Evidence for the involvement of β-adrenergic receptors in conditioned immunomodulation

Journal of Neuroimmunology, 38 (1992) 209-220 209 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

JNI 02185

Evidence for the involvement of/3-adrenergic receptors in conditioned immunomodulation

Linda J. L u e c k e n and D o n a l d T. Lysle

Department of Psychology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

(Received 30 September 1991) (Revised, received and accepted 22 January 1992)

Key words: /3-Adrenergic receptor; Immunomodulation; Conditioned aversive stimulus; Atenolol; ICI 118,551

Summary

This study investigates the role of /3-adrenergic receptors in the immunomodulatory effects of a conditioned aversive stimulus (CS). A CS is an environmental event that is not inherently aversive, but acquires aversive properties through pairings with a stimulus such as electric shock. This study evaluated the effects of administration of the /3~-receptor selective antagonist atenolol, and the /32-receptor antagonist ICI 118,551 on conditioned immune alterations. Administration of either antagonist prior to presentation of the CS resulted in a dose-dependent attenuation of the CS-induced suppression of splenic T-cell proliferative response to concanavalin A, phytohemagglutinin, and the combination of ionomycin/phorbol-myris ta te-aceta te . Furthermore, both antagonists dose-dependently attenuated the CS-induced suppression of y-interferon production by concanavalin-A (ConA)-stimulated splenocytes. In contrast, neither antagonist significantly attenuated the CS-induced suppression of the B-cell mitogenic response to LPS, interleukin-2 production, natural killer cell activity, or mitogenic responsiveness of blood lymphocytes. Thus it is likely that multiple mechanisms are involved in CS-induced immune alterations and these results clearly implicate/3~ and/32-adrenergic receptors in a subset of immunomodulatory effects.

Introduction

It has become increasingly clear that a variety of psychological and environmental factors can modulate the functioning of the immune system. For example, stressful situations such as marital disruption have been associated with suppressed

Correspondence to: D.T. Lysle or L.J. Luecken, Department of Psychology, Davie Hall, CB No. 3270, University of North Carolina, Chapel Hill, NC 27599-3270, USA.

immune responses as measured by mitogenic response of lymphocytes (Kiecolt-Glaser et al., 1987). In animal studies, exposure to physical stressors such as electric shock have been shown to suppress in-vitro immune responsiveness (Keller et al., 1981; Lysle et al., 1987).

Recent studies have demonstrated that conditioned aversive stimuli can induce immunomodulatory effects. A conditioned aversive stimulus (CS) is an environmental event (such as a visual, auditory, or contextual stimulus) that is not inherently aversive, but acquires that property through

21{I

pairing it with a stimulus that is inherently aver- give. Studies have shown that presentation of a CS developed through pairings with electric shock induces pronounced suppression of the responsiveness of lymphocytes to T- and B-cell mitogens, and suppression of natural killer cell activity (Lysle et al., 1988, 1990). These studies pro- vided controls allowing the conclusion that the immune alterations were the result of a conditioned state induced by the conditioned stimulus, and were not due to the prior shock experience, handling, or exposure to the conditioned stimulus alone. Furthermore, in evidence of a conditioned effect, the suppression of lymphocyte responsiveness was attenuated by extinction and pre-exposure manipulations (Lysle et aI., 1988).

The mechanisms by which a conditioned aversive stimulus modulates the immune response are less clear. However, there is growing evidence that catecholamines have an important role in the modulation of the immune system. Noradrenergic nerve fibers innervate both primary and secondary lymphoid tissues, including the bone mar- row, thymus, spleen, lymph nodes, and gut-associated lymphoid tissue (Bulloch, 1985; Felten et al., 1987). Numerous studies have identified adrenergic receptors on the surface of lymphocytes, monocytes, and neutrophils (Hadden et al., 1970; Williams et al., 1976; Abrass et al., 1985; Fuchs et al., 1988), and norepinephrine has been shown to be released at lymphoid sites and present in specific compartments of primary and secondary lymphoid tissues (Felten et al., 1987).

There is also some evidence suggesting that catecholamines play a role in the immunomodulatory effect of a conditioned stimulus. In a prior study conducted in our laboratory, administration of the non-selective /3-adrenergic antagonist pro- pranolol was shown to dose-dependently attenu- ate the CS-induced suppression of the mitogenic proliferative response of splenic lymphocytes (Lysle et al., 1991). Similar findings have been reported using electric shock as the immunosup- pressive stimulus (Cunnick et al., 1990).

