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The Journal of Immunology, 2002, 169: 3630-3636.
Copyright © 2002 by The American Association of Immunologists

µ-Opioid Receptor Mediates Chronic Restraint Stress-Induced Lymphocyte Apoptosis1

Jinghua Wang*, Richard Charboneau{dagger},{ddagger}, Roderick A. Barke{dagger},{ddagger}, Horace H. Loh* and Sabita Roy2,*,{dagger},{ddagger}

* Department of Pharmacology, University of Minnesota, Minneapolis, MN 55455; {dagger} Department of Surgery, Veterans Affairs Medical Center, Minneapolis, MN 55417; and {ddagger} North Memorial Medical Center, Robbinsdale, MN 55422


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Psychological stress is associated with immunosuppression in both humans and animals. Although it was well established that psychological stressors stimulate the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system, resulting in the release of various hormones and neurotransmitters, the mechanisms underlying these phenomena are poorly understood. In this study, µ-opioid receptor knockout (MORKO) mice were used to investigate whether the µ-opioid receptor mediates the immunosuppression induced by restraint stress. Our results showed that wild-type (WT) mice subjected to chronic 12-h daily restraint stress for 2 days exhibited a significant decrease in splenocyte number with a substantial increase in apoptosis and CD95 (Fas/APO-1) expression of splenocytes. The effects are essentially abolished in MORKO mice. Furthermore, inhibition of splenic lymphocyte proliferation, IL-2, and IFN-{gamma} production induced by restraint stress in WT mice was also significantly abolished in MORKO mice. Interestingly, both stressed WT and MORKO mice showed a significant elevation in plasma corticosterone and pituitary proopiomelanocortin mRNA expression, although the increase was significantly lower in MORKO mice. Adrenalectomy did not reverse restraint stress-induced immunosuppression in WT mice. These data clearly established that the µ-opioid receptor is involved in restraint stress-induced immune alterations via a mechanism of apoptotic cell death, and that the effect is not mediated exclusively through the glucocorticoid pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It is well established that psychosocial manipulations influence a variety of immune parameters (1, 2). In humans, behavioral and psychosocial factors, including stressful life experience, mediate changes in immunity and are considered cofactors in susceptibility to infection and immune-related disorders (3, 4). Stress can in fact promote or enhance tumor development, autoimmunity, and infectious diseases (5, 6, 7). Conversely, psychosocial well being (e.g., social support) has been shown to increase NK cell function and improve survival in cancer patients (8). Behavioral manipulations have also been exploited to modify allergic reactions, the course of system lupus erythematosus, and the symptoms of multiple sclerosis (9). Studies on laboratory animals, in which environmental stimuli, genetic background, and immune challenge can be controlled, can potentially provide insight into the molecular and cellular processes that underlie the complex interactions among brain, behavior, and the immune system that influence pathogenic processes in humans. Numerous studies have revealed that adhesion molecules, various hormones (e.g., corticosteroids), and neurotransmitters (e.g., catecholamines) may be involved in stress-induced immune system changes, but the exact mechanisms by which stress affects the immune response have not been identified and characterized (10, 11).

Restraint stress, a key animal model, is believed to be largely psychological in nature, and induces the production of various immunosuppressive mediators. Among these, corticosteroids and endogenous opioids are the best-recognized mediators modulating the immune response (12, 13). The immunoregulation of corticosteroids is mediated by specific binding of glucocorticoids to glucocorticoid receptors that are expressed in all leukocytes. Corticosteroids have been shown to promote the immune response during acute stress and to inhibit the immune response during chronic stress (14). Restraint stress alters lymphocyte sensitivity to corticosterone, and restraint stress-induced corticosterone secretion was shown to affect only delayed-hypersensitivity reactions (12). Endogenous opioid peptides, in contrast, are known to be elevated by both acute and chronic stress, and play a critical role in regulating stress-induced changes of the immune system (15). Blockade of endogenous opioids with naloxone results in attenuation or reversal of stress-induced immune alterations (13, 14). Although the role of endogenous opioids in mediating stress-induced immune alterations is thus well accepted, the mechanisms responsible for the phenomenon remain controversial. Three classic opioid receptors (µ, {delta}, and {kappa}) have been identified in the mammalian brain, all of which have also been found in lymphocytes (16, 17). The presence of these receptors in these cells indicates that the immune system is sensitive to endogenous opioids.

