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(The FASEB Journal. 2009;23:1161-1167.)
© 2009 FASEB

Calcineurin inhibition in splenocytes induced by pavlovian conditioning

Gustavo Pacheco-López*,1,2, Carsten Riether*,1, Raphael Doenlen*, Harald Engler*,{dagger}, Maj-Britt Niemi*, Andrea Engler*, Annemieke Kavelaars{ddagger}, Cobi J. Heijnen{ddagger} and Manfred Schedlowski{dagger}

* Department of Psychology and Behavioral Immunobiology, Institute for Behavioral Sciences, ETH Zurich, Zurich, Switzerland;

{dagger} Division of Medical Psychology and Behavioral Immunobiology, Medical Faculty, University of Duisburg-Essen, Essen, Germany; and

{ddagger} Laboratory of Psychoneuroimmunology, University Medical Center Utrecht, Utrecht, The Netherlands

2Correspondence: Institute for Behavioral Sciences, Psychology and Behavioral Immunobiology, TUR B21.1, Turnerstrasse 1, 8092 Zurich, Switzerland. E-mail: pacheco{at}ifv.gess.ethz.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pavlovian conditioning is one of the major neurobiological mechanisms of placebo effects, potentially influencing the course of specific diseases and the response to a pharmacological therapy, such as immunosuppression. In our study with behaviorally conditioned rats, a relevant taste (0.2% saccharin) preceded the application of the immunosuppressive drug cyclosporin A (CsA), a specific calcineurin (CaN) inhibitor. Our results demonstrate that through pavlovian conditioning the particular pharmacological properties of CsA can be transferred to a neutral taste, i.e., CaN activity was inhibited in splenocytes from conditioned rats after reexposure to the gustatory stimulus. Concomitant immune consequences were observed on ex vivo mitogenic challenge (anti-CD3). Particularly, Th1-cytokine, but not Th2-cytokine, production and cell proliferation were impeded. Appropriate pharmacological and behavioral controls certify that all these changes in T-lymphocyte reactivity are attributable to mere taste reexposure. Furthermore, the underlying sympathetic-lymphocyte interaction was revealed modeling the conditioned response in vitro. CaN activity in CD4+ T lymphocytes is reduced by β-adrenergic stimulation (terbutaline), with these effects antagonized by the β-adrenoreceptor antagonist nadolol. In summary, CaN was identified as the intracellular target for inducing conditioned immunosuppression by CsA, contributing to our understanding of the intracellular mechanisms behind "learned placebo effects."—Pacheco-López, G., Riether, C., Doenlen, R., Engler, H., Niemi, M.-B., Engler, A., Kavelaars, A., Heijnen, C. J., Schedlowski, M. Calcineurin inhibition in splenocytes induced by pavlovian conditioning.


Key Words: cyclosporin A • immunosuppression • associative learning • placebo • taste


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ACTIVATED IMMUNE CELLS in periphery secrete soluble messengers such as cytokines, neurotransmitters, and prostaglandins that affect the activity of the central nervous system (CNS; refs. 1 2 3 4 ). Reciprocally, the CNS modulates the responsiveness of immune cells in the periphery via neural and humoral pathways (5 6 7) . Behavioral conditioning of immune functions is one of the most intriguing and exquisite examples of the intense bidirectional communication between the CNS and the immune system (8 9 10 11) . We have documented behaviorally conditioned immunosuppression in humans (12) and in corresponding rodent models (13 , 14) . In this paradigm, the immunosuppressive drug cyclosporin A (CsA) as the unconditioned stimulus is contingently paired with a distinctive taste as the conditioned stimulus. The mere reexposure to the taste stimulus decreased splenocyte proliferation and reduced Th1-cytokine production and mRNA expression after ex vivo mitogenic stimulation. Notably, this conditioning paradigm may have potential clinical relevance, since in rodents it prolonged heart allograft survival, attenuated the course of experimental autoimmune disease, and delayed type hypersensitivity reaction after conditioning (13 , 15 16 17) .