The consequences of manipulation of adrenergic agonists and antagonists on the in-vitro immune response have been the focus of many studies, with what often appear to be conflicting results. For example, the mitogenic response of

lymphocytes was shown to be suppressed after the addition of /32 agonists, norepinephrine, or epinephrine to cultures (Johnson et al., 1981). Similarly, isoproterenol added to cell cultures was found to inhibit proliferation of blood lymphocytes in the presence of PHA, as well as inhibit- ing interleukin-2 receptor expression (Feldman et al., 1987). Other studies, however, have shown enhanced in-vitro immune responses following the addition of norepinephrine or terbutaline to cells (e.g., Sanders and Munson, 1984a, b; Good- win et al., 1979; Hadden et al., 1970). What is clear from the results of these and many similar studies is that catecholamines are capable of modulating the immune response, although the direction of the response and the subpopulation of lymphoid cells affected varies depending on the conditions under investigation.

The purpose of the present research was to extend our prior investigations of the role of catecholamines, specifically the /3-adrenergic system, in immune modulation resulting from exposure to a CS based on electric shock. The present study evaluated the effect of more selective /3- adrenergic antagonists on the CS-induced immune alterations. The/3e-selective antagonist ICI 118,551 was chosen to determine whether the immune modulation is primarily mediated by action at /32 adrenergic receptors. The /31-selective antagonist atenolol was similarly investigated for its ability to block CS-induced immune alterations. The immunological measures were also extended beyond those used in prior investigations to include assessment of lymphocyte proliferation using blood and spleen lymphocytes, natural killer cell activity, T-interferon and interleukin-2 production.

Materials and methods

Subjects Male Lewis rats approximately 65 day old were

obtained from Charles-River Laboratories (Ra- leigh, NC). The rats were individually caged in a colony room in which a reverse day-night cycle was maintained. Upon arrival, subjects were left undisturbed for a 2-week acclimation period. All

rats received free access to food and water throughout the experiment. All manipulations were initiated 1 h into the dark phase of the cycle.

Conditioning apparatus Eight identical rodent chambers ( B R S / L V E

Model RTC-020) were used as the conditioning apparatuses. Timer circuitry to the output of a shock generator and scrambler ( B R S / L V E Mod- els SG-903 and SC-902) was used to provide the unconditioned aversive stimulus of a 5.0 s, 1.6 mA footshock. The chambers were individually housed in identical sound-attenuating cubicles.

Conditioning procedure All subjects were given 2 days of exposure to

the chambers and the footshock. On each day, a series of 10 shocks were delivered on a 4-min variable interval schedule. Previous studies have demonstrated that this procedure effectively es- tablishes the cues from the chamber environment as a conditioned aversive stimulus (Lysle et al., 1990). Following the second day of conditioning, the subjects were left undisturbed in their home cages for 12 days. On the test day, subjects were randomly assigned to one of eight groups in a 4 x 2 factorial design. All subjects received an injection of either saline or a dose of 0.125, 0.5, 2.0, or 8.0 m g /kg ICI 118,551. 30 min following the injection, half of the subjects were re-exposed to the conditioning chambers and the other half were left in their home cages. 40 min later all subjects were rapidly killed by cervical disloca- tion. Blood was drawn from the abdominal aorta and collected into heparinized syringes through 21 gauge needles. Each subject's spleen was re- moved and placed in 7 ml of RPMI-1640 tissue culture medium supplemented with 10 mM Hepes, 2 mM glutamine, and 50 ~g genta- micin/ml (Gibco). The assessment of the effects of administration of atenolol followed identical procedures as described for ICI 118,551.

Mitogen assay A mitogen stimulation assay was performed

with the splenic leukocytes using the T-cell mito-

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gens concanavalin-A (ConA), phytohemagglutinin (PHA), and the B-cell mitogen lipopolysaccharide (LPS). In addition, the proliferative response to the combination of ionomycin and phorbol-myri- state-acetate (PMA) was also evaluated. Recent studies have shown a remarkable synergy between ionomycin and phorbol ester leading to the proliferation of lymphocytes (Truneh et al., 1985). The combination of ionomycin and PMA acti- vates intracytoplasmic second messengers leading to the proliferation of T-lymphocytes, and possi- bly B-lymphocytes (Fresa et al., 1989).