Previous studies by Yin et al. (13, 18) have shown that chronic restraint stress results in decreased splenic cellularity by a mechanism associated with CD95-mediated apoptosis. Naloxone (an opioid antagonist) and Fas antagonists attenuated this effect, implicating endogenous opioids involvement in CD95-mediated splenocyte apoptosis. Based on these findings, we further investigated whether the µ-opioid receptor plays a major role in the immune alterations induced by restraint stress. We have recently generated a µ-opioid receptor knockout (MORKO)3 mouse line, which lacks the µ-opioid receptor (19), and is therefore a very useful tool in determining the role of these receptors in various physiological functions. We have previously shown that morphine-mediated modulation of immune parameters is significantly attenuated in the MORKO mice (20). In this study, we use these animals to determine the role of the µ-opioid receptor in immune changes induced by restraint stress, and in particular, for evaluating its role in the elevation of endogenous opioids that has been shown to occur following restraint stress. Our results show that chronic 12-h daily restraint for 2 days promotes expression of pituitary proopiomelanocortin (POMC) and circulating plasma corticosterone levels in both wild-type (WT) and MORKO mice. Stress-induced splenocyte reduction and apoptosis, CD95 overexpression, and inhibition of lymphocyte proliferation, and of IL-2 and IFN-{gamma} synthesis, however, were essentially abolished in MORKO mice. These results establish for the first time the involvement of the µ-opioid receptor in chronic restraint stress-mediated lymphocyte apoptosis and immunosuppression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
MORKO mice

MORKO mice (BALB/c x C57BL/6 genetic background) were produced as previously described by Loh and colleagues (19). Briefly, a XhoI/XbaI fragment, which spans the entire sequence of exons 2 and 3 of the µ-opioid receptor, was replaced with a Neor cassette, followed by the ligation of a thymidine kinase expression cassette to the 3' end of this segment. WT mice (CB6F1/J, BALB/c female x C57BL/6 male), 8 wk of age, were obtained from The Jackson Laboratory (Bar Harbor, ME). A maximum of four mice was housed per cage. Food and tap water were available ad libitum. The animal room was maintained on a 12-h light/dark cycle, with constant temperature (72 ± 1 °F) and 50% humidity.

Restraint stress procedures

WT and MORKO mice were subjected to an established chronic physical restraint protocol (13). They were placed in a 50-ml conical centrifuge tube with multiple ventilation holes. Mice were restrained horizontally in the tubes for 12 h, followed by a 12-h rest, during which food and water were provided ad libitum. The control mice were kept in their original cages, receiving food and water only during the rest interval of the experimental groups. Mice were restrained for two cycles, as described. This restraint procedure was approved by the Institutional Animal Care and Use Committee of The Minneapolis Veterans Affairs Medical Center.

Naltrexone or placebo pellet implantation

MORKO mice were anesthetized with methoxyflurane (Mallinckodt Veterinary, Mundelein, IL). Naltrexone (generously provided by the National Institute on Drug Abuse) or placebo pellets were placed into pockets formed to the left of the dorsal midline. The skin incision was closed with a surgical clip.