Advances have also been made in understanding the central processing and peripheral routes involved in this conditioned immunosuppressive response. In particular, the insular cortex and the ventromedial hypothalamic nucleus were identified as indispensable forebrain structures during the evocation phase (18) , a discrete neural network that has been ascribed to peripheral sympathetic facilitation, in particular within the spleen (19 20 21) . In addition, the conditioned response was demonstrated to be mediated via sympathetic innervation of the spleen and β-adrenoreceptor-dependent mechanisms (22 , 23) . However, the cellular signaling pathways mediating this phenomenon are still poorly understood.

The immunosuppressive activity of CsA reflects its ability to inhibit the protein phosphatase calcineurin (CaN). In activated T lymphocytes, CaN dephosphorylates the nuclear factor of activated T cells and promotes its translocation into the nucleus and the up-regulation of early T-lymphocyte genes such as the Th1-cytokine interleukin (IL)-2 (24 , 25) . The synthesis and release of IL-2 by activated T lymphocytes results in effective clonal expansion (26) . Thus, inhibition of CaN by CsA suppresses expansion of T lymphocytes on antigenic challenge (in vivo and/or ex vivo) mainly due to reduced IL-2 synthesis and release (27) . Against this background, we analyzed the cellular mechanisms of conditioned immunosuppression by CsA assessing CaN activity in the spleen, Th1- and Th2-cytokine production, and splenocyte proliferation ex vivo after challenge with the T-cell stimulus anti-CD3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male dark agouti rats (between 220 and 250 g) were obtained from Harlan Netherlands (Horst, The Netherlands). The animals were individually housed under an inverted 12:12-h light-dark schedule (lights off at 7 AM) with food available ad libitum. Water was available ad libitum until the water deprivation regimen started. The experiments are in accordance with the Swiss Federal Act on Animal Protection and Swiss Animal Protection Ordinance and were approved by the local animal ethics committee (Cantonal Veterinary Office Zurich).

Behavioral protocol
All animals were allowed to acclimatize to the new surroundings for 3 wk before any experimental procedure was started. Five days before the first association trial, animals were placed on a water deprivation regimen, allowing them 15 min of drinking at 8 AM and 5 PM each day. On experimental day 6, conditioning started with 72 h intervals between association trials and 24 h intervals between evocation trials (Fig. 1 ). The time period between the last association and the first evocation trial for all groups was 72 h. In the morning of association days, conditioned animals (CS; n=16) and conditioned not-evoked animals (CSo; n=14) received a 0.2% w/v saccharin solution (sodium saccharin; Sigma, Schnelldorf, Germany) as conditioned stimulus. Immediately after the bottles were removed, animals received an intraperitoneal injection of 20 mg/kg CsA (Sandimmune; Novartis, Basel, Switzerland). In the afternoon session, water (tap water) intake preceded an intraperitoneal saline injection (0.9% NaCl; B. Braun Medical, Emmenbrücke, Switzerland). Noncontingent paired rats (NP; n=14) received the same stimuli but in inverse order: water preceding CsA injection in the morning and saccharin in combination with a saline injection in the afternoon of the 3 acquisition days. During evocation trials, conditioned rats were reexposed to saccharin in the morning sessions and water in the afternoon sessions, whereas the CSo group received water in both sessions. The order was reversed for the NP group, which received water in the mornings and saccharin in the afternoons. In addition, a pharmacological control (US; n=14) group was included, which was treated similarly to the NP group but received an additional CsA injection after water presentation during morning sessions on evocation days. This group allowed a comparison between the conditioned immune effect and the pharmacological effect. Finally, a group of untreated rats (N; n=12) was included. These animals remained undisturbed during the whole conditioning procedure, except that they were subjected to the water deprivation regimen. On the last evocation day, 1 h after finishing the morning drinking session, animals were anesthetized with isoflurane (Attane; Minrad, Buffalo, NY, USA), blood was collected by cardiac puncture, spleens were removed, and the animals were finally euthanized.