Each spleen was dissociated to a single cell suspension by gently pressing the tissue between sterile frosted microscope slides in supplemented RPMI enriched with 10% fetal bovine serum. Each subject's splenocytes were counted using a NOVA Celltrak II cell analyzer and cell suspen- sions were adjusted to 5 x 106/ml. The mitogens were prepared to a concentration of 0, 1.0, and 10.0 /xg/ml to provide background, suboptimal, and optimal concentrations of each mitogen. 100 /xl of the mitogen preparations were pipetted in triplicate into the wells of a 96-well, flat-bottom microtiter plate (Costar No. 3595). Ionomycin was dissolved in dimethylsulphoxide and then diluted to a concentration of 1.0, 3.0, 5.0, 7 .0 /xg/ml in supplemented RPMI, and 100 /xl were pipetted in triplicate into the wells of the microtiter plate. In addition, 10 /xl of PMA (20 /xg/ml) were added to each well. Our preliminary investigations, and the work of others, indicated that both ionomycin and PMA are required to induce lymphocyte proliferation, and that neither alone is sufficient to induce proliferation (Truneh et al., 1985). 100/xl of the adjusted cell suspension were pipetted into each well to provide a final concentration of ConA, PHA, and LPS of 0, 0.5, and 5.0 /xg/ml, and a final concentration of 0.5, 1.5,2.5, and 3.5 /xg/ml of ionomycin with 1.0 /xg/ml of PMA. The plates were incubated at 37°C in a humidified incubator, with 5% CO 2. The cultures were pulsed with 1 /xCi [3H]thymidine (specific activity = 6.7 Ci mmol, ICN) in 50 /xl of RPMI- 1640 during the last 5 h of a 48-h incubation period. The cultures were harvested onto glass fiber filter paper using a Skatron harvester. The incorporation of [3H]thymidine was determined with a liquid scintillation counter (Beckman

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Model LS 1701) and expressed as disintegrations per rain (DPM).

A whole blood assay was also performed. The blood was diluted 1 : 5 with supplemented RPMI- 1640, and leukocyte counts were determined using a Coulter counter. 100/xl of the diluted blood were pipetted in triplicates into the cells of a 96-well plate. The mitogens ConA and PHA were prepared at concentrations of 0, 10.0, and 20 /xg /ml and 100 /xl were added to the cells to yield a final concentration of 0, 5.0, and 10.0 p .g/ml . The incubation and harvest procedures were the same as outlined for the spleen cell assays, except that the total incubation period was extended to 120 h and the cultures were pulsed during the last 18 h. In addition, in the whole blood assay, DPMs were adjusted to counts per minute per 5 × 105 leukocytes to account for variations in the number of leukocytes plated.

Natural killer cell assay A natural killer cell assay was performed using

splenic leukocytes. A murine T-cell lymphoma, YAC-1, was maintained in supplemented RPMI plus 10% FBS in a CO 2 incubator. On the day of the assay, YAC-1 targets were prepared by la- belling them for 70 rain with 200 #Ci of sodium chromate-51 (Dupont-New England Nuclear). The YAC-1 ceils were then washed three times to remove exogenous Cr sl. Splenic leukocytes, prepared as previously described, were used as effectors and were plated in triplicate at 10, 5, 2.5, and 1.25 x l0 s cells/well of a 96-well plate. The labelled targets were diluted and plated at 1 x 104 cells/well. Effector : target ratios of 100 : 1, 50 : 1, 25 : 1, and 12.5 : 1 were obtained. Control wells containing only labelled targets were also plated to determine the spontaneous release and maxi- mum possible release. The plates were centrifuged at 600 rpm for 4 rain and incubated 4 h at 37°C in a humidified CO 2 incubator. Immedi- ately prior to harvest, the targets in one set of control wells were lysed with 10% trichloroacetic acid and the microtiter plates were centrifuged. The supernatant was harvested using a Titertek multichannel pipette. The amount of Cr 51 released in the supernatant was counted using a LKB gamma counter (Model 1272 CliniGamma).