Analysis of splenic cellularity and lymphocyte subtypes

Immediately after two cycles of 12-h restraint stress, the stressed or control animals were then sacrificed by cervical dislocation. Each spleen was removed with sterile forceps, placed on a metal sieve (Sigma-Aldrich, St. Louis, MO), and submerged in cold 10% newborn calf serum RPMI 1640 medium. The cell suspension was prepared by forcing the tissue through the sieve with a sterile syringe plunger. Cells were washed twice with PBS and then counted in a Brightline hemacytometer. Lymphocyte subtypes were analyzed by flow cytometry. Briefly, splenic cell suspensions were preincubated for 30 min at 4°C with an anti-mouse FcRII/III Ab (clone 2.4G2), to prevent background signals from nonspecific binding. The cells were then washed once with PBS containing 2% FCS plus 0.1% NaN3, and incubated at 1 x 106 cells/sample for 30 min at 4°C with FITC-conjugated anti-CD4, PE-conjugated anti-CD8, and FITC anti-CD19 (all from BD PharMingen, San Diego, CA). After washing, a flow cytometric analysis of 104 cells was performed using a FACSCalibur. Residual erythrocytes, dead cells, and debris were excluded from the analysis. Quantification of each subpopulation was determined by multiplying the white blood cell count by the specific subpopulation percentage.

Determination of apoptosis by TUNEL assay

Apoptotic nuclear DNA fragments were determined using the TUNEL ApoAlert DNA Fragmentation Assay kit (Clontech Laboratories, Palo Alto, CA), according to manufacturer’s instructions. Briefly, splenic cells (5 x 105 cells) from WT and MORKO mice were fixed in 1% formaldehyde/PBS for 20 min at 4°C. Cells were then permeabilized with 70% ice-cold ethanol for at least 4 h at -20°C. Each sample was then washed twice with PBS, incubated for 1 h at 37°C with TdT enzyme and FITC-dUTP in a reaction buffer. Cells were washed with PBS and resuspended in 0.5 ml propidium iodide/RNase/PBS, then incubated for 30 min at room temperature. The frequency of apoptotic cells was determined by detecting fragmented nuclear DNA measured by flow cytometry. Data were analyzed using CellQuest software (BD Biosciences, San Jose, CA).

Splenic lymphocyte proliferation assay

The resulting splenic cell suspension was washed in cold RPMI 1640 medium and adjusted to a concentration of 2 x 106 cells/ml. Triplicate samples were plated onto 96-well plates containing Con A (5 µg/ml) and incubated for 72 h at 37°C, 5% CO2. Cells were pulsed with 0.2 µCi methyl-[3H]thymidine (Amersham, Piscataway, NJ) in a 20-µl vol and incubated for 8 h. Samples were lysed with distilled water and harvested onto glass filters using an automatic 96-well cell harvester (Skayron Instrument, Lier, Norway). The amount of labeled DNA was determined with a liquid scintillation counter (1900CA; Packard, Downers Grove, IL).

IL-2 and IFN-{gamma} measurement by ELISA

Splenic lymphocytes from MORKO and WT mice were adjusted to a final concentration of 2 x 106 cells/ml in 24-well plates. Cells were incubated in the presence of Con A (5 µg/ml) for 24 h at 37°C in a humidified 5% CO2 incubator. Culture supernatants were analyzed using cytokine-specific sandwich ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

Adrenalectomy

Bilateral adrenalectomy was performed on WT and MORKO mice. Mice were anesthetized by i.p. injection of 2.5% tribromoethanol (250 mg/kg). A 0.5-cm skin incision was made on the back. The skin on both sides of the incision was moved to the side, and a muscle incision was made on the top of each adrenal gland. The entire adrenal gland was removed with a pair of sterile fine forceps. The skin incision was closed with a surgical clip. After recovery, the mice were maintained by providing food and drinking water containing 0.9% sodium chloride ad libitum. Control animals (sham group) underwent the same surgical procedure as the adrenalectomized animals, except their adrenal glands were not removed. Experiments were performed with these mice 2–3 wk after adrenalectomy.

Corticosterone RIA

Animals were sacrificed, and plasma samples were obtained and stored at -70°C until time of analysis. Plasma corticosterone levels were determined using a 125I-coupled double Ab RIA (ICN Biochemicals, Costa Mesa, CA). Corticosterone concentration was expressed as ng/ml.