Figure 1
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  		Figure 1. Taste-CsA conditioning paradigm. a) Conditioning protocol consisting of 3 association and 3 evocation trials. Na-saccharin solution (0.2%, Sac) was used as conditioned stimulus, and CsA (20 mg/kg i.p.) was used as unconditioned stimulus. Stimuli order presented to induce or to control for a conditioned response are illustrated (for details see Materials and Methods). Wat, water. b) Fluid consumption (open symbols, water; solid symbols, saccharin) of experimental and control groups during association and evocation phases. Data are expressed as means ± SE. *P ≤ 0.05 vs. CSo and NP; ANOVA with Tukey’s post hoc test.

Splenocyte isolation
Single-cell suspensions of the spleen were obtained by mechanically disrupting the tissue with a syringe plunger in cold Hanks’ balanced salt solution (Invitrogen, Basel, Switzerland). Red blood cells were removed using diluted PharM Lyse (BD Pharmingen, Allschwil, Switzerland). Splenocytes were washed in cell culture medium (RPMI 1640 supplemented with GlutaMAX I, 25 mM HEPES, 10% FBS, and 50 µg/ml gentamicin; Invitrogen) and filtered through a 70 µm nylon cell strainer. Cell concentrations were determined with an automatic animal cell counter (Vet abc; Medical Solution, Steinhausen, Switzerland), and splenocytes were adjusted to a final concentration of 5 x 106 cells/ml.

Ex vivo proliferation assay
Splenocytes were stimulated in 96-well flat-bottom microtiter plates with 1 µg/ml of anti-rat CD3 monoclonal antibody (clone: G4.18; BD Pharmingen) for 72 h in a humidified incubator (37°C, 5% CO2). Cell proliferation was determined with a colorimetric assay (CellTiter96 AQueous One Solution Cell Proliferation Assay; Promega, Madison, WI, USA) according to the manufacturer’s instructions. The tetrazolium substrate solution was added for the last 4 h of incubation, and absorbance was measured at 492 nm. To account for differences in background activity, the mean absorbance of the triplicate set of wells with unstimulated cells was subtracted from the mean absorbance of the three corresponding anti-CD3-stimulated wells.

Flow cytometry
Blood (20 µl) or splenocyte suspensions (2.5x105 cells) were incubated at 4°C for 45 min with the following fluorochrome-conjugated monoclonal antibodies: anti-rat CD3 (clone 1F4; BD Pharmingen), anti-rat CD4 (clone OX-35; BD Pharmingen), anti-rat CD8a (clone OX-8; BD Pharmingen), anti-rat CD45 (clone OX-1; BD Pharmingen), anti-rat CD45RA (clone OX-33; BD Pharmingen), anti-rat CD161 (clone 10/78; AbD Serotec, Düsseldorf, Germany), and anti-rat CD172a (clone ED9; AbD Serotec). Antibody labeling was performed by a standard lyse-wash procedure using FACS lysing solution (BD Immunocytometry Systems, Allschwil, Switzerland) and supplemented with PBS (Dulbecco’s PBS, 2% FBS, 0.1% NaN3). Lymphocytes were identified by forward and sideward scatter characteristics and the expression level of CD45. Ten thousand lymphocytes from each sample were analyzed on a LSR II flow cytometer using FACS Diva software (BD Immunocytometry Systems).

Cytokine analyses
Culture supernatants of unstimulated and anti-CD3-stimulated splenocytes were collected after 48 h of incubation, and cytokine concentrations were measured by ELISA according to the manufacturer’s instructions (IL-2 and IFN-{gamma} from Biosource, Invitrogen; IL4 from BD Pharmingen). Detection limits were 15.6 pg/ml for IL-2, 21.8 pg/ml for IFN-{gamma}, and 1.5 pg/ml for IL-4.

Protein determination
Protein levels in splenic lysates for CaN activity analysis were determined using the BCA protein assay according to the manufacturer’s instructions (Pierce, Rockford, IL, USA; ref. 28 ). For Western blot analysis, the protein concentration was determined by the Bradford method using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) and bovine serum albumin as the standard (29) .