Lytic uni ts (LU) were calculated using a computer program based on the equations of Pross and Maroun (1984). The percent cytotoxicity at all e f fec tor : ta rget ratios was utilized. Lytic units were based on the number of leukocytes per 107 effectors necessary to lyse 20% of the targets.

y-Interferon assay To test the production of y-interferon by

ConA-stimulated splenocytes, 5 x 10 6 splenocytes in 1 ml of media were incubated with 5 /xg /ml ConA for 48 h in 12-well microculture plates. The supernatants were harvested and stored at - 8 0 ° C until assay. The supernatants were tested for the presence of y-interferon using a commercial ELISA assay for rat interferon-7 (Holland Biotechnology). The assay was conducted using an automated microtiter plate washer (Biotek, Model EL403) and absorbance was determined using a Biotek plate reader (Model EL312). The interferon concentration of each sample was determined by a standard curve obtained using pure recombinant rat y-interferon. The data are expressed in units, with each unit corresponding to the absorbance obtained using 250 pg of pure recombinant rat y-interferon.

lnterleukin-2 assay Interleukin-2 (IL-2) is a T-cell growth factor

produced by stimulated T lymphocytes. To test the IL-2-producing ability of T-cells, 5 × l06 splenocytes in 1 ml of media were incubated with 5 /xg/ml ConA for 48 h in 12-well microculture plates. The supernatants were then harvested, stored at -80°C, and subsequently tested for the presence of IL-2. The supernatants and a positive IL-2 control were serially diluted 1 : 2 (total of six dilutions), with a starting dilution of 1:8, in du- plicate in a 96-well microtiter plate using a 100-txl octapet (Titertek). The IL-2-dependent cell line CTLL-2 was washed to remove any exogenous IL-2, and was then diluted in RPMI-1640 plus 10% FBS to 1 x 10S/ml. Then 100 txl of the CTLL-2 cell suspension was plated in each well and in six control wells containing no IL-2. The plates were incubated for 24 h at 37°C in a humidified atmosphere of 5% CO 2. Each well

was pulsed with 1 /xCi of [3H]thymidine (50 ~zl) for the last 5 h of the incubation. The cultures were then harvested using a Skatron harvester, and the thymidine incorporation was determined using a scintillation counter. The data were expressed as half-max units, as calculated using a computer program (Lilly Research laboratories).

Statistical treatment of the data Statistical analysis of the data was calculated

using computerized analysis of variance software (Statistix, NH Analytical Software). A two-way analysis of variance was used to assess the effect of treatment and ICI 118,551 (or atenolol) on the mitogenic proliferative response of splenic and blood lymphocytes, natural killer cell activity, interferon and interleukin-2 production. In the analysis, the first factor was the type of treatment received on test day (home cage, or exposure to the conditioning chambers). The second factor was the dose of ICI 118,551 or atenolol adminis- tered. Most importantly, the analysis assessed the interaction between treatment and drug. The level of significance for the F-test was set at P < 0.05.

Results

ICI 118,551

Mitogen stimulation assays The mitogen stimulation assay using splenic

lymphocytes-showed comparable effects for all concentrations of mitogen. Figure 1 displays the mean DPM for the optimal concentration of ConA (5.0 /~g/ml final concentration), LPS (5.0 /xg/ml final concentration), and ionomycin (1.5 izg/ml final concentration). Analysis of variance of the results for the optimal concentration of ConA showed a pronounced suppressive effect of exposure to the CS relative to home-cage animals, as evidenced by a highly significant treatment effect (F(1,50) = 34.65, P < 0.0001). There was also a significant interaction between ICI 118,551 and treatment (F(4,50)= 5.46, P < 0.001). Polynomial contrasts showed a highly significant linear component to the interaction (F(1,50) = 17.96, P < 0.0001). There were no sig-

213

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ICI I18,551 (rng/kgl Fig. 1. Effects of different doses of ICI 118,551 on the mitogenic response of splenic lymphocytes to the optimal concentration of ConA (5.0 /zg/ml), ionomycin/PMA (1.5 /zg/ml), and LPS (5.0 p,g/ml) for CS-exposed and home cage

control animals. Data expressed as mean DPM + SE.

nificant differences between responses for home- cage animals at varying doses of the drug. Thus, the significant linear component to the interaction demonstrated that the suppressive effect of CS-exposure was dose-dependently attenuated by administration of ICI 118,551.

The results for the optimal concentration of PHA were similar. A significant treatment effect (F(1,48) = 72.89, P < 0.0001), a significant interaction (F(4,48)= 3.16, P < 0.022), and a significant linear effect (F(1,48) = 10.3, P < 0.002) were found. The PHA data from two subjects were lost due to technical error.

