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(The FASEB Journal. 2002;16:1869-1873.)
© 2002 FASEB

Behavioral conditioning of immunosuppression is possible in humans

MARION U. GOEBEL*, ALMUTH E. TREBST*, JAN STEINER*, YU F. XIE*, MICHAEL S. EXTON*, STILLA FREDE{dagger}, ALI E. CANBAY{ddagger}, MARTIN C. MICHEL{ddagger}, UWE HEEMANN{ddagger} and MANFRED SCHEDLOWSKI*1

Departments of
* Medical Psychology,
{dagger} Physiology and
{ddagger} Medicine, University of Essen, Essen, Germany

1Correspondence: Department of Medical Psychology, University of Essen, Hufelandstr. 55, 45122 Essen, Germany. E-mail: manfred.schedlowski{at}uni-essen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Behavioral conditioned immunosuppression has been described in rodents as the most impressive demonstration of brain-to-immune system interaction. To analyze whether behavioral conditioned immunosuppression is possible in humans, healthy subjects in this double-blind, placebo-controlled study were conditioned in four sessions over 3 consecutive days, receiving the immunosuppressive drug cyclosporin A as an unconditioned stimulus paired with a distinctively flavored drink (conditioned stimulus) each 12 h. In the next week, re-exposure to the conditioned stimulus (drink), but now paired with placebo capsules, induced a suppression of immune functions as analyzed by the IL-2 and IFN-{gamma} mRNA expression, intracellular production, and in vitro release of IL-2 and IFN-{gamma}, as well as lymphocyte proliferation. These data demonstrate for the first time that immunosuppression can be behaviorally conditioned in humans.—Goebel, M. U., Trebst, A. E., Steiner, J., Xie, Y. F., Exton, M. S., Frede, S., Canbay, A., Michel, M. C., Heemann, U., Schedlowski, M. Behavioral conditioning of immunosuppression is possible in humans.


Key Words: conditioned reaction • interleukin-2 • cyclosporin • behavioral conditioning • cyclosporin A


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CHARACTERIZATION OF THE effect of behavior on immune function may represent an innovative therapeutic approach to immune-related disease. Specifically, the endogenous pharmacology of the brain may potentially be used as a supplement to traditional drug delivery. The capacity of brain pharmacology has been experimentally demonstrated in rodents by behaviorally conditioned immune suppression (1 2 3) . We have established a conditioning model in the rat using the novel stimulus saccharin as the conditioned stimulus (CS) and the immunosuppressive drug cyclosporin A as the unconditioned stimulus (UCS) (4) , which affects activated T lymphocytes and selectively reduces their secretion of interleukin-2 (IL-2) and interferon-{gamma} (IFN-{gamma}) on a transcriptional level (5 , 6) . After several CS-UCS pairings, re-exposition to the CS in the absence of the drug elicits a conditioned suppression of splenocyte proliferation and IL-2 and IFN-{gamma} production, which was mediated via neural innervation of the spleen (2 , 7) . Demonstrating that conditioned immunosuppression prolongs the survival time of transplanted heart allografts and inhibits contact sensitivity in rats, we showed that the behaviorally conditioned immunosuppression is biologically relevant (4) . Since extrapolation of this paradigm from rodents to humans is all but certain, we investigated in this double-blind and placebo-controlled study whether behaviorally conditioned immunosuppression is inducible in humans.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and protocol
Thirty-four healthy male volunteers (26.7±4.2 years) were tested during a 2 wk period (Fig. 1 A). Starting on day 1 at 1800, the experimental group (n=18) received 4 doses of cyclosporin A (CsA, 2.5 mg/kg) every 12 h in capsule form (Sandimmun®, Novartis, Basel, Switzerland) together with a green-colored, novel-tasting drink (150 mL strawberry milk aromatized with 1 drop lavender oil). Five days later, subjects were re-exposed to the drink but received identical looking placebo capsules. The control group (n=16) was treated similarly but received placebo throughout the study. Blood was drawn on the first day at 0800 and at 1000 on day 3 and 10 for determination of immunological parameters. We monitored CsA plasma levels by measuring the trough level at 0800 and maximum level at 1000 on day 3 and at 0800 on days 8 and 10 (Fig. 1A ). To reduce subjects’ expectations, participants were told that should they receive CsA, it may occur in 1 of the 2 wk but were not informed as to which week. To avoid conditioned responses to stimuli in the experimental setting, we conducted the protocol in two separate rooms, changing the location for each drinking session. The study protocol was approved by the ethics committee for human investigation of the University of Essen.



