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Immune deviation following stress odor exposure: role of endogenous opioids
Journal of Neuroimmunology 102 Ž2000. 145–153 www.elsevier.comrlocaterjneuroim
Immune deviation following stress odor exposure: role of endogenous opioids Jan A. Moynihan a
a,)
, Jonathan D. Karp b, Nicholas Cohen c , Robert Ader
a
Department of Psychiatry, UniÕersity of Rochester Medical Center, 300 Crittenden BlÕd., Rochester, NY 14642, USA b Department of Biological Sciences, Rider UniÕersity, LawrenceÕille, NJ, USA c Department of Microbiology and Immunology, UniÕersity of Rochester Medical Center, Rochester, NY, USA Received 13 May 1999; received in revised form 21 July 1999; accepted 1 September 1999
Abstract Olfactory cues can alter immune function. BALBrc mice exposed to odors produced by footshock stressed donor mice have increased antibody responses and increased splenic interleukin ŽIL.-4 production following immunization relative to recipients of odors from unstressed animals. Here we document that exposure to stress odors results in analgesia that is blocked by the non-selective opioid receptor antagonist naltrexone. The stress odor-induced increase in antigen-driven IL-4 and antibody is also blocked by oral administration of naltrexone. Thus, we provide evidence that immune deviation can occur following a psychosocial stressor, and that the deviation appears to be mediated by endogenous opioid production. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Th2 cytokines; Psychosocial stress; Opioids; Antibody; Mice
1. Introduction Clinical studies provide evidence that behavioral and psychosocial factors, including stressful life experiences, are associated with altered immune function ŽKiecolt and Glaser, 1991; Kiecolt-Glaser et al., 1994. and susceptibility to infection ŽCohen, 1994.. Other studies document that psychological well-being Že.g., social support. is associated with changes in immune function, e.g., increased natural killer ŽNK. cell function ŽLevy et al., 1990; Payne et al., 1996. and increased survival in cancer patients ŽSpiegel et al., 1989; Fawzy and Fawzy, 1994.. Because stress may be a risk factor for human disease ŽCohen, 1994; Sheridan et al., 1994., the neurochemical and subsequent immunological consequences of stressors must be elucidated. Animal studies in which environmental stimuli, genetic background, and immunological challenge can be controlled provide data that are essential for understanding the complex mechanisms involved in brain, behavior, immune system interactions that influence pathogenic processes in humans. )
Corresponding author. Tel.: q1-716-275-4648; fax: q1-716-2711279; e-mail: [email protected]
Like humans, rodents make behavioral and physiological adjustments to ethologically relevant signals from their environment. Olfactory cues have been shown by our laboratory ŽCocke et al., 1993; Moynihan et al., 1994. and others ŽMatthes, 1963; Rogers et al., 1980; Shibata et al., 1990; Zalcman et al., 1991. to alter immune function. For example, BALBrc mice exposed to odors produced by footshock stressed mice have increased antibody responses and increased splenic interleukin ŽIL.-4 production following immunization with the T cell-dependent antigen keyhole limpet hemocyanin ŽKLH. relative to recipients of odors from unstressed animals ŽMoynihan et al., 1994.. Preferential skewing toward a T helper ŽTh. 1- or Th2-dominant response is important for the generation of an adaptive immune response. Th1 responses Žcharacterized by production of IL-2 and interferon ŽIFN.-g . are often critical for viral clearance, whereas Th2 responses Žincluding production of IL-4. are important in generation of humoral immunity ŽO’Garra and Murphy, 1996; Pawelec et al., 1996; Romagnani, 1996, 1997.. A number of factors such as antigen concentration, route of inoculation ŽBrenner et al., 1994; Bretscher et al., 1994; Golding et al., 1994; Paul and Seder, 1994; Hosken et al., 1995; Guery et al., 1996., and the strain of the experimental subject
0165-5728r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 7 3 - 3
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ŽHeinzel et al., 1989; Sadick et al., 1990; Bretscher et al., 1994. can influence production of Th1 vs. Th2 cytokines. Few studies to date have shown that psychosocial factors can alter Th1 and Th2 cytokine responses Že.g., Moynihan et al., 1994; Decker et al., 1996; Dobbs et al., 1996; Elenkov et al., 1996; Maes et al., 1998.. Stress odor exposure ŽSOE. provides a unique naturalistic model for determining the effects of endogenous hormones on the process of immune deviation toward an increased Th2 state of activation. In this paper, we examine the role of endogenous opioids in stress odor-induced cytokine production and antibody responses. Centrally and peripherally produced opioids constitute an important set of chemical messengers connecting the immune and nervous systems. Exogenously administered opioids Že.g., morphine., as well as endogenous opioids Že.g., b-endorphins., have a wide array of immunomodulatory effects that result from their direct andror indirect action on cells of the immune system ŽCarr and France, 1993; Fuchs and Pruett, 1993; Hernandez et al., 1993; Freier and Fuchs, 1994; Sutton et al., 1994; Maity et al., 1995; Yeager et al., 1995; Fecho et al., 1996a; Lockwood et al., 1996; Panerai and Sacerdote, 1997; Eisenstein and Hilburger, 1998; Madden et al., 1998; Mellon and Bayer, 1998; Peterson et al., 1998; Sharp et al., 1998.. Data suggest that endogenous b-endorphin plays a physiological inhibitory role in immunity ŽPanerai and Sacerdote, 1997; Panerai et al., 1994, 1995; Wiedermann et al., 1994..