214

Figure 1 also shows the results for the optimal concentration of ionomycin /PMA. Analysis of those results showed a highly significant treatment effect, (F (1 ,50)= 45.15, P < 0.0001), a significant interaction (F(4,50) = 2.56, P < 0.05), and a significant linear component to the interaction (F(1,50) = 9.85, P < 0.003).

The results for the optimal conce~tration of LPS (Fig. 1) also showed a significant effect of t reatment (F(1,50) = 14.49, P < 0.001). However, in contrast to the effects for the other mitogens, the LPS data did not show a significant interaction (F(4,50) = 0.95, P < 0.45), indicating that the administration of ICI 118,551 did not significantly at tenuate CS-induced suppression of LPS proliferative response.

Collectively, these results show a pronounced suppressive effect of the conditioned aversive stimulus on the mitogenic responsiveness to ConA, PHA, LPS, and the combination of ionomycin and PMA. More importantly, the administration of ]CI 118,551 close-dependently dimin- ished the CS-induced suppression of the response to ConA, PHA, and ionomycin /PMA. In contrast, the administration of ICI 118,551 did not significantly affect the CS-induced suppression of the response to LPS.

For the whole blood assay, analysis of variance for both ConA and PHA at the optimal concentration of 10 # g / m l showed a significant effect of t reatment (ConA: F(1,50) = 16.18, P < 0.0002, PHA: F(1,50) = 54.98, P < 0.0001). The mean DPM values for ConA and PHA were 60 420 and 2064 for CS-exposed animals, and 102420 and 19 090 for home-cage animals. There was no evidence of an interaction between treatment and ICI 118,551 administration (F(4 ,50)=0.48, P < 0.76). In addition, there were no significant differences between home cage animals, and like- wise no significant differences between CS exposed animals. Thus, administration of ICI 118,551 had no significant effect on CS-induced suppression of the mitogenic responsivity of blood lymphocytes.

Natural killer cell assay The results for the natural killer cell assay are

shown in Fig. 2. Analysis of lytic units showed a

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30

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20 15

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NK CELL ACTIVITY 0

i i 5 0 . l i 2 5 • 2i,0 81.0

HC CS

ICI 118,551 ( m g / k g ) Fig. 2. Effects of different doses of ]CI 118,551 on natural killer cell activity for CS-exposed and home cage control

animals. Data expressed as mean lytic unit + SE.

highly significant effect of t reatment (F(1 ,50)= 86.39, P < 0.0001) representing a potent suppressive effect of CS-exposure. However, there was no significant interaction between ICI 118,551 and treatment (F(4 ,50)= 1.08, P < 0.38). Thus, the administration of ICI 118,551 did not attenu- ate the CS-induced suppression of natural-killer cell activity.

Interferon- y assay The analysis of units of y-interferon showed a

pronounced decrease in the production of y-interferon in animals exposed to the CS relative to home-cage animals, as evidenced by a highly significant t reatment effect (F(1 ,50)= 26.91, P < 0.0001). Although the overall interaction between ICI 118,551 and treatment did not achieve significance (F(4,50) = 1.91, P = 0.12), polynomial contrasts showed a significant linear component to the interaction (F(1,50) = 4.80, P < 0.05). The significant linear component to the interaction provides evidence that the suppressive effect of CS-exposure was dose-dependently at tenuated by administration of ICI 118,551.

Interleukin-2 assay The assay for IL-2 using the dependent cell

line CTLL-2 showed significant suppression of IL-2 production in subjects exposed to the conditioned aversive stimulus (F(1,50) = 8.15, P <

350

300

250 ( / ) I - - 200

Z 150

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INTERFERON

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0 I I 0 . l l 2 5 .5 21.0 8 . 0

ICl 118-551 (mg/kg) Fig. 3. Effects of different doses of ICI 118,551 on y-interferon production by splenic lymphocytes for CS-exposed and home cage control animals. Data expressed as mean

unit _+ SE.

215

data did not reach significance for the interaction.

In the whole-blood assay, analysis of variance for the optimal concentration of both ConA and PHA showed a significant effect of t reatment (ConA: F(1,50) = 9.69, P < 0.004, PHA: F(1,50) = 72.09, P < 0.0001). The mean DPM values for ConA and P H A were 57 460 and 3924 for CS-exposed animals, and 96230 and 26 090 for home- cage controls. There was no evidence of an interaction between t reatment and atenolol administration. In addition, there were no significant differences between home cage animals, and like- wise no significant differences between CS animals.