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  		Figure 1. On days 1–3, subjects received CsA (2.5 mg/kg/12 h) paired with a distinctively flavored drink; participants were re-exposed to the drink, now paired with placebo capsules on days 8–10 (A). Results of quantitative PCR for IL-2 (B) and IFN-{gamma} (C) on days 3, 8, and 10. RNA levels were measured in fg/µL. Relative changes in RNA levels from baseline were calculated for conditioned subjects ({blacksquare}) and placebo-treated controls ({square}). Data are expressed as relative changes from baseline cytokine expression on day 1. Bars represent mean ± SE; *P < 0.05, **P < 0.01,***P < 0.001 compared with placebo-treated controls. Representative ethidium bromide stained gels of qualitative PCR for IL-2 (D) and IFN-{gamma} (E) cDNA measured on days 1, 3, and 10.

IL-2 and IFN-{gamma} mRNA expression
Peripheral mononuclear cells were isolated according to a standard Ficoll protocol, resuspended in cell culture medium (RPMI with 5% fetal calf serum), and adjusted to 5 x 106 cells/mL. Cells were incubated with 25 µg/mL PHA (ICN, Frankfurt, Germany) for 4 h. Cells were harvested by centrifugation at 13,000 rpm for 5 min. Guanidine thiocyanate (GTC) (+0.1% ß-mercaptoethanol) was added to the pellet and samples were stored at -20°C. Total RNA was isolated by acid phenol chloroform (21) and redissolved in water. RNA concentration was determined by measurement of the optical density at 260 nm. Total RNA (1 µg) was reverse transcribed into cDNA using oligodT15 as a primer for reverse transcriptase (AMV reverse transcriptase; Promega, Heidelberg, Germany). Expression states of IL-2 and IFN-{gamma} mRNA were determined by qualitative PCR. Specific PCR products were run on ethidium bromide-stained agarose gel (2.5%) and visualized under UV light.

Quantification of IL-2 and IFN-{gamma} cDNA was conducted by real-time PCR (Gene Amp 5700, PE Applied Biosystems, Weiterstadt, Germany) using the SYBR Green PCR Master Mix (PE Applied Biosystems). Sense and antisense for human IL-2 were 5'-CCTCAACTCCTGCCACAATG and 3'-TTGCTGATTAAGTCC CTG GG and for IFN-{gamma} 5'-TCGTTTTGGGTTCTCTTGGC and 3'-GCAGGCAGGACAACCATTAC. Serial dilutions of the human IL-2 and IFN-{gamma} specific cDNA fragment were used as standard. Each sample was quantified in triplicate. RNA levels were measured in fg/µL. Relative changes in RNA levels from baseline were calculated.

Intracellular IL-2 and IFN-{gamma} expression in activated lymphocytes
For detection of intracellular IL-2 and IFN-{gamma} in activated lymphocytes, we used flow cytometry (FACSCalibur, Becton-Dickinson, San Jose, CA). We cultured peripheral mononuclear cells from heparinized blood for 4 h in medium (RPMI with 10% fetal calf serum and 1% antibiotics) with ionomycin (1 µg/mL) and PMA (10 ng/mL), adding brefeldin A (10 µg/mL) after 1 h. Cells were centrifuged and fixated with paraformaldehyde (ICN) and perforated with saponin (ICN). Positive two- and three-color staining was used with monoclonal antibodies conjugated to either fluorescein isothiocyanate, phycoerythrin, or peridinin chlorophyll protein (Becton-Dickinson, PharMingen, and DAKO, Hamburg, Germany). Appropriate isotypic controls were used for each assay to determine nonspecific staining. Fluorescence compensation was performed using CaliBRITE beads (Becton-Dickinson) and FACSComp software. Optimal amounts of antibodies were used, counting 10,000 events per tube using CellQuest software. A complete blood count was performed and used to quantify lymphocyte, monocyte, and granulocyte populations.