CaN activity
Cellular CaN phosphate activity in rat splenic lysates was assessed following the manufacturer’s instructions (EMD Chemicals, San Diego, CA, USA). Briefly, spleens were disrupted using a tissue homogenizer (Tissue-terror; Biospec Products, Bartlesville, UK) in the presence of lysing buffer including a mixture of protease inhibitors provided by the manufacturer. Extracts were centrifuged at 16,000 g for 1 h at 4°C, and free phosphate was eliminated by passing the supernatant through a chromatography column. Effective removal of phosphate was qualitatively tested by the addition of green reagent to the flow through. To determine CaN activity, 5 µl of flow through was incubated with RII phosphopeptide, a known substrate for CaN, and the free phosphate released was detected based on the Malachite green assay (30) . Since EGTA inhibits calcineurin, total CaN phosphatase activity was reported as the difference between phosphatase activity (total) and phosphatase activity (EGTA buffer).

Western blotting
Splenic cells were sonicated twice for 15 s on ice-cold lysis RIPA buffer [20 mM HEPES, pH 7.5; 1% Triton X-100; 150 mM NaCl; 10 mM EDTA; 2 mM 4-(2-aminoethyl) benzenesulphonyl fluoride; 20 µg/ml leupeptin; and 200 µg/m benzamidine], and lysates were clarified at 15,000 g for 30 min at 4°C. The supernatants were collected, and the protein concentration was determined by the Bradford assay. Fifty micrograms of lysates was boiled for 5 min in the SDS sample buffer and analyzed by SDS-PAGE in 10% separating gel. After electrophoresis, the proteins were transferred to Hybond-P polyvinylidene difluoride membranes (Hybond, Uppsala, Sweden) by electroblotting. Efficiency of transfer was verified by Ponceau red staining. Membranes were blocked overnight in Tris-buffered saline-Tween containing 5% of nonfat milk and then incubated with a specific antibody against the catalytic {alpha}-subunit of CaN (CaN{alpha}; 1:1000; Cell Signaling Technology, Danvers, MA, USA). Membranes were washed 3 times with Tris-buffered saline and incubated with peroxidase-conjugated donkey anti-rabbit IgG (1:5000; Amersham, Buckinghamshire, UK). Blots were developed with an ECL kit (Amersham), and bands were visualized after exposing blots to X-ray films. Band density was determined using a GS-700 Imaging Densitometer and analyzed by Molecular Analyst Software v. 1.5 (Bio-Rad Laboratories). Quantification of CaN was normalized to the amount of β-actin present.

Isolation of CD4+ T lymphocytes and adrenergic stimulation
Purified CD4+ T lymphocytes from naive rats were obtained using a two-step magnetic cell separation protocol. First, CD4+ monocytes/macrophages and B cells were eliminated by positive selection using indirect magnetic labeling with purified mouse anti-rat CD172a (clone ED9, isotype IgG1; AbD Serotec) and anti-rat CD45RA (clone OX-33, isotype IgG1; BD Pharmingen) in combination with rat anti-mouse IgG1 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Second, T-helper cells were positively selected from the CD172a/CD45RA-depleted splenocytes using rat CD4 MicroBeads (Miltenyi Biotec). The purity of the isolated CD4+ T cells was typically 92–95%, as verified by flow cytometry. Isolated CD4+ T lymphocytes (1x106) were stimulated for 60 min in sterile polystyrene tubes (BD Pharmingen) in the presence or absence of indicated concentrations of terbutaline and/or nadolol (Sigma). CaN activity was determined in CD4+ T-cell lysates.