2. Materials and methods 2.1. Animals Four-week-old BALBrc male mice were purchased from the Jackson Laboratories ŽBar Harbor, ME.. Mice were housed in standard ‘‘shoe box’’ plastic cages in groups of four for 2–4 weeks prior to experimentation. Purina rodent chow and water were available ad libitum, and mice were maintained on a 12:12 h light:dark cycle, with lights on at 0600 h. All mice were sacrificed by rapid cervical dislocation or decapitation in a room separate from where the mice were housed. 2.2. SOE paradigm Plexiglas chambers located in a sound-deadened room separate from the main animal quarters were used in these experiments ŽMoynihan et al., 1994.. Controlled air flow through these airtight chambers was achieved by passing compressed breathing air from gas cylinders into donor chambers. Airflow from the donor chambers was exhausted to the recipient chambers via vacuum pressure. Each donor chamber was connected to two recipient cham-
bers Žin parallel. with Tygon tubing. The air flow was maintained at approximately 4 lrmin. The experimental paradigm began by placing cages of mice Žfour micercage. into the recipient chambers at 0900 h. After a 3-h adaptation, cages of mice Žfour micercage. were placed into shock boxes in the donor chambers. The shock boxes ŽLafayette Instruments, Easton, PA. were programmed by computer to deliver one unsignaled footshock Ž10 s at 0.5 mA. every 30 min for 24 h. Control odor donor mice were also placed in donor shock boxes, but these boxes were not connected to the computer. Following 24 h, all mice were removed from the odor-exposure apparatus and housed 1rcage Žto prevent stress-induced fighting. in the original colony room. 2.3. Immunization Mice were injected 24 h prior to the onset of odor exposure with 100 mg of KLH in 0.2 ml of sterile saline via intraperitoneal Ži.p.. injection. KLH was obtained as a slurry from Pacific Biomarine ŽVenice, CA. and was centrifuged, dialyzed, and filtered prior to storage at y208C. 2.4. Measurement of analgesia and antagonism Odor-mediated changes in analgesia were assessed by tail-flick ŽHoran et al., 1992.. Mice were removed from their home cage and held over a beaker of water maintained at 568C. The distal 1r3 of the tail of each mouse was immersed in the water and the latency for the mouse to remove its tail from the water was recorded. To determine if odor-induced changes in tail-flick latency were opioid-mediated, some mice were administered either naloxone or naltrexone ŽSigma, St. Louis, MO. prior to tail-flick testing. 2.5. Spleen cell cultures Spleens were removed aseptically from decapitated mice and dissociated into single cell suspensions using a Stomacher Lab Blender ŽTekmar, Cincinnati, OH.. Cells were washed twice in Hanks Balanced Salt Solution HBSS, counted using a Coulter Counter ŽCoulter Electronics, Hialeah, FL., and adjusted to a final concentration of 2.5 = 10 6rml in a 1:1 mixture of RPMI 1640:high glucose DMEM and 1% Nutridoma-NS medium ŽBoehringer Mannheim, Indianapolis, IN. supplemented with 10 mM HEPES, 2 mM L-glutamine, 50 Urml penicillin, 50 mgrml streptomycin, and 5 = 10y5 M 2-mercaptoethanol Žall from GIBCO, Grand Island, NY.. Cells were cultured in 24-well tissue culture-treated plates at 378C in a 5% CO 2 humidified atmosphere with 80–120 mgrml of KLH. Supernatants ŽSN. were harvested after 24 h to measure IL-2, and at 48 or 72 h to measure IL-4 and IFN-g and stored at y708C prior to assay.
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2.6. Enzyme linked immunosorbent assay (ELISA) for IL-2, IL-4, and IFN-g Levels of cytokines in supernatants were determined by ELISA using monoclonal antibodies ŽmAb. and our published protocol ŽMoynihan et al., 1994.. Briefly, 96-well enhanced protein binding ELISA plates ŽCorning, Corning, NY. were coated overnight at 48C with 100 mlrwell of purified anti-cytokine capture mAb Ž2 mgrml, PharMingen, San Diego, CA.. Between each step, plates were washed with PBSrTween 20. Plates were blocked for 2 h with PBS–10% fetal equine serum ŽFES, Sigma. at room temperature. Recombinant cytokine standards Ž in Unitsrml, PharMingen. and samples were added to plates in 100 ml and allowed to incubate overnight at 48C. Biotinylated anti-cytokine detecting mAb Ž1 mgrml, PharMingen. was added in 100 ml and the plates were incubated at room temperature for 45 min. Avidin-peroxidase at 2.5 mgrml in PBS–10% FES was added in 100 ml and the plates were incubated for 30 min at room temperature. Finally, 100 mlrwell of substrate Ž2,2X-azinobisŽ3-ethylbensthiazoline-6-sulfonic acid., Sigma. was added. Absorbance at 405 nm was determined using a Biotek automated plate reader ŽWinooski, VT.. 2.7. ELISA for serum anti-KLH antibody responses IgM and IgG anti-KLH antibody titers in sera were determined using a standard ELISA ŽMoynihan et al., 1994.. Wells of 96-well microtiter plates were coated overnight with 10 mgrml of KLH in a carbonate coating buffer, pH 9.6, and post-coated for 1 h with PBS containing 1% bovine serum albumin ŽBSA. at 378C. Experimental sera were diluted 1:25, 1:75, and 1:225 in PBSrTween containing 1 M NaCl and incubated for 3 h at 378C. The plates were incubated overnight at 48C with alkaline phosphatase-conjugated goat anti-mouse IgM or IgG antibody Žm- and g-chain specific, respectively, Cappel, Durham, NC. diluted appropriately in PBSrTween followed by addition of the substrate, p-nitrophenyl phosphate ŽSigma., at 1 mgrml in carbonate buffer, pH 9.6. Washes with PBSrTween followed each step. Absorbance at 405 nm was measured. A standard curve was generated using sera from mice hyperimmunized with KLH. Sera from non-immunized mice served as a negative control. 2.8. Statistical analysis When appropriate, data were analyzed by analysis of variance ŽANOVA. with treatment conditions and replication as factors using StatView Žversion 5.0. on a Macintosh PowerPC computer. Post-hoc comparisons of significant interactions were made with Duncans Multiple range test. Planned comparisons were made using the two-tailed Student Neuman–Keuls t-test.