ELISA for IL-2 and IFN-{gamma} production
Whole blood was diluted (1:10) in medium (RPMI with 1% antibiotics) and incubated with 25 µg/mL PHA (ICN) for 24 h. Supernatants were collected and stored at -20°C. IL-2 and IFN-{gamma} production was determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN). Samples were analyzed in duplicate.

Lymphocyte proliferation
Peripheral mononuclear cells were collected from EDTA blood and washed several times in PBS. Cell numbers were adjusted to 106 cells/mL and cultured for 72 h in the presence of ConA (5 µg/mL). Cells were then pulsed with 10 µL/well [3H] thymidine (1 µCi) and harvested 24 h later. Radioactivity was measured using a ß-counter.

Statistical analysis
Data were analyzed using 2-factor (group x time) repeated measures analysis of variance (SPSS, Chicago, IL). If not otherwise stated, P values represent post hoc test results for group comparison.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We analyzed mRNA expression, cytokine synthesis, and lymphocyte proliferation at baseline (day 1), after the conditioning phase to analyze the CsA effects (day 3), and after re-exposition to the conditioned stimulus (CS) to determine the conditioned effects (day 10) (Fig. 1A ). Analyzing IL-2 and IFN-{gamma} mRNA expression with real-time PCR, subjects receiving CsA showed a significant reduction of IL-2 mRNA expression compared to controls (P<0.001) (Fig. 1B ). More important, re-exposure to the novel drink (CS) alone significantly reduced IL-2 mRNA (P<0.05) and IFN-{gamma} mRNA (P<0.05) expression in the conditioned group vs. the control group (Fig. 1B , conditioned effect). To determine whether these effects were due to behaviorally conditioned suppression and not to residual effects of the drug, mRNA analysis performed in a subset of subjects (n=21) on day 8 revealed that IL-2 and IFN-{gamma} had returned to baseline levels (Fig. 1B, C ). This concurs with CsA plasma levels monitored at each time point. CsA administration resulted in peak and trough levels of 1109.8 (±217.6 SD) ng/mL and 144.5 (±33.4 SD) ng/mL, respectively, on day 3. However, at the time of re-exposition to the novel drink (CS) on day 8, 5 days after the last CsA administration (Fig. 1A ), no CsA was detectable in circulation. Thus, re-exposure to the conditioned stimulus (novel drink) induced a suppression of IL-2 and IFN-{gamma} mRNA expression in peripheral blood lymphocytes in conditioned subjects.

To further analyze these behaviorally conditioned effects, IL-2 and IFN-{gamma} production of activated CD3+CD4+ lymphocytes was analyzed by flow cytometry. In the conditioned group, re-exposure to the novel drink (CS) significantly decreased IL-2 and IFN-{gamma} production of CD3+CD4+ lymphocytes (P<0.001 and P<0.01, respectively), as was observed after CsA treatment (P<0.001; P<0.01, respectively) (Fig. 2 ). These findings demonstrate that behavioral conditioning reduces not only cytokine gene expression, but also IL-2 and IFN-{gamma} production of CD3+CD4+ lymphocytes.



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  		Figure 2. Reduction of IL-2 (A) and IFN-{gamma} (B) production of CD3+CD4+ lymphocytes as measured at baseline (day 1), after 4 pairings of CsA (2,5 mg/kg/12 h) with a novel drink (CS) (CsA effect on day 3), and 4 re-exposures every 12 h to the novel drink (CS) without CsA (conditioned effect on day 10). Data are expressed as absolute numbers of helper T cells producing the cytokine for CsA-treated subjects ({blacksquare}) and for placebo-treated controls ({square}). Bars represent mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001 compared with placebo-treated controls.