Statistical analyses
Differences between groups were analyzed using ANOVA. Statistically significant differences were reported when P < 0.05. Post hoc analyses were completed using Tukey’s test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fluid intake
The first encounter with saccharin taste induced neophobia, a stereotypic rodent behavior to novel food (Fig. 1b , first association trial). Stimuli contingency allowed conditioned animals (CS and CSo groups) to associate the sweet taste of saccharin with CsA administration. Clear avoidance behavior was elicited in these groups by later representations of the taste. In contrast, no avoidance behavior was shown when the same stimuli were inverted and the interstimuli interval was extended to 9 h (NP group). For conditioned animals, two further association trials resulted in strong and stable associative learning, as demonstrated by the strong resistance to extinction (CS group; Fig. 1b ), in agreement with previous reports (31) .

Ex vivo T-cell responsiveness
In the presence of CsA, transcription of Th1-cytokine genes such as IL-2 and IFN-{gamma} is strongly inhibited in T lymphocytes, concomitantly reducing cell proliferation (26) . In contrast, it is known that Th2 cytokines such as IL-4 are not affected by CsA (32 , 33) . Therefore, we assessed ex vivo IL-2, IFN-{gamma}, and IL-4 production by splenocytes after 48 h of stimulation with the T-cell stimulus anti-CD3 (Fig. 2a-c ). Cytokine analyses confirmed the expected reduction in IL-2 and IFN-{gamma} production in CsA-treated animals (US group). More important, the CS group also displayed a pronounced and significant reduction in Th1-cytokine production compared with both noncontingent paired and conditioned not-evoked animals. Both control groups received exactly the same pharmacological treatment but differed in the conditioning assignment. In contrast, production of the Th2-cytokine IL-4 remained unaffected in all groups. These changes in T-lymphocyte reactivity were not due to alterations in T-lymphocyte subsets in the spleen, as confirmed by flow cytometry (Supplemental Fig. 2). The data demonstrate the specificity of behavioral conditioning on ex vivo Th1-cytokine production.


Figure 2
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  		Figure 2. Conditioned immunosuppression. a–c) Ex vivo production of Th1. (a,b) and Th2 cytokines (c) after 48 h of stimulation with anti-CD3. d) Splenic T-lymphocyte proliferation after 72 h of ex vivo stimulation with anti-CD3. Data are expressed as means +se. *P ≤ 0.05 vs. CSo and NP, #P ≤ 0.05 vs. N; ANOVA with Tukey’s post hoc test. T-lymphocyte subset composition of the spleen and blood was not affected (see Supplemental Fig. 2).

To further confirm the functional significance of the conditioned cytokine reduction, we also analyzed ex vivo T-lymphocyte proliferation after anti-CD3 stimulation. Both the CS and US groups showed a pronounced reduction in T-cell proliferation (Fig. 2d ). Notably, the conditioned effects on T-lymphocyte proliferation and cytokine production are independent of any residual CsA effect, as confirmed by the results of the CSo and NP groups. Although receiving the same pharmacological treatment as the CS group, the CSo and NP groups showed neither a reduction in Th1-cytokine production nor a suppression of cell proliferation (Fig. 2 ). Furthermore, we (13 , 34) previously reported that CsA plasma levels were undetectable at evocation time in conditioned animals. In addition, conditioned immunosuppression cannot be attributed to the immunosuppressive properties of glucocorticoids, since plasma corticosterone was unaffected in the evocation phase (Supplemental Fig. 1; reported in ref. 13 ). Finally, peripheral blood cell composition was also not affected by conditioning (Supplemental Fig. 2).

CaN activity and CaN{alpha} protein expression
To further explore the cellular mechanism underlying the conditioned immunosuppression by CsA, we assessed CaN activity in splenic homogenates. Pharmacological CaN inhibition by CsA impaired Th1-cytokine production and reduced T-lymphocyte proliferation after antigenic challenges in vivo and ex vivo (25 , 27 , 35) . As expected, pharmacological controls (US group) displayed a strong reduction in splenic CaN activity (Fig. 3a ). Notably, however, conditioned animals (CS group) also showed a pronounced and statistically significant reduction in CaN activity compared with behavioral controls (CSo and NP groups). Western blot analysis of the expression of the catalytic subunit CaN{alpha} certifies that neither CsA administration nor conditioned response affected the protein level of the catalytic subunit CaN{alpha} (Fig. 3b, c ), demonstrating that the observed alterations in CaN activity were independent of changes in CaN protein. Thus, for the first time, these results identify CaN as the target molecule of conditioned immunosuppression by CsA.