Analysis of POMC expression by Northern blot

Northern blot analysis was performed according to standard procedures. Total RNA was isolated from pituitary with the ToTALLY RNA isolation kit (Ambion, Austin, TX), according to the manufacturer’s instructions. A quantity amounting to 15 µg total RNA was analyzed by separation on a 1% agarose formaldehyde gel, followed by transfer to a Hybond-N membrane (Amersham). The mouse POMC and GADPH probes were labeled with [{alpha}-32P]dCTP according to instruction in random primed DNA labeling kit (Roche Molecular Biochemicals, Indianapolis, IN). Hybridization signals were detected by PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Analysis of CD95 expression by Western blot

Splenocytes were lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), supplemented with protease inhibitor mixture) for 15 min on ice. Lysates were centrifuged at 10,000 rpm at 4°C for 5 min. Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA) using BSA as the standard. A quantity amounting to 5 µg to tal protein was loaded on 10% SDS-PAGE gels and transferred onto nitrocellulose membrane. Incubating the membrane in Superblock (Pierce, Rockford, IL) for 1 h blocked nonspecific binding. Membranes were incubated overnight at 4°C in primary Ab (anti-Fas polyclonal Ab (M-20); Santa Cruz Biotechnology, Santa Cruz, CA). The blots were washed three times with TBST buffer and then incubated for 1 h at room temperature with anti-rabbit secondary Ab conjugated with HRP. Western blot analysis was conducted according to standard procedures using Supersignal Chemiluminescence Detection Substrate (Pierce).

Statistical analysis

For comparison of mean values between two groups, the unpaired t test was used. To compare values among multiple groups, ANOVA was applied. All values are mean ± SEM, except where otherwise indicated. Statistical significance was accepted at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effect of restraint stress on splenic cellularity and lymphocyte subset in WT and MORKO mice

WT and MORKO mice were subjected to chronic restraint stress according to the protocol described in the experimental design. Our results show that restraint stress dramatically affects splenic cellularity. Data from stressed WT mice showed about a 35% reduction in splenic lymphocytes when compared with unstressed controls. This reduction was abolished in MORKO mice. Furthermore, a drastic reduction in the number of splenic lymphocyte subsets (CD4, CD8, and CD19) was found in the stress group when compared with the control group in WT mice. This effect was not seen in MORKO mice (Fig. 1GoA). In contrast, no significant changes in the proportion of splenic lymphocyte subsets were found between either group (p > 0.05, Fig. 1GoB). These results suggest that chronic restraint stress exerts a profound effect on the number of lymphocyte subsets rather than on the proportion of lymphocyte subsets in the spleen. In addition, our data clearly established that stress-induced lymphocyte reduction appears to require the µ-opioid receptor.



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  		FIGURE 1. Effect of chronic restraint stress on absolute number (A) and proportion (B) of splenocyte subsets in WT and MORKO mice. The animals were restrained (stress group, n = 6) or deprived of food and water (control group, n = 6) 12 h daily for 2 days. The number and percentages of various cell subsets were then assessed as described in Materials and Methods. All data are expressed as the mean ± SEM. Statistical differences were determined by a factorial ANOVA followed by the unpaired t test. **, Significance at level p < 0.01 compared with the control group. Similar results were obtained in three independent experiments.

 
Stress-induced splenocyte apoptosis and CD95 overexpression are mediated by the µ-opioid receptor