In the next step, we determined the effect of behavioral conditioning on cytokine secretion by analyzing cytokine concentration by ELISA after 24 h phytohemagglutinin (PHA) stimulation in whole-blood cell cultures. Behaviorally conditioned subjects displayed significantly lower IL-2 (P<0.05) and IFN-{gamma} (P<0.05) production than controls after CS re-exposition (Fig. 3 A, B). To confirm the functional relevance of conditioned changes of cytokine levels in the supernatant, we analyzed the proliferative response of the lymphocytes with a 3H-thymidine assay, demonstrating a significant behaviorally conditioned suppression of the functional capacity of T lymphocytes (P<0.01) (Fig. 3C ).



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  		Figure 3. Reduction of IL-2 (A) and IFN-{gamma} (B) production after 24 h PHA stimulation in whole-blood cell cultures and 3H-thymidine uptake (C) after 72 h ConA stimulation as measured at baseline (day 1), after 4 pairings of CsA (2.5 mg/kg/12 h) with a novel drink (CsA effect, day 3), and after 4 re-exposures every 12 h to the novel drink without CsA (conditioned effect, day 10). Data are expressed as cytokine concentrations in the supernatant (A, B) and counts/min (C) for CsA-treated subjects ({blacksquare}) and placebo-treated controls ({square}). Bars represent mean ± SE. *P < 0.05, **P < 0.01 compared with placebo-treated controls.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These data for the first time demonstrate in humans that after repetitive pairings of an immunosuppressant drug (UCS) with a novel and distinctively flavored drink (CS), re-exposition to the novel drink alone is sufficient to elicit an immune suppression that mirrors the actual drug effect. Since the original demonstration of conditioned immunosuppression in rodents (1) , behavioral conditioning has been shown to modulate humoral and cellular immune functions such as antibody responses (8) , graft-vs.-host reaction (9) , natural killer cell activity (10) , and the delayed-type hypersensitivity response (11) . In several studies our group has consistently demonstrated that conditioning procedures using CsA as an UCS reduces the in vitro capacity of isolated rat splenocytes to proliferate and produce IL-2 in response to a mitogen (12 , 13) .

Moreover, the biological relevance of behavioral conditioned immunosuppression has been demonstrated in animal models (3) . Specifically, we have shown that conditioning CsA-like effects prolonged the survival time of a transplanted heart allografts (2) . Conditioned immunosuppression also ameliorated the progression of the disease in animal models of contact sensitivity (11) , lupus (14) , and rheumatoid arthritis (15) .

Despite the wealth of evidence describing an intensive bidirectional communication among the immune, neuroendocrine, and central nervous systems, (16 17 18 19 20) , the efferent and afferent mechanisms of behaviorally conditioned immunosuppression remain unclear (2) . Several neuroendocrine pathways have been suggested to be involved in the acquisition and recall of the conditioned immune response, such as the sympathetic nervous system (12) and the hypothalamus-pituitary-adrenal axis (21) . To analyze whether the conditioning procedure used in this study is associated with neuroendocrine changes, we determined epinephrine, norepinephrine, and cortisol concentrations in plasma at each time point. Subjects showed no significant changes in these parameters (data not shown). This suggests that neural mechanisms rather than humoral pathways seem to regulate the behaviorally conditioned immunosuppressive effects in this study, concurring with the neural route of conditioned CsA immune suppression in the rat (7) .

This study demonstrates for the first time in humans in a double-blind, placebo-controlled design that behavioral conditioning is able to mimic the immunological effects of an immunosuppressive drug. This effect is specific to the effects of the drug and is observed on levels of cellular gene expression, intracellular, and extracellular cytokine production of activated T lymphocytes. These findings may have therapeutic implications for the treatment of immune-related diseases by implementing behavioral conditioning paradigms as supplementary therapy to traditional drug regimes.


   ACKNOWLEDGMENTS
 
This study was supported by the Grant I/75100 from the Volkswagen Foundation and by a Grant from the Deutsche Forschungsgemeinschaft (Sche 341/9–1).

Received for publication April 24, 2002. Accepted for publication August 8, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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