Figure 3
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  		Figure 3. CaN inhibition induced by conditioning. a) Phosphatase activity of CaN. b, c) Relative protein expression of the catalytic subunit CaN{alpha} (61 kDa) to β-actin (42 kDa) was assessed in splenic homogenates. d) CaN activity measurement in lysates of freshly isolated naive CD4+ T lymphocytes (1x106) exposed to terbutaline (Ter) in the presence and absence of nadolol (Nad). Data are expressed as means ± SE (a, b); means ± SE of triplicates (d). *P ≤ 0.05 vs. CSo and NP, #P ≤ 0.05 vs. N; ANOVA with Tukey’s post hoc test.

Adrenergic modulation of CaN activity in vitro
CD4+ T lymphocytes express β2-adrenoceptors (36 , 37) . In addition, experimental findings demonstrate that the conditioned immunosuppression by CsA is mediated via the sympathetic innervation of the spleen and is β-adrenoreceptor dependent (13 , 22 , 23) . To elaborate a potential link between conditioned induced β2-adrenoreceptor stimulation and inhibition of CaN activity, splenic CD4+ T cells of naive rats were exposed to different concentrations of the β-adrenoreceptor agonist terbutaline in the presence or absence of the nonselective β-adrenoreceptor antagonist nadolol. CaN activity in T-lymphocyte lysates was assessed after 60 min of incubation (Fig. 3d ). β2-Adrenoreceptor stimulation dose dependently reduced CaN activity in isolated CD4+ T lymphocytes, with these effects being reversed by the nonselective adrenoreceptor antagonist nadolol (Fig. 3d ). These data document that β2-adrenoreceptor stimulation affects CaN activity in enriched T cells and suggest that sympathetic-T-lymphocyte interaction is mediating the conditioned CaN inhibition in splenocytes.

In summary, the CsA treatment as well as behavioral conditioning induced similar immunosuppressive statuses in T lymphocytes by specifically impeding relevant Th1-cytokine production and T-lymphocyte proliferation after ex vivo stimulation with anti-CD3. More important, CaN phosphatase activity is inhibited in both splenocytes from conditioned animals after saccharin reexposure and CsA-treated animals, illustrating for the first time the intracellular target responsible for the conditioned immunosuppression by CsA. In addition, these observations were further confirmed by modeling the sympathetic-T-lymphocyte interaction in vitro.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The functionality of CNS-immune system interaction has been extensively documented by behavioral studies (8 , 10 , 11) describing how immunosuppression can be elicited by pavlovian conditioning. However, the intracellular mechanisms responsible for this behavioral conditioned immunosuppression were still unclear. Here we demonstrate, in a model using CsA as an unconditioned stimulus in a learned taste avoidance paradigm, that inhibition of CaN activity in T lymphocytes by β-adrenoreceptor stimulation seems to be the underlying intracellular mechanism mediating these "learned placebo effects" in splenocytes.

Our data document splenic CaN to be modulated by associative learning processes. After saccharin-CsA association, reexposure to the taste stimulus results in a strong inhibition of CaN activity in splenocytes, subsequently inducing a specific inhibition of Th1-cytokine, but not Th2-cytokine, production and T-lymphocyte proliferation on anti-CD3 challenge. Recent experimental evidence indicates that this conditioned response relies on neocortical-sympathetic regulation of splenic immunocompetent cells. Complete disruption of the conditioned immunosuppression has been observed as result of the following: 1) lesioning the insular cortex or the ventromedial hypothalamic nucleus before evocation (18) , 2) surgical denervation of the spleen before association or chemical sympathectomy before evocation (13 , 22) , and 3) β-adrenoreceptor blockade during evocation (22) . The present data further contribute to identify the main players and mechanisms mediating this learned immune phenomenon. Here we document that both CsA treatment as well as pavlovian conditioning specifically suppress splenic T-lymphocyte reactivity by targeting CaN as common intracellular mediator acting through different pathways. CaN phosphatase activity was significantly inhibited in splenic homogenates after taste evocation (saccharin reexposure). Furthermore, modeling in vitro the sympathetic-T-lymphocyte interaction, occurring in the spleen during evocation, we could confirm that CaN activity can be inhibited in CD4+ T lymphocytes by stimulation of β-adrenoceptors (i.e., terbutaline), with these suppressive effects being antagonized by the β-adrenoreceptor antagonist nadolol. These data support the hypothesis that sympathetic-immune interaction is responsible for behavioral conditioned CaN inhibition in lymphocytes.