0.007). There was no evidence, however, that the administration of ICI 118,551 had any effect in blocking the conditioned immune suppression (F(4,50) = 0.21, P < 0.94). The mean half-max values were 63.43 for the CS-exposed subjects and 84.9 for the home-cage subjects.

Atenolol

Mitogen stimulation assays The results for atenolol were strikingly similar

to those for ICI 118,551. Analysis of variance of ConA data (Fig. 4) showed a highly significant effect of t reatment (F(1,50) = 68.15, P < 0.0001), a significant interaction between atenolol and t reatment effects (F(4,50) = 8.17, P < 0.0001), and a significant linear component to the interaction (F(1,50) = 26.75, P < 0.0001).

The results for P H A were similar. A significant t reatment effect (F (1 ,48 )= 76.15, P < 0.0001), a significant interaction (F(4 ,48)= 2.73, P < 0.04), and a significant linear component to the interaction (F(1,48) = 6.78, P < 0.013) were found. I o n o m y c i n / P M A results (Fig. 4) showed a significant t reatment effect (F(1 ,50)= 21.2, P < 0.00001), a significant interaction (F(4,50) --- 3.09, P < 0.024), and a significant linear component to the interaction, (F(1,50) -- 10.59, P < 0.002). LPS data also showed a significant effect of t reatment (F(1,50) = 27.49, P < 0.0001), however, the LPS

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Fig. 4. Effects of different doses of atenolol on the mitogenic response of splenic lymphocytes to the optimal concentration of ConA (5.0 ~g/ml), ionomycin/PMA (1.5/xg/ml), and LPS (5.0/zg/ml) for CS-exposed and home cage control animals.

Data expressed as mean DPM _+ SE.

216

Natural killer cell assay The results for the natural killer cell assay are

shown in Fig. 5. Analysis of lytic units showed a highly significant effect of treatment (F(1 ,50)= 31.9, P < 0.0001) representing a potent suppressive effect of CS-exposure. There was no significant overall interaction between between atenolol and treatment, (F(4,50) = 2.05, P < 0.10), and no significant polynomial contrasts.

lnterferon- y assay Analysis of variance of the units y-interferon

showed a pronounced suppressive effect of exposure to the CS relative to home-cage animals (Fig. 6), as evidenced by a highly significant treatment effect (F(1,50) = 260.37, P < 0.0001). In a manner similar to ICI 118,551, the overall interaction between atenolol and treatment did not achieve significance (F(4 ,50)= 1.81, P=0 .14 ) , but polynomial contrasts showed a significant linear component to the interaction (F(1,50) = 5.03, P < 0.05). The significant linear component to the interaction provides evidence that the suppressive effect of CS-exposure was dose-dependently attenuated by administration of atenolol.

Interleukin-2 assay The assay for IL-2 showed significant suppres-

sion of IL-2 production in subjects exposed to the CS (F(1,50) = 45.41, P < 0.0001). There was no

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ATENOLOL ( m g / k g ) Fig. 5. Effects of different doses of atenolol on natural killer cell activity for CS-exposed and home cage control animals.

Data expressed as mean lytic unit + SE.

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ATENOLOL (rng/kg) Fig. 6. Effects of different doses of atenolol on y-interferon production by splenic lymphocytes for CS-exposed and home

cage control animals. Data expressed as mean unit _+ SE.

evidence, however, that the administration of atenolol was effective in blocking the CS-induced suppression of IL-2 production (F(4 ,50)= 0.79, P < 0.54). The mean half-max values were 35.9 for the CS-exposed animals and 51.9 for the home-cage animals.

Discussion

The present results show that a conditioned aversive stimulus can induce a pronounced reduction in the proliferative response of blood and spleen lymphocytes to mitogens. Moreover, the conditioned stimulus also induces a suppression of natural killer cell activity, and a reduction of y-interferon and interleukin-2 production. More importantly, the present research provides insight into the mechanisms that play a role in the CS-induced immune modulation. The results show that both the /32-adrenergic antagonist ICI 118,551 and the /3~-adrenergic antagonist atenolol were highly effective at blocking the CS-induced suppression of the mitogenic response of splenic T cells to ConA and PHA, and the proliferative response to the combination of ionomycin and PMA. Furthermore, both antagonists dose-dependently blocked the CS-induced reduction in y-interferon production. However, neither drug was effective at blocking suppression of the mitogenic response of splenic B-cells to LPS, splenic

217

natural killer cell activity, splenic lymphocyte interleukin-2 production, or the proliferative response of blood lymphocytes to ConA and PHA.