The reduction of splenic cellularity induced by stress could be mediated by two possible mechanisms: lymphocyte migration or cell death. We previous reported that the exogenous opioid morphine can induce lymphocyte apoptosis through the µ-opioid receptor. This effect was completely abolished in lymphocytes from MORKO mice (20). To investigate whether lymphocyte reduction was a result of apoptosis and CD95 overexpression, we assessed splenic lymphocyte apoptosis by TUNEL assay and CD95 expression using Western blot. As shown in Fig. 2GoA, a significant number of apoptotic splenocytes was found in stressed WT mice (WT, stress 39.8 ± 4.2%**; **, p < 0.01 compared with control group, n = 6), whereas only a few apoptotic splenocytes were detected in stressed MORKO, and unstressed WT and MORKO mice (MORKO, stress 13.3 ± 1.3%; WT, control 10.3 ± 0.9%; MORKO, control 11.6 ± 1.5%). We next examined the CD95 protein levels in splenocytes using Western blot. In agreement with changes of splenic cellularity and apoptosis, stress-induced overexpression of CD95 only occurred in WT, but not in MORKO mice (Fig. 2GoB). These data suggest that stress-induced apoptosis results in the reduction of splenocytes, and this effect may be mediated by CD95-induced apoptosis, in which the µ-opioid receptor plays a prominent role.



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  		FIGURE 2. The µ-opioid receptors mediated chronic restraint stress-induced apoptosis and CD95 expression in splenocytes. A, Restraint stress-induced splenocyte apoptosis requires the µ-opioid receptors. WT and MORKO mice were subjected to a 12-h restraint daily for 2 days. Splenocytes from stress or control WT and MORKO mice were fixed in 1% paraformaldehyde. Splenocyte apoptosis was detected using TUNEL staining and flow cytometry analysis. The results shown are representative of three such experiments. B, Restraint up-regulated CD95 express was abolished in MORKO mice. Immediately after restraint stress, animals were sacrificed and proteins were extracted from splenocytes. CD95 expression was examined using Western blot analysis. To verify equality of loading, the blots were reprobed with anti-tubulin Ab (H-300; Santa Cruz Biotechnology). The results shown are representative of three independent experiments. Results were quantified using Image-Pro Plus software and presented as a ratio of CD95/anti-tubulin. Data are expressed as mean ± SEM of six animals per group. Statistical differences were determined by a factorial ANOVA followed by the unpaired t test. **, Significance at level p < 0.01 compared with the control group.

 
Restraint stress induced inhibition of lymphocyte proliferation; IL-2 and IFN-{gamma} synthesis is through the µ-opioid receptor

We next evaluated the effects of chronic restraint stress on splenic lymphocyte proliferation and the Th1-like cytokine production in WT and MORKO mice. Chronic stress suppressed splenic lymphocyte proliferation (38.5% compared with control, p < 0.01) in WT mice. The suppressive effect of lymphocyte proliferation induced by restraint stress was completely abolished in MORKO mice (Fig. 3GoA). Culture supernatants from Con A-stimulated cells were assayed for the Th1-like cytokines, IL-2 and IFN-{gamma}, by ELISA. As shown in Fig. 3GoB, splenic lymphocytes from stressed WT mice produced significantly less IL-2 and IFN-{gamma} than lymphocytes from unstressed WT mice. In contrast, chronic stress did not result in any significant decrease in Th1 cytokines in the MORKO mice when compared with unstressed animals (p > 0.05). These results clearly show that inhibition of splenic lymphocyte proliferation and inhibition of Th1 cytokine synthesis induced by chronic restraint stress are mediated by the µ-opioid receptor. These results support the conclusion that the endogenous opioid production induced by restraint stress acts through the µ-opioid receptor and dramatically enhances lymphocyte apoptosis and reduces splenic cellularity, thereby affecting immune function.



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  		FIGURE 3. Effect of chronic restraint stress on splenic lymphocyte proliferation (A) and IL-2 and IFN-{gamma} production (B) induced by Con A in WT and MORKO mice. Both WT and MORKO mice were subjected to the stress protocol and then were sacrificed. Each spleen was removed and splenocytes were prepared aseptically. Splenic lymphocyte proliferation and Th1-like cytokine production were analyzed by [methyl-3H]thymidine incorporation and ELISA analysis. Data are expressed as mean ± SEM of six animals per group. Statistical differences were determined by a factorial ANOVA followed by the unpaired t test. **, Significance at level p < 0.01 compared with the control group. Similar results were obtained in three independent experiments.