Apart from immunosuppression, CsA induces additional effects. Of notice for the present results is the well-known CsA-induced hypertension (38) . This increased sympathetic activity follows (as a reflex) vagal afferent activation (39) . Therefore, are all CsA pharmacological properties consequently transferred to the gustatory conditioned stimulus? So far, no empirical data are available, but theoretically two main alternatives exist (9 , 40) . A first possibility is fully consistent with a specific pavlovian conditioning. The association between sensory (saccharin taste) and immunological perturbations (immunosuppression by CsA) is encoded and stored in the brain, inducing a particular and independent engram. An alternative model (41) proposes that a generic memory triggered by a specific gustatory stimulus transmits just a part of the pattern to which some immuncompetent cells are selective responders due to "acquisition residual" effects to the periphery. Employing conditioning protocols using non-CaN inhibitors that elicit a conditioned sympathetic response (42 , 43) would be of great value to dissect the specificity aspect behind the observed conditioned effects on splenic CaN activity. So far, our data support the hypothesis of a specific conditioned immunological response, since CsA-induced hypercortisolemia (34 , 44) was not conditioned (Supplemental Fig. 1). However, conclusive evidence can only come from experiments designed to assess passive forgetting, discriminative conditioning, and conditioning generalization to other immunosuppressants.

Behavioral conditioning paradigms may have therapeutic potential in clinical settings where immunosuppression is desirable. For instance, in a murine model of systemic lupus erythematosus, a partial reinforcement conditioning protocol (taste paired with the cytotoxic drug cyclophosphamide) retarded proteinurea and mortality in conditioned mice (45) . Similarly, repeated recalls of a reinforced taste-CsA conditioning, together with the administration of subtherapeutical doses of CsA during the evocation phase, significantly prolonged the rejection time of heterotopic heart allografts, with 25% of the grafts fully functional 100 days after transplantation (16) . More important, behaviorally conditioned immunosuppression has also been documented in humans (46) . Using a conditioning protocol analogous to the one described here for rodents, we (12) recently reported ex vivo proliferation, Th1-cytokine production, and mRNA expression to be suppressed in peripheral blood mononuclear cells of healthy male subjects. Taking into consideration that behavioral conditioning and expectations have been identified as major neurobiological mechanisms of the placebo response (47 , 48) , it is now becoming clear why, how, and when its effects significantly influence the course of a disease, as well as the response to a therapy (49 50 51) . The documented mechanism underlying conditioned immunosuppression by CsA promotes such behavioral protocols to be used as supportive therapies together with the standard pharmacological regimens with the aim of maximizing therapeutic efficacy for the patient’s benefit, reducing unwanted side effects, and, last but not least, saving costs.

In summary, these data identify CaN as the intracellular target for inducing conditioned immunosuppression by CsA in T lymphocytes involving β-adrenergic mechanisms, highlighting the potential underlying association (i.e., taste-CaN inhibition) and contributing to our understanding of the mechanisms behind "learned placebo effects."


   ACKNOWLEDGMENTS
 
This work was supported by ETH Fund grant 0-20297-05 and by the Dr. Donald C. Cooper Fund (ETH Zurich).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication July 14, 2008. Accepted for publication November 20, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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