLMES

TABLE 2 of Primary Antibody Response in Vitro with Psychoactive Drugs ConcentrationU

None Diazepam

~.

.____

AFCiculture” 1360 ” 130

(2 ngiml) ( IO ngiml) (50 ngiml)

1290 2 160 1310 ” 120 1.560 t 140

Amitriptyline

(10 rig/ml) (50 rig/ml) (250 rig/ml)

1210 i 180 1190 * 130 1150 * 150

Chlorpromazine

(20 rig/ml) (100 ngiml) (500 ngiml)

1260 k 130 1070 2 160 1120 2 160

Met-enkephalin

(10 Ml (10 M) (10 M)

660 k 160 490 2 210 890 ? 170

a Spleen cells were pooled from six normal BALBiC (10 weeks of age) and 10 X 1Ohcells cultured in Marbrook chambers with 20 x 10’ SE, with/without added drugs at the concentrations shown. All groups were set up in triplicate and cultured at 37°C in a humidified atmosphere with 5% CO,. ’ Arithmetic mean AFCiculture (?SEM) at 5 days.

(Rowland, Chukwuocha, & Tokuda, 1987), was added to some groups as a control to ensure that the cell pool used could respond to previously documented neuromodulators. Data in Table 2 (one of four experiments) show clearly that there was no significant change in immune response measured from cultures containing diazepam, amitriptyline, or chlorpromazine at these concentrations. Significant immunosuppression was seen with Met-enkephalin, especially at lOA7 A4 concentration, in keeping with other reports. Further studies (not shown) have indicated that even when responding spleen cells are prepared from conditioned mice there is no change in in vitro antibody responses measured in the presence of these drugs. DISCUSSION

By associative learning, mice can be conditioned to show suppressed antibody responses to SE in the context of a novel taste (Sacc), if they had previously experienced that taste in the presence of treatment with an immunosuppressive drug (Cy). In earlier studies (Gorczynski, 1987a, b), it was documented that this suppression was a function both of the cell populations within conditioned mice and of the environment in which those cells interacted. Given the evidence from the behavioral literature that psychoactive drugs can influence conditioned behaviors, it was of interest to ask whether these drugs would affect the conditioned immune response in a way not explained simply by direct effects on the immune system. Data in Tables I and 2 suggest indeed that amitriptyline and chlorpromazine, while having no direct effect on antibody responses in vivo or in vitro, can

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abolish the conditioned immunosuppression seen when conditioned mice (Cy + Sacc) are reexposed to Sacc in the presence of SE. There are a number of possible explanations for these data. Drugs may influence the processes involved in learning a behavior (the motivation and/or the memory processes) or those mechanisms involved in regulating the ability to perform a learned behavior (stimulus perception; regulation of “response output capacity”). From the psychological literature it seems that the direction (increase/ decrease) of the effect of a drug on a rate of responding depends, too. on the predrug rate for the behavior under consideration, regardless of whether the behavior is positively or negatively reinforced. Thus, if, prior to drug exposure, the rate of response is low, then after exposure to low levels of drug, the response rate is increased, while higher drug levels decreased the response rate. However, if the rate of response before drug exposure is high, then all drug doses used caused a subsequent decrease in response rate (Kelleher & Morse, 1964; Thompson & Dews, 1977). Adding complexity to these studies is the evidence that a low rate of punished responding is increased more by drug than a low rate of unpunished responding; i.e., the motivational state of the responding animal seems to be an important additional variable. Note too that the drug dosages chosen were extrapolated from those used in man, and we have no evidence that in mouse these would be equivalently neuroleptic or psychoactive. Other data indicate that decreased levels of noradrenaline/dopamine uptake (such as might be expected in animals treated with amitriptyline and chlorpromazine, respectively) can actually increase the rate of learning (by increasing attention?). However, drugs with primarily anti-cholinergic effects (a known sideeffect of amitriptyline and chlorpromazine in man) can decrease memory and learning (Kalant, Roschlau. & Sellers, 1985). Specifically, chlorpromazine, a centrally acting dopamine D2R blocker, has been shown to increase CNS turnover of dopamine and to attenuate the response altering properties of peripheral stimuli (i.e., while conditioned animals still escape from the shock itself, they no longer avoid shock in response to a buzzer, a CS (Kelleher & Morse, 1964)). Within the CNS. decreased arousal (neuronal firing) in the limbic system and the reticular activating system of the brainstem and hypothalamus has been documented. Amitriptyline, by decreasing uptake of 5hydroxytryptamine (serotonin) and noradrenaline centrally, has been shown to decrease motor behavior activity (antihistamine sedative effect’?) and to decrease the learning and/or performance of conditioned behavior. Diazepam has also been reported to interfere with memory organization and/or recall, presumably like other benzodiazepines, via some alterations in GABA-ergic transmission (and altered Cl- channel conductance?). Already referred to are those studies which have shown CNS changes in neurotransmitters following immune stimulation. There is evidence that lymphocytes themselves possess receptors for a variety of neurotransmitters (Blalock & Smith, 1985). One could build a model in which psychoactive drugs acted directly on lymphocytes to regulate immunity. However, our data are, we feel, better explained by a drug-mediated central blockade of those CNS-immune system interactions that control conditioned immunosuppression. To the extent that some degree of drug receptor specificity can be assumed, our data suggest that these