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3. Results 3.1. SOE increases production of IL-4 by spleen cells from odor recipients Spleen cells from stress odor and control odor exposed ŽCOE. mice were obtained 6 days following immunization Ž5 days following odor exposure. and were cultured with KLH as described. Supernatants were harvested from antigen-stimulated cultures and the Th1-like cytokines IL-2 and IFN-g, as well as the Th2-like cytokine IL-4 were assayed by ELISA. As shown in Fig. 1a, spleen cells from stress odor-exposed mice produced significantly more KLH-induced IL-4 than cells from COE mice Ž F Ž1,70. s 11.9, p - 0.01., confirming our previous observations ŽMoynihan et al., 1994.. As shown in Fig. 1b and c, however, no significant differences in KLH-stimulated Th1 cytokine production ŽIL-2 and IFN-g . were observed. Calculating the ratio of type 1: type 2 cytokines provides a useful measure of the balance between the two types of cytokines and is reflective of the immunological state of the animal; that is, the ratio reflects the ability of an animal to produce a cell-mediated vs. humoral immune response to antigen. The ratio of IFN-g: IL-4 in supernatants was observed to be 2.1 for spleen cells from COE mice and 1.2 for spleen cells from SOE mice, a significant 1.75-fold difference Ž p - 0.05.. These data indicate that SOE is associated with a pronounced immune deviation or shift in the balance between antigen-stimulated type 1 and type 2 cytokine production. 3.2. SOE induces opioid-mediated analgesia Olfactory information Žpheromones. is particularly important for reproduction, neuroendocrine responses, and the recognition of conspecifics, predators and prey. Production of endogenous opioids in the nervous system is particularly responsive to pheromonal influences and, as a result, opioids are strong candidates for mediating both the behavioral and immune responses to psychosocial stress. Because others have reported that odor stimuli can induce endogenous opioid secretion in animals ŽHarris and Pierson, 1964; Lichtman and Fanselow, 1990., and since opioids have a role in immune modulation as discussed above, the possibility that SOE induces endogenous opioid secretion was examined. This was first examined by investigating whether SOE induced analgesia, such as can be produced by opioids. Mice were tested for tail-flick latency, a standard measure of analgesia, as described above. The analgesic effect of putative opioids was determined at the end of 24 h of SOE. SOE was associated with a longer tail-flick latency compared to COE mice ŽFig. 2, t Ž1,14. s 2.7, p s 0.017.. In a separate experiment, SOE-induced analgesia was de-
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Fig. 2. SOE induces analgesia. Following 24 h of SOE or COE, mice Ž ns8rgroup. were subjected to tail-flick measures as described in Section 2. Asterisk indicates ps 0.017.
termined to be opioid mediated; administration of naloxone Ž10 mgrkg administered i.p.. 30 min prior to the end of the odor-exposure session blocked SOE-induced increases in tail-flick latency ŽFig. 3, t Ž1,14. s 2.5, p s 0.024..
Fig. 1. SOE results in increased Th2-type cytokine production. BALBrc male mice were immunized with 100 mg of KLH 24 h prior to SOE or control odor exposure ŽCOE.. Mice were sacrificed 6 days following immunization. Spleen cells were cultured with 80 mgrml of KLH for 24 h ŽIL-2. or 48 h ŽIL-4 and IFN-g .. Supernatants were harvested and assayed for cytokine production by ELISA. Figures represent three combined experiments with ns 36rgroup. Asterisk indicates p- 0.05.
Fig. 3. Naloxone abrogates SOE-induced analgesia. Mice Ž ns8rgroup. were injected i.p. with naloxone or saline following 24 h of SOE. Tail flick latency was assessed 15 min following injection. Asterisk indicates ps 0.024.
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Fig. 4. SOE induces opioid-mediated analgesia. BALBrc mice were exposed to SOE or COE for 24 h with either water or naltrexone Ž0.5 mgrml in water. available for the entire period of odor exposure. Tail-flick latency was measured at the end of the odor exposure. N s 16 for both SOE groups and n s 4 for both COE groups. SOE q water group is different from all other groups Ž p - 0.05..
3.3. Naltrexone in the drinking water of SOE mice blocks opioid-induced analgesia
induced analgesia that was antagonized by naltrexone availability in the drinking water ŽFig. 4, F Ž2,35. s 6.0, p s 0.002..
Although tail-flick latency was significantly decreased by antagonist administered at the end of the 24 h of SOE, alterations in immune responses are likely to be blocked only when antagonist is present throughout the 24-h odor exposure period. To test the hypothesis that opioid receptor antagonism would abrogate the stress odor-induced increase in immune function, we first determined that the longer acting antagonist naltrexone could be effectively administered in the drinking water of odor exposed mice, thereby avoiding both handling of mice and multiple injections of the drug. COE and SOE mice were given drinking water containing 0.5 mgrml of naltrexone 3 h before being placed in the odor-exposure apparatus and throughout the 24-h odor-exposure session. Analysis of tail-flick latencies at the end of the odor exposure period indicated that SOE
3.4. Oral naltrexone abrogates SOE induced increases in IL-4 and both IgM and IgG anti-KLH antibody production After determining that the analgesic effects of stress odor-induced opioids could be antagonized by access to oral naltrexone, we examined whether the immunological effects of SOE might also be antagonized. Beginning 3 h before, and lasting throughout the 24 h of odor exposure, KLH-immunized mice were given access to drinking water with or without naltrexone Ž0.5 mgrml.. Five days following the end of the odor exposure session, mice were sacrificed, splenocytes were stimulated with KLH in vitro, and supernatants were assayed for IL-4 Žat 72 h., IFN-g Žat 72 h. and IL-2 Žat 24 h.. IL-4 values from two experiments were combined into a single ANOVA; an interaction between odor exposure
Fig. 5. Naltrexone blocks the SOE-induced increase in IL-4 production by BALBrc splenocytes. Mice were given either plain water or water containing 0.5 mgrml of naltrexone in water to drink during the period of odor exposure. N s 12-16 micergroup. SOE q water group was different from all other groups Ž p - 0.05..