The fact that both drugs antagonized some of the CS-induced immune alterations suggests that /3-adrenergic receptors are involved in CS-induced immune alterations. However, the lack of an effect of these drugs on other immune measures indicates that multiple mechanisms are involved in CS-induced immune alterations. In sup- port of this conclusion, prior work has suggested that the mechanism involved in shock-induced suppression of lymphocyte responsiveness to ConA is dependent on the compartment of the immune system evaluated (e.g., Cunnick et al., 1990). In that study, the administration of the non-selective /3-adrenergic antagonists, propra- nolol and nadolol, attenuated shock-induced suppression of ConA responsiveness of splenic, but not blood lymphocytes. Moreover, adrenalectomy attenuated the shock-induced suppression of the proliferative response of blood lymphocytes to ConA, but did not affect the shock-induced suppression of splenic lymphocytes. These results suggest that the suppression of blood lymphocytes is induced by the release of an adrenal hormone such as corticosterone. However, a more complex conclusion is probably warranted, for the present results indicate that multiple immunomodulatory mechanisms can operate simultane- ously within a specific compartment (e.g., spleen) of the immune system.

The present results indicate that both 131 and /32 receptors are involved in conditioned immunomodulatory effects. Previous attempts to identify the subtype of fl-receptor present on lymphocytes have tended to implicate /32 receptors as the major type involved in immunoregula- tion. For example~ fl-adrenergic receptors on the surface of B- and T-lymphocytes have been reported to be primarily of the/32 subtype (Conolly and Greenacre, 1977; Ikegami, 1977; Galant et al., 1978; Loveland et al., 1981; Felten et al., 1990). Other studies, however, have indicated that /31-receptor activity is also involved in im- munoregulation. For example, Sanders and col- leagues (1984a, b) found that while norepinephrine-induced enhancement of murine anti-

body response was closely mimicked by the /32 - agonist terbutaline, the /31-agonist dobutamine also modulated the immune response. Thus, there is some evidence suggesting that both /31 and /32-adrenoceptors are involved in immunomodulation. This suggestion is consistent with those from other studies showing that /31 and /32-receptors occasionally coexist in the same tissues and can mediate the same physiological activities (Weiner and Molinoff, 1989). In our results, the fact that both a /31 and a /32-receptor selective antagonist were equally effective at blocking the CS-induced immune alterations suggests that neuro-immune regulation may require synergistic activity at both /31 and /32-receptor sites.

The present results also found that although both /3-antagonists were capable of blocking immune suppression in the splenic T-cell population (as measured by the mitogen assays ConA, PHA, and ionomycin/PMA),they did not significantly affect suppression of the proliferative response of B-cells (as measured by the mitogen LPS). This difference may reflect a differential sensitivity to /3-adrenergic stimulation between T- and B- lymphocytes. For example, considerable heterogeneity has been shown to exist among lymphocyte subpopulations in studies of cyclic AMP responses to isoproterenol (e.g., Bach, 1975). Hu- man T cells from the blood, tonsils, or adenoids have been shown to have greater responsiveness to isoproterenol than B-cells (Niaudet et al., 1976; Galant et al., 1978). In addition, differences in /3-receptor density between T- and B-cells have been noted (Pochet et al., 1979; Pochet and De- lespesse, 1983). Similarly, in the present study, differential sensitivity to/3-adrenergic stimulation may account for the antagonism of a subset of the conditioned alterations in T-cell function.

The current findings provide further characterization of sympathetic nervous system modulation of immune function by showing antagonism of CS-induced immune alterations by administration of either ICI 118,551 or atenolol prior to exposure to the CS. These results indicate that the immunomodulatory effects of a conditioned aversive stimulus are partially mediated by /3-adrenergic activity at both fl~ and fl2-receptor sites.

218

Acknowledgements

We are grateful to Elizabeth H. Bennett and Kimberly A. Maslonek for their technical assis- tance on this project. This work was supported by a grant to D.T.L. from the National Institute of Mental Health (MH46284).

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Evidence for the involvement of β-adrenergic receptors in conditioned immunomodulation