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effects may be mediated by dopaminergic/serotonergic (noradrenergic) changes in the CNS. There are a number of issues which must be addressed in order to explore this model. Does the relative binding avidity of different neuroleptic/ anti-depressant drugs for their respective CNS-receptors parallel their efficacy in modulating conditioned immunosuppression? What concentration of these drugs is achieved in mice (the metabolism is likely to be quite different from man), and do the effects observed follow some predictable dose-response relationship‘? What are the possible interpretations of the disparity between modulation of conditioned immunosuppression and taste aversion by different drugs (see Table l), and how can these differences be used to improve our understanding of the mechanisms behind conditioned immunity? Does drug treatment inhibit expression of the conditioned response (i.e., the mice do retain a memory of their “conditioned immunosuppressed” state) or has retention of the learned behavior been affected? These questions are the subject of ongoing studies. REFERENCES Ader, R., & Cohen. N. (1975). Behaviorally conditioned immunosuppression.

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Sci. 8, 379-395.

Besedovsky, H. O., del Rey. A., & Sorkin, E. (1983). What do the immune system and the brain know about each other? lmmunol. Today 4, 342-346. Besodovsky, H. 0.. Felix, D., & Haas, H. (1977). Hypothalamic changes during the immune response. Eur. J. Immunol. 7, 323-332. Blalock. J. E., & Smith, E. M. (1985). The immune system: Our mobile brain? Immunol. Today 6, 115-122. Bovbjerg, D., Ader, R.. & Cohen, N. (1982). Behaviorally conditioned suppression of a graft-vs-host response. Proc. Nati. Acad. Sci. USA 79, 583-587. Bullock, K., & Pomerantz, W. (1984). Autonomic nervous system innervation of thymic-related lymphoid tissue in wild-type and nude mice. .I. Camp. Neural. 228, 57-68. Carlson, S. L., Felten, D., Livnat, S., & Felten, S. Y. (1987). Alteration of monoamines in specific central autonomic nuclei following immunization in mice. Brain Behav. Immun. 1, 52-63. Cross, R. J.. Markesberry, W. R., Brooks, W. H., & Roszman, T. L. (1980). Hypothalamic-immune interactions. 1. The acute effects of anterior hypothalamic lesions on the immune response. Brain Res. 196, 79-88. Felten, D. L., Overhange, .I. M., Felten, S. Y., & Schmedtje, J. F. (1981). Noradrenergic sympathetic innervation of lymphoid tissue in the rabbit appendix: Further evidence of a link between the nervous and immune systems. Brain Res. Bull. 7, 595412. Gorczynski, R. M. (1987a). Analysis of lymphocytes in, and host environment of, mice showing conditioned immunosuppression to cyclophosphamide. Brain Behav. Immun. 1, 21-35. Gorczynski, R. M. (1987b). Conditioned immunosuppression in young versus aged mice: Differences in cells and responses to environmental stimuli lead to altered conditioning in aged animals. Brain Behav. Immun. 1, 306317. Gorczynski, R. M., & Cunningham, A. J. (1978). Requirement for matching T-cell and B-cell subsets in secondary anti-hapten antibody responses. Eur. .I. Immunol. 8, 753-755. Gorczynski, R. M., & Kennedy, M. (1984). Associative learning and regulation of immune responses. Prog.

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Gorczynski, R. M., Kennedy, M., & Ciampi, A. (1985). Cimetidine reverses tumor growth enhancement of plasmacytoma tumors in mice demonstrating conditioned immunosuppression. J. Immuno/. 134, 4261-4266. Johnson, H. M., Torres, B. A., Smith, E, M.. Dion, L. D., & Blalock, J. E. (1984). Regulation of lymphokine (n-interferon) production by corticotropin. J. Immunol. 132, 246-252.

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Kalant. H., Roschlau, W. H. E., &Sellers, E. M. (1985). Principles ofmedicalpharmacology, 4thed. Toronto: Univ. of Toronto Press. Kelleher, R. T., & Morse, W. H. (1964). Escape behaviour and punished behaviour. Fed. Proc. 23, 808-817. Kusnecov, A. W., Husband, A. J.. King, M. G.. Pang, G., & Smith. R. (1987). In viva effects of B-endorphin on lymphocyte proliferation and interleukin-2 production. Bruin Behav. Immun. 1, 88-97. Rowland, R. R. R., Chukwuocha, R.. & Tokuda, S. (1987). Modulation of the in vitro murine immune response by met-enkephalin. Bruin Behuv. Immun. 1, 342-348. Thompson, T., & Dews. P. B. (1977). Advances in behuviourul pharmacology, Vols. l-3. New York: Academic Press. Received February 9, 1989