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ŽSOE vs. COE. and fluid consumed Žnaltrexone vs. water. was observed Ž F Ž1,102. s 6.7, p s 0.011., as well as a main effect of odor exposure Ž F Ž1,102. s 7.2, p s 0.008.. Post hoc analyses indicated that splenocytes from SOE q water mice produced more IL-4 than splenocytes from COE q water mice and both naltrexone groups Ž p - 0.05, Fig. 5.. In these cultures there was an unexplained paucity of KLH-stimulated IFN-g production Žaveraging only 3.6 unitsrml.; therefore, the ratio of IFN-g:IL-4 could not be determined with any confidence. Naltrexone had no effect on COE mice compared to COE mice given water without naltrexone for 24 h. In contrast to the data shown in Fig. 1, analysis of IL-2 levels in these supernatants from the two experiments revealed a marginally significant main effect of IL-2 produced by cells from SOE compared to COE Ž3.6 " 0.2 compared to 4.25 " 0.21 unitsrml, respectively; F Ž1,108. s 3.90, p s 0.051., with IL-2 production decreased in cultures of spleen cells from SOE mice. IgM and IgG serum anti-KLH antibody titers in serum from mice were examined in another group of mice 6 days
Fig. 6. Naltrexone abrogates the SOE-induced increase in IgM and IgG anti-KLH antibody responses. Mice were immunized 24 h prior to odor exposure and were bled 7 days later. Sera samples were assayed for IgM and IgG anti-KLH antibody responses by ELISA. N s10–14 micergroup. SOEqwater group responses were greater than all other groups Ž p0.05..
following odor exposure Ž7 days following immunization with 100 mg of KLH.. ANOVA indicated an interaction of odor exposure and treatment for both IgM Ž F Ž3,188. s 8.73, p - .01. and IgG Ž F Ž3,188. s 5.2, p - 0.01.. Planned comparisons indicated that the group of mice receiving SOE q water had significantly elevated IgM anti-KLH antibody responses compared to all other groups Ž p - 0.05, Fig. 6a.. The group receiving SOE q water also had higher IgG anti-KLH antibody responses compared to all other groups Ž p - 0.05, Fig. 6b.. ANOVA indicated no interaction with serum dilution; data are shown collapsed across three dilutions.
4. Discussion Here we have demonstrated that a psychosocial stressor, exposure to the odors of footshocked mice, is associated with immune deviation and increased antigen-specific antibody responses to the protein antigen KLH. Further, this stressor induces changes in nociception that are blocked by administration of the opioid receptor antagonists naloxone or naltrexone, and naltrexone also blocks the stressor-induced changes in immune function. Thus, we have evidence that endogenous opioids are immunomodulatory, resulting in increased Th2 cytokines and antibody titers. Opioid peptides Žproopiomelanocortin peptides, the enkephalins and the dynorphins. are derived from the hypothalamus, pituitary, and adrenal medulla, as well as from lymphocytes Žreviewed in Weigent and Blalock, 1994; Carr et al., 1996; Stefano et al., 1996.. The best characterized opiate receptors in the nervous system are the m, k, and d receptors; evidence also exists for expression of these receptors on cells of the immune system Žreviewed in Stefano et al., 1996; Madden et al., 1998; Mellon and Bayer, 1998; Sharp et al., 1998.. Opioids, which can be viewed as cytokines acting within the CNS and immune system ŽPeterson et al., 1998., are clearly immunomodulatory and their effects on immune function are dependent upon concentration as well as the immune parameter being investigated. The in vivo effects of exogenous opioids are generally immunosuppressive. Phagocytic and other monocytermacrophage function Žreviewed in Stefano et al., 1996; Eisenstein and Hilburger, 1998.; NK cell activity ŽShavit, 1991; Carr and France, 1993; Lysle et al., 1993; Yeager et al., 1995; Hsueh et al., 1996., mitogen-induced proliferation ŽHernandez et al., 1993; Lysle et al., 1993; Fecho et al., 1996a; Mellon and Bayer, 1998., CTL activity ŽCarpenter and Carr, 1995., IL-2 and IFN-g production ŽLysle et al., 1993. were all observed to be decreased following injection of morphine. The effects of stress-induced endogenous opioids on immune function have largely focused on suppressed NK cell activity and increased tumor growth Žreviewed by
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Shavit, 1991.. Other data suggest that endogenous b-endorphin normally plays a physiological role in immune regulation, suppressing NK cell activity and mitogen-induced proliferation, and increasing allograft survival time ŽPanerai et al., 1995.. In addition, b-endorphin may be involved in downregulation of Th1-induced autoimmune diseases, including experimental allergic encephalomyelitis ŽEAE. in rats ŽPanerai et al., 1994. and Crohn’s disease in humans ŽWiedermann et al., 1994.. Panerai and his colleagues have hypothesized that opioids, in particular b-endorphin that might be elevated during a stress response, result in increased production of Th2 cytokines, exerting a tonic inhibitory effect on the immune system ŽPanerai and Sacerdote, 1997.. Our data support this hypothesis; however, more detailed investigation is needed to understand the regulation of immune function by endogenous opioids. There is evidence of direct receptor-mediated actions of opioids on leukocytes Žreviewed in Carr et al., 1996; Stefano et al., 1996; Eisenstein and Hilburger, 1998; Sharp et al., 1998. and ample evidence for the expression of opioid receptors on lymphocytes ŽCarr et al., 1996; Stefano et al., 1996; Sharp et al., 1998.. In contrast, the effects of exogenous morphine on immune function have been frequently demonstrated to be centrally-mediated ŽHernandez et al., 1993; Fecho et al., 1996a; Mellon and Bayer, 1998., acting predominantly through m opioid receptors expressed in the periacqueductal gray of the mesencephalon. Evidence exists for either peripheral b-adrenergic ŽFecho et al., 1993; Fecho et al., 1996b. andror glucocorticoid mechanisms in morphine-induced changes in immune function ŽFreier and Fuchs, 1994; Fecho et al., 1996b.. Morphine injection has been associated with an increase in glucocorticoids ŽLockwood et al., 1996; Mellon and Bayer, 1998.; however, the glucocorticoid increase does not always appear to correlate with decreased immune responses ŽLockwood et al., 1996.. Of relevance to our study are the findings of Sutton et al. Ž1994. who showed that opioidmediated analgesia induced by acute inescapable tail shock in rats was attenuated by adrenalectomy ŽADX.. Basal corticosterone ŽCort. replacement in the drinking water of the ADX rats restored the opioid-induced analgesia; therefore, basal Cort was permissive for the shock-induced analgesia, but shock-induced increases in Cort were not necessary. This is of interest because previous studies from our laboratory indicated that some of the immunological effects of SOE Ži.e., increased IL-4 production. could be abrogated by pretreatment of mice with the glucocorticoid receptor antagonist RU486 ŽMoynihan et al., 1994. even though glucocorticoids were not significantly elevated in the SOE paradigm Žunpublished data.. In conclusion, our studies demonstrate that the balance between Th1 and Th2 cytokines is a potential target for neuroendocrine influences on immunity. Future studies will be directed at determining the consequence of immune deviation due to neuroendocrine perturbations for disease pathogenesis and in clinical populations.