 
Restraint stress induced elevated levels of plasma corticosterone in both WT and MORKO mice

To determine whether the µ-opioid receptor is involved in the alteration of plasma corticosterone levels induced by chronic restraint stress, plasma from both restrained WT and MORKO animals was collected and analyzed. Both WT and MORKO mice showed significantly elevated levels of corticosterone after restraint stress (243 and 134% compared with controls). However, the plasma corticosterone levels in the MORKO mice were 2-fold lower following stress when compared with WT mice (Fig. 4GoA). These results suggest that a 12-h restraint stress daily for 2 days activates the hypothalamic-pituitary-adrenal (HPA) axis and that the µ-opioid receptor plays a significant role in this activation.



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  		FIGURE 4. The role of the µ-opioid receptor in HPA axis activity induced by chronic restraint stress. A, The effect of chronic restraint stress on plasma corticosterone in WT and MORKO mice. Mice were stressed as described in Materials and Methods. Plasma was collected from stressed or unstressed (control) WT and MORKO mice. The concentration of plasma corticosterone was determined by RIA. B, The effect of naltrexone pellet implantation on HPA axis response in MORKO mice. MORKO mice were implanted with naltrexone or placebo pellet 24 h before the beginning of restraint stress. Data (mean ± SEM) were obtained from six animals per group. Statistical differences were determined by a factorial ANOVA followed by the unpaired t test. **, Significance at level p < 0.01 compared with the control group. Similar results were obtained in three independent experiments.

 
The role of {delta} and {kappa} opioid receptors in stress-induced activation of HPA axis

Because our results showed that stress-induced elevated corticosterone levels were reduced, but not completely abolished in MORKO mice, we next investigated the role of {delta} and {kappa} opioid receptors. MORKO mice were implanted with naltrexone or placebo pellets, as described in Materials and Methods. Twenty-four hours after pellet implantation, mice were given 12-h restraint stress daily for 2 days. Our results show that HPA axis activation produced by restraint stress in the MORKO was completely abolished in naltrexone-pelleted stressed MORKO mice (Fig. 4GoB). Naltrexone is a classical opioid antagonist and blocks all three (µ, {delta}, and {kappa}) opioid receptor types. This result suggests that the HPA activation produced by restraint stress is mediated by opioid receptors. Although the µ-opioid receptor appears to be the major receptor type involved, the {delta} and {kappa} receptor may play a contributing role.

Restraint stress enhanced pituitary POMC mRNA expression

We found that pituitary POMC mRNA expression, which is directly influenced by hypothalamic corticotropin-releasing hormone release, was significantly increased in both WT (316.7%, compared with WT controls, p < 0.01) and MORKO (158.3%, compared with MORKO controls, p < 0.01) mice after chronic restraint stress. However, POMC mRNA levels were significantly higher in WT mice compared with MORKO mice in the stressed groups (p < 0.01, Fig. 5Go). These results are in agreement with observed changes in plasma corticosterone levels, and further support the conclusion that the µ-opioid receptor is involved in stress-induced HPA axis activity, but may not be the sole pathway in this response.



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  		FIGURE 5. The effect of chronic restraint stress on expression of POMC mRNA in WT and MORKO mice. POMC transcription was analyzed by Northern blot. After exposure to detect POMC, the blots were stripped with 0.1x SSC and 1% SDS buffer, and then reblotted for GAPDH, which provides a control for RNA loading. The representative autoradiograms are shown. Similar results were obtained from three independent experiments. Results were quantified using Image-Pro Plus software and presented as a ratio of POMC mRNA:GAPDH mRNA. Data (mean ± SEM) were obtained from six animals per group. Statistical differences were determined by a factorial ANOVA followed by the unpaired t test. **, Significance at level p < 0.01 compared with the control group.