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Acknowledgements The authors wish to thank Tracey Smith for her excellent technical assistance. This research was supported by research grants from the PHS ŽMH-45681. and The Fetzer Institute.
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Immune deviation following stress odor exposure: role of endogenous opioids Jan A. Moynihan a
a,)
, Jonathan D. Karp b, Nicholas Cohen c , Robert Ader
a
Department of Psychiatry, UniÕersity of Rochester Medical Center, 300 Crittenden BlÕd., Rochester, NY 14642, USA b Department of Biological Sciences, Rider UniÕersity, LawrenceÕille, NJ, USA c Department of Microbiology and Immunology, UniÕersity of Rochester Medical Center, Rochester, NY, USA Received 13 May 1999; received in revised form 21 July 1999; accepted 1 September 1999
Abstract Olfactory cues can alter immune function. BALBrc mice exposed to odors produced by footshock stressed donor mice have increased antibody responses and increased splenic interleukin ŽIL.-4 production following immunization relative to recipients of odors from unstressed animals. Here we document that exposure to stress odors results in analgesia that is blocked by the non-selective opioid receptor antagonist naltrexone. The stress odor-induced increase in antigen-driven IL-4 and antibody is also blocked by oral administration of naltrexone. Thus, we provide evidence that immune deviation can occur following a psychosocial stressor, and that the deviation appears to be mediated by endogenous opioid production. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Th2 cytokines; Psychosocial stress; Opioids; Antibody; Mice
1. Introduction Clinical studies provide evidence that behavioral and psychosocial factors, including stressful life experiences, are associated with altered immune function ŽKiecolt and Glaser, 1991; Kiecolt-Glaser et al., 1994. and susceptibility to infection ŽCohen, 1994.. Other studies document that psychological well-being Že.g., social support. is associated with changes in immune function, e.g., increased natural killer ŽNK. cell function ŽLevy et al., 1990; Payne et al., 1996. and increased survival in cancer patients ŽSpiegel et al., 1989; Fawzy and Fawzy, 1994.. Because stress may be a risk factor for human disease ŽCohen, 1994; Sheridan et al., 1994., the neurochemical and subsequent immunological consequences of stressors must be elucidated. Animal studies in which environmental stimuli, genetic background, and immunological challenge can be controlled provide data that are essential for understanding the complex mechanisms involved in brain, behavior, immune system interactions that influence pathogenic processes in humans. )
Corresponding author. Tel.: q1-716-275-4648; fax: q1-716-2711279; e-mail: [email protected]
Like humans, rodents make behavioral and physiological adjustments to ethologically relevant signals from their environment. Olfactory cues have been shown by our laboratory ŽCocke et al., 1993; Moynihan et al., 1994. and others ŽMatthes, 1963; Rogers et al., 1980; Shibata et al., 1990; Zalcman et al., 1991. to alter immune function. For example, BALBrc mice exposed to odors produced by footshock stressed mice have increased antibody responses and increased splenic interleukin ŽIL.-4 production following immunization with the T cell-dependent antigen keyhole limpet hemocyanin ŽKLH. relative to recipients of odors from unstressed animals ŽMoynihan et al., 1994.. Preferential skewing toward a T helper ŽTh. 1- or Th2-dominant response is important for the generation of an adaptive immune response. Th1 responses Žcharacterized by production of IL-2 and interferon ŽIFN.-g . are often critical for viral clearance, whereas Th2 responses Žincluding production of IL-4. are important in generation of humoral immunity ŽO’Garra and Murphy, 1996; Pawelec et al., 1996; Romagnani, 1996, 1997.. A number of factors such as antigen concentration, route of inoculation ŽBrenner et al., 1994; Bretscher et al., 1994; Golding et al., 1994; Paul and Seder, 1994; Hosken et al., 1995; Guery et al., 1996., and the strain of the experimental subject
0165-5728r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 1 7 3 - 3
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ŽHeinzel et al., 1989; Sadick et al., 1990; Bretscher et al., 1994. can influence production of Th1 vs. Th2 cytokines. Few studies to date have shown that psychosocial factors can alter Th1 and Th2 cytokine responses Že.g., Moynihan et al., 1994; Decker et al., 1996; Dobbs et al., 1996; Elenkov et al., 1996; Maes et al., 1998.. Stress odor exposure ŽSOE. provides a unique naturalistic model for determining the effects of endogenous hormones on the process of immune deviation toward an increased Th2 state of activation. In this paper, we examine the role of endogenous opioids in stress odor-induced cytokine production and antibody responses. Centrally and peripherally produced opioids constitute an important set of chemical messengers connecting the immune and nervous systems. Exogenously administered opioids Že.g., morphine., as well as endogenous opioids Že.g., b-endorphins., have a wide array of immunomodulatory effects that result from their direct andror indirect action on cells of the immune system ŽCarr and France, 1993; Fuchs and Pruett, 1993; Hernandez et al., 1993; Freier and Fuchs, 1994; Sutton et al., 1994; Maity et al., 1995; Yeager et al., 1995; Fecho et al., 1996a; Lockwood et al., 1996; Panerai and Sacerdote, 1997; Eisenstein and Hilburger, 1998; Madden et al., 1998; Mellon and Bayer, 1998; Peterson et al., 1998; Sharp et al., 1998.. Data suggest that endogenous b-endorphin plays a physiological inhibitory role in immunity ŽPanerai and Sacerdote, 1997; Panerai et al., 1994, 1995; Wiedermann et al., 1994..
2. Materials and methods 2.1. Animals Four-week-old BALBrc male mice were purchased from the Jackson Laboratories ŽBar Harbor, ME.. Mice were housed in standard ‘‘shoe box’’ plastic cages in groups of four for 2–4 weeks prior to experimentation. Purina rodent chow and water were available ad libitum, and mice were maintained on a 12:12 h light:dark cycle, with lights on at 0600 h. All mice were sacrificed by rapid cervical dislocation or decapitation in a room separate from where the mice were housed. 2.2. SOE paradigm Plexiglas chambers located in a sound-deadened room separate from the main animal quarters were used in these experiments ŽMoynihan et al., 1994.. Controlled air flow through these airtight chambers was achieved by passing compressed breathing air from gas cylinders into donor chambers. Airflow from the donor chambers was exhausted to the recipient chambers via vacuum pressure. Each donor chamber was connected to two recipient cham-
bers Žin parallel. with Tygon tubing. The air flow was maintained at approximately 4 lrmin. The experimental paradigm began by placing cages of mice Žfour micercage. into the recipient chambers at 0900 h. After a 3-h adaptation, cages of mice Žfour micercage. were placed into shock boxes in the donor chambers. The shock boxes ŽLafayette Instruments, Easton, PA. were programmed by computer to deliver one unsignaled footshock Ž10 s at 0.5 mA. every 30 min for 24 h. Control odor donor mice were also placed in donor shock boxes, but these boxes were not connected to the computer. Following 24 h, all mice were removed from the odor-exposure apparatus and housed 1rcage Žto prevent stress-induced fighting. in the original colony room. 2.3. Immunization Mice were injected 24 h prior to the onset of odor exposure with 100 mg of KLH in 0.2 ml of sterile saline via intraperitoneal Ži.p.. injection. KLH was obtained as a slurry from Pacific Biomarine ŽVenice, CA. and was centrifuged, dialyzed, and filtered prior to storage at y208C. 2.4. Measurement of analgesia and antagonism Odor-mediated changes in analgesia were assessed by tail-flick ŽHoran et al., 1992.. Mice were removed from their home cage and held over a beaker of water maintained at 568C. The distal 1r3 of the tail of each mouse was immersed in the water and the latency for the mouse to remove its tail from the water was recorded. To determine if odor-induced changes in tail-flick latency were opioid-mediated, some mice were administered either naloxone or naltrexone ŽSigma, St. Louis, MO. prior to tail-flick testing. 2.5. Spleen cell cultures Spleens were removed aseptically from decapitated mice and dissociated into single cell suspensions using a Stomacher Lab Blender ŽTekmar, Cincinnati, OH.. Cells were washed twice in Hanks Balanced Salt Solution HBSS, counted using a Coulter Counter ŽCoulter Electronics, Hialeah, FL., and adjusted to a final concentration of 2.5 = 10 6rml in a 1:1 mixture of RPMI 1640:high glucose DMEM and 1% Nutridoma-NS medium ŽBoehringer Mannheim, Indianapolis, IN. supplemented with 10 mM HEPES, 2 mM L-glutamine, 50 Urml penicillin, 50 mgrml streptomycin, and 5 = 10y5 M 2-mercaptoethanol Žall from GIBCO, Grand Island, NY.. Cells were cultured in 24-well tissue culture-treated plates at 378C in a 5% CO 2 humidified atmosphere with 80–120 mgrml of KLH. Supernatants ŽSN. were harvested after 24 h to measure IL-2, and at 48 or 72 h to measure IL-4 and IFN-g and stored at y708C prior to assay.
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2.6. Enzyme linked immunosorbent assay (ELISA) for IL-2, IL-4, and IFN-g Levels of cytokines in supernatants were determined by ELISA using monoclonal antibodies ŽmAb. and our published protocol ŽMoynihan et al., 1994.. Briefly, 96-well enhanced protein binding ELISA plates ŽCorning, Corning, NY. were coated overnight at 48C with 100 mlrwell of purified anti-cytokine capture mAb Ž2 mgrml, PharMingen, San Diego, CA.. Between each step, plates were washed with PBSrTween 20. Plates were blocked for 2 h with PBS–10% fetal equine serum ŽFES, Sigma. at room temperature. Recombinant cytokine standards Ž in Unitsrml, PharMingen. and samples were added to plates in 100 ml and allowed to incubate overnight at 48C. Biotinylated anti-cytokine detecting mAb Ž1 mgrml, PharMingen. was added in 100 ml and the plates were incubated at room temperature for 45 min. Avidin-peroxidase at 2.5 mgrml in PBS–10% FES was added in 100 ml and the plates were incubated for 30 min at room temperature. Finally, 100 mlrwell of substrate Ž2,2X-azinobisŽ3-ethylbensthiazoline-6-sulfonic acid., Sigma. was added. Absorbance at 405 nm was determined using a Biotek automated plate reader ŽWinooski, VT.. 2.7. ELISA for serum anti-KLH antibody responses IgM and IgG anti-KLH antibody titers in sera were determined using a standard ELISA ŽMoynihan et al., 1994.. Wells of 96-well microtiter plates were coated overnight with 10 mgrml of KLH in a carbonate coating buffer, pH 9.6, and post-coated for 1 h with PBS containing 1% bovine serum albumin ŽBSA. at 378C. Experimental sera were diluted 1:25, 1:75, and 1:225 in PBSrTween containing 1 M NaCl and incubated for 3 h at 378C. The plates were incubated overnight at 48C with alkaline phosphatase-conjugated goat anti-mouse IgM or IgG antibody Žm- and g-chain specific, respectively, Cappel, Durham, NC. diluted appropriately in PBSrTween followed by addition of the substrate, p-nitrophenyl phosphate ŽSigma., at 1 mgrml in carbonate buffer, pH 9.6. Washes with PBSrTween followed each step. Absorbance at 405 nm was measured. A standard curve was generated using sera from mice hyperimmunized with KLH. Sera from non-immunized mice served as a negative control. 2.8. Statistical analysis When appropriate, data were analyzed by analysis of variance ŽANOVA. with treatment conditions and replication as factors using StatView Žversion 5.0. on a Macintosh PowerPC computer. Post-hoc comparisons of significant interactions were made with Duncans Multiple range test. Planned comparisons were made using the two-tailed Student Neuman–Keuls t-test.