 
Adrenalectomy did not attenuate restraint stress-induced immunosuppression

Although restraint stress has been associated with activation of the HPA axis and elevated corticosterone levels, the role endogenous glucocorticoids play in stress-associated immunosuppression is not completely understood. It has been shown that opioids can exert their effects by modulating the production of corticosteroid via the HPA axis. In this study, we performed chronic restraint stress in adrenalectomized mice. Our results show that adrenalectomy did not attenuate the stress-induced reduction of splenocytes, nor affect inhibition of lymphocyte proliferation (Fig. 6Go). These results strongly suggest that endogenous opioids, induced by restraint stress, act directly on the µ-opioid receptor present in cells of immune system. This agrees with our previous finding that morphine-induced lymphocyte apoptosis occurs through the activation of caspase-3 and 8 and NO, thereby inhibiting the immune response in cultured lymphocytes (20).



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  		FIGURE 6. The effect of adrenalectomy on stress-induced reduction of splenocytes and inhibition of lymphocyte proliferation in stressed WT and MORKO mice. WT and MORKO mice were adrenalectomized and subjected to restraint stress. Adrenalectomy or shammed WT and MORKO mice were restrained for 12 h daily for 2 days. Mice were sacrificed at the end of the stress cycle. Splenic cellularity and lymphocyte proliferation were measured. Data (mean ± SEM) were calculated from six mice. Statistical differences were determined by a factorial ANOVA followed by the unpaired t test. The results were compared with unstressed WT or MORKO controls and show no significant difference between groups (p > 0.05). Similar results were obtained in three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Stress exists in our daily life. Chronic stress such as long-term emotional stress can decrease immune function and increase disease susceptibility. The mechanisms involved in this process still remained controversial. Numerous studies support the idea that the endogenous opioid peptide system contributes to the mediation, modulation, and regulation of stress responses by either: 1) activation of the endocrine (HPA axis), 2) modulation of the autonomic nervous system, or 3) a direct effect on immune cells (13, 21, 22). The families of endogenous opioids identified to date are the endorphins, the enkephalins, and the dynorphins. Each is derived from a different multifunctional precursor: POMC, proenkephalin, and prodynorphin, respectively (23). Endogenous opioid peptides have been shown to be presented in the hypothalamus, pituitary, and adrenal medulla, as well as in lymphocyte (24). The opioids produce their biological effect through three main types of G protein-coupled receptors referred to as µ (endorphins), {delta} (enkephalins), and {kappa} (dynorphins) (30). The different types of opioid receptors have been defined based on pharmacological and radioligand-binding experiments, and more recently by cloning (25). The opioid receptors are located in varying densities throughout the central, peripheral, and autonomic nervous system as well as in several endocrine tissues and cells of the immune system (26, 27). Endomorphins (endomorphin-1 and endomorphin-2) are endogenous opioid tetrapeptides that have been recently identified in the CNS and immune tissues with high selectivity and affinity for the µ-opioid receptor (28, 29). Carrigan et al. (30) reported that endomorphin-1 produces significant and naltrexone-reversible antinociception, but does not induce immunomodulatory effects in rats. Coventry et al. (31) investigated the effect of endomorphins on activation of the HPA axis, and their results show that neither endomorphin-1 nor endomorphin-2 stimulates the HPA axis. These studies argue against an important role for endomorphins in mediating HPA axis activity and immunomodulatory effects. Several studies, including ours, have shown that thymocytes, splenocytes, lymph node cells, and Jurkat T cells treated with morphine, in vitro, resulted in T cell apoptosis and inhibition of T lymphocyte proliferation (20, 32, 33, 34). In addition, exposure of human PBLs to {beta}-endorphin has been shown to induce apoptotic cell death (35). Furthermore, it has been shown that up-regulated Fas may mediate the morphine-induced T cell apoptosis (18). Blocking of endogenous opioids with naloxone has been demonstrated to attenuate or reverse stress-induced immune alterations (13, 14). Although these studies strongly support the hypothesis that endogenous opioids mediate stress-induced immune alterations, the effector mechanisms of endogenous opioids are still not clearly defined. To investigate whether and/or which opioid receptors are involved in restraint stress-induced immunosuppression, and to further define the role of endogenous opioids, MORKO mice were subjected to restraint stress. This animal model, in which the µ-opioid receptor is completely absent, is a very useful tool to evaluate the role of classical opioid receptors (naloxone sensitive), especially the µ-opioid receptor, in stress-induced immune alterations. Our results show that chronic restraint stress resulted in: 1) reduction of splenic cellularity and the number of lymphocyte subsets through an apoptotic mechanism; 2) inhibition of lymphocyte proliferation; and 3) inhibition of Con A-induced IL-2 and IFN-{gamma} synthesis. These effects were completely abolished in MORKO mice. These results show that the µ-opioid receptor is involved in stress-induced immune alterations.