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3. Results 3.1. SOE increases production of IL-4 by spleen cells from odor recipients Spleen cells from stress odor and control odor exposed ŽCOE. mice were obtained 6 days following immunization Ž5 days following odor exposure. and were cultured with KLH as described. Supernatants were harvested from antigen-stimulated cultures and the Th1-like cytokines IL-2 and IFN-g, as well as the Th2-like cytokine IL-4 were assayed by ELISA. As shown in Fig. 1a, spleen cells from stress odor-exposed mice produced significantly more KLH-induced IL-4 than cells from COE mice Ž F Ž1,70. s 11.9, p - 0.01., confirming our previous observations ŽMoynihan et al., 1994.. As shown in Fig. 1b and c, however, no significant differences in KLH-stimulated Th1 cytokine production ŽIL-2 and IFN-g . were observed. Calculating the ratio of type 1: type 2 cytokines provides a useful measure of the balance between the two types of cytokines and is reflective of the immunological state of the animal; that is, the ratio reflects the ability of an animal to produce a cell-mediated vs. humoral immune response to antigen. The ratio of IFN-g: IL-4 in supernatants was observed to be 2.1 for spleen cells from COE mice and 1.2 for spleen cells from SOE mice, a significant 1.75-fold difference Ž p - 0.05.. These data indicate that SOE is associated with a pronounced immune deviation or shift in the balance between antigen-stimulated type 1 and type 2 cytokine production. 3.2. SOE induces opioid-mediated analgesia Olfactory information Žpheromones. is particularly important for reproduction, neuroendocrine responses, and the recognition of conspecifics, predators and prey. Production of endogenous opioids in the nervous system is particularly responsive to pheromonal influences and, as a result, opioids are strong candidates for mediating both the behavioral and immune responses to psychosocial stress. Because others have reported that odor stimuli can induce endogenous opioid secretion in animals ŽHarris and Pierson, 1964; Lichtman and Fanselow, 1990., and since opioids have a role in immune modulation as discussed above, the possibility that SOE induces endogenous opioid secretion was examined. This was first examined by investigating whether SOE induced analgesia, such as can be produced by opioids. Mice were tested for tail-flick latency, a standard measure of analgesia, as described above. The analgesic effect of putative opioids was determined at the end of 24 h of SOE. SOE was associated with a longer tail-flick latency compared to COE mice ŽFig. 2, t Ž1,14. s 2.7, p s 0.017.. In a separate experiment, SOE-induced analgesia was de-
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Fig. 2. SOE induces analgesia. Following 24 h of SOE or COE, mice Ž ns8rgroup. were subjected to tail-flick measures as described in Section 2. Asterisk indicates ps 0.017.
termined to be opioid mediated; administration of naloxone Ž10 mgrkg administered i.p.. 30 min prior to the end of the odor-exposure session blocked SOE-induced increases in tail-flick latency ŽFig. 3, t Ž1,14. s 2.5, p s 0.024..
Fig. 1. SOE results in increased Th2-type cytokine production. BALBrc male mice were immunized with 100 mg of KLH 24 h prior to SOE or control odor exposure ŽCOE.. Mice were sacrificed 6 days following immunization. Spleen cells were cultured with 80 mgrml of KLH for 24 h ŽIL-2. or 48 h ŽIL-4 and IFN-g .. Supernatants were harvested and assayed for cytokine production by ELISA. Figures represent three combined experiments with ns 36rgroup. Asterisk indicates p- 0.05.
Fig. 3. Naloxone abrogates SOE-induced analgesia. Mice Ž ns8rgroup. were injected i.p. with naloxone or saline following 24 h of SOE. Tail flick latency was assessed 15 min following injection. Asterisk indicates ps 0.024.
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Fig. 4. SOE induces opioid-mediated analgesia. BALBrc mice were exposed to SOE or COE for 24 h with either water or naltrexone Ž0.5 mgrml in water. available for the entire period of odor exposure. Tail-flick latency was measured at the end of the odor exposure. N s 16 for both SOE groups and n s 4 for both COE groups. SOE q water group is different from all other groups Ž p - 0.05..
3.3. Naltrexone in the drinking water of SOE mice blocks opioid-induced analgesia
induced analgesia that was antagonized by naltrexone availability in the drinking water ŽFig. 4, F Ž2,35. s 6.0, p s 0.002..
Although tail-flick latency was significantly decreased by antagonist administered at the end of the 24 h of SOE, alterations in immune responses are likely to be blocked only when antagonist is present throughout the 24-h odor exposure period. To test the hypothesis that opioid receptor antagonism would abrogate the stress odor-induced increase in immune function, we first determined that the longer acting antagonist naltrexone could be effectively administered in the drinking water of odor exposed mice, thereby avoiding both handling of mice and multiple injections of the drug. COE and SOE mice were given drinking water containing 0.5 mgrml of naltrexone 3 h before being placed in the odor-exposure apparatus and throughout the 24-h odor-exposure session. Analysis of tail-flick latencies at the end of the odor exposure period indicated that SOE
3.4. Oral naltrexone abrogates SOE induced increases in IL-4 and both IgM and IgG anti-KLH antibody production After determining that the analgesic effects of stress odor-induced opioids could be antagonized by access to oral naltrexone, we examined whether the immunological effects of SOE might also be antagonized. Beginning 3 h before, and lasting throughout the 24 h of odor exposure, KLH-immunized mice were given access to drinking water with or without naltrexone Ž0.5 mgrml.. Five days following the end of the odor exposure session, mice were sacrificed, splenocytes were stimulated with KLH in vitro, and supernatants were assayed for IL-4 Žat 72 h., IFN-g Žat 72 h. and IL-2 Žat 24 h.. IL-4 values from two experiments were combined into a single ANOVA; an interaction between odor exposure
Fig. 5. Naltrexone blocks the SOE-induced increase in IL-4 production by BALBrc splenocytes. Mice were given either plain water or water containing 0.5 mgrml of naltrexone in water to drink during the period of odor exposure. N s 12-16 micergroup. SOE q water group was different from all other groups Ž p - 0.05..