We also found that stress-induced alterations in HPA axis response were significantly reduced, although not abolished, in MORKO mice. Our data indicate that at least 60% of the stress-induced alterations in HPA axis response is dependent on the µ-opioid receptors, with the balance dependent on the {delta} and/or {kappa} opioid receptors. Our previous work has shown that injection of {kappa} agonist U50-488H in MORKO mice results in a significant increase in plasma corticosterone, whereas injection of the Enkephalin, [D-Pen2,5]-(DPDPE) {delta}1 opioid receptor agonist did not result in an induction of corticosterone (36). Taken together with our present study, these results suggest that stress-induced HPA axis activity may be mediated through the µ and {delta} opioid receptors, but not the {delta} opioid receptors. Further investigation into the role of {kappa} and {delta} opioid receptors in restraint stress-induced HPA axis activity is currently underway in our laboratory by use of {kappa} and {delta} receptor antagonists.

Restraint stress has been associated with an increase in glucocorticoids (37, 38). However, these increases do not always appear to correlate with a decrease in immune response (39). Perhaps a reasonable explanation is that stressor re-exposure, following chronic intermittent restraint stress, rapidly alters splenocyte sensitivity to glucocorticoids (40). In addition, it has been reported that morphine induced immunosuppression in rat via a mechanism that is not mediated by corticosterone, despite a rise in serum corticosterone (41). Our findings were in accordance with these results. Chronic restraint stress induced elevated levels of plasma corticosterone, but did not result in immune alterations in MORKO mice. Adrenalectomy did not significantly attenuate restraint stress-induced immunosuppression. Thus, the HPA axis is unlikely to be involved in mediating the decreased immune response in this chronic restraint stress model. It has been shown that endogenous opioids can exert their effect by modulating the production of corticosteroid via the HPA axis in an endocrine manner (42). Our observation that adrenalectomy did not significantly attenuate chronic stress-induced immune response strongly suggests that the effect of endogenous opioids is most likely autocrine or paracrine in nature, and is exerted directly by the µ-opioid receptors expressed on lymphocytes.

In conclusion, this study to our knowledge is the first to clearly demonstrate that the µ-opioid receptor is involved in chronic restraint stress-induced immune alterations through an apoptotic mechanism. We believe that this finding provides a mechanism by which stress modulates the immune system and should help us develop therapeutic methods to reduce stress-induced immunosuppression by modulating the functions of the µ-opioid receptor.


   Acknowledgments
 
We thank Dr. William C. Engeland (Department of Surgery, University of Minnesota) for providing invaluable technical advice on adrenalectomy in mice.


   Footnotes
 
1 This work was supported by Grants RO-1 DA 12104 and DA 11806-03 (to S.R.) from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Sabita Roy, Veterans Affairs Medical Center, Research RT 151, Room 3N 107, One Veterans Drive, Minneapolis, MN 55417. E-mail address: royxx002{at}tc.umn.edu Back

3 Abbreviations used in this paper: MORKO, µ-opioid receptor knockout; HPA, hypothalamic-pituitary-adrenal; POMC, proopiomelanocortin; WT, wild type. Back

Received for publication April 1, 2002. Accepted for publication July 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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