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ŽSOE vs. COE. and fluid consumed Žnaltrexone vs. water. was observed Ž F Ž1,102. s 6.7, p s 0.011., as well as a main effect of odor exposure Ž F Ž1,102. s 7.2, p s 0.008.. Post hoc analyses indicated that splenocytes from SOE q water mice produced more IL-4 than splenocytes from COE q water mice and both naltrexone groups Ž p - 0.05, Fig. 5.. In these cultures there was an unexplained paucity of KLH-stimulated IFN-g production Žaveraging only 3.6 unitsrml.; therefore, the ratio of IFN-g:IL-4 could not be determined with any confidence. Naltrexone had no effect on COE mice compared to COE mice given water without naltrexone for 24 h. In contrast to the data shown in Fig. 1, analysis of IL-2 levels in these supernatants from the two experiments revealed a marginally significant main effect of IL-2 produced by cells from SOE compared to COE Ž3.6 " 0.2 compared to 4.25 " 0.21 unitsrml, respectively; F Ž1,108. s 3.90, p s 0.051., with IL-2 production decreased in cultures of spleen cells from SOE mice. IgM and IgG serum anti-KLH antibody titers in serum from mice were examined in another group of mice 6 days
Fig. 6. Naltrexone abrogates the SOE-induced increase in IgM and IgG anti-KLH antibody responses. Mice were immunized 24 h prior to odor exposure and were bled 7 days later. Sera samples were assayed for IgM and IgG anti-KLH antibody responses by ELISA. N s10–14 micergroup. SOEqwater group responses were greater than all other groups Ž p0.05..
following odor exposure Ž7 days following immunization with 100 mg of KLH.. ANOVA indicated an interaction of odor exposure and treatment for both IgM Ž F Ž3,188. s 8.73, p - .01. and IgG Ž F Ž3,188. s 5.2, p - 0.01.. Planned comparisons indicated that the group of mice receiving SOE q water had significantly elevated IgM anti-KLH antibody responses compared to all other groups Ž p - 0.05, Fig. 6a.. The group receiving SOE q water also had higher IgG anti-KLH antibody responses compared to all other groups Ž p - 0.05, Fig. 6b.. ANOVA indicated no interaction with serum dilution; data are shown collapsed across three dilutions.
4. Discussion Here we have demonstrated that a psychosocial stressor, exposure to the odors of footshocked mice, is associated with immune deviation and increased antigen-specific antibody responses to the protein antigen KLH. Further, this stressor induces changes in nociception that are blocked by administration of the opioid receptor antagonists naloxone or naltrexone, and naltrexone also blocks the stressor-induced changes in immune function. Thus, we have evidence that endogenous opioids are immunomodulatory, resulting in increased Th2 cytokines and antibody titers. Opioid peptides Žproopiomelanocortin peptides, the enkephalins and the dynorphins. are derived from the hypothalamus, pituitary, and adrenal medulla, as well as from lymphocytes Žreviewed in Weigent and Blalock, 1994; Carr et al., 1996; Stefano et al., 1996.. The best characterized opiate receptors in the nervous system are the m, k, and d receptors; evidence also exists for expression of these receptors on cells of the immune system Žreviewed in Stefano et al., 1996; Madden et al., 1998; Mellon and Bayer, 1998; Sharp et al., 1998.. Opioids, which can be viewed as cytokines acting within the CNS and immune system ŽPeterson et al., 1998., are clearly immunomodulatory and their effects on immune function are dependent upon concentration as well as the immune parameter being investigated. The in vivo effects of exogenous opioids are generally immunosuppressive. Phagocytic and other monocytermacrophage function Žreviewed in Stefano et al., 1996; Eisenstein and Hilburger, 1998.; NK cell activity ŽShavit, 1991; Carr and France, 1993; Lysle et al., 1993; Yeager et al., 1995; Hsueh et al., 1996., mitogen-induced proliferation ŽHernandez et al., 1993; Lysle et al., 1993; Fecho et al., 1996a; Mellon and Bayer, 1998., CTL activity ŽCarpenter and Carr, 1995., IL-2 and IFN-g production ŽLysle et al., 1993. were all observed to be decreased following injection of morphine. The effects of stress-induced endogenous opioids on immune function have largely focused on suppressed NK cell activity and increased tumor growth Žreviewed by
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Shavit, 1991.. Other data suggest that endogenous b-endorphin normally plays a physiological role in immune regulation, suppressing NK cell activity and mitogen-induced proliferation, and increasing allograft survival time ŽPanerai et al., 1995.. In addition, b-endorphin may be involved in downregulation of Th1-induced autoimmune diseases, including experimental allergic encephalomyelitis ŽEAE. in rats ŽPanerai et al., 1994. and Crohn’s disease in humans ŽWiedermann et al., 1994.. Panerai and his colleagues have hypothesized that opioids, in particular b-endorphin that might be elevated during a stress response, result in increased production of Th2 cytokines, exerting a tonic inhibitory effect on the immune system ŽPanerai and Sacerdote, 1997.. Our data support this hypothesis; however, more detailed investigation is needed to understand the regulation of immune function by endogenous opioids. There is evidence of direct receptor-mediated actions of opioids on leukocytes Žreviewed in Carr et al., 1996; Stefano et al., 1996; Eisenstein and Hilburger, 1998; Sharp et al., 1998. and ample evidence for the expression of opioid receptors on lymphocytes ŽCarr et al., 1996; Stefano et al., 1996; Sharp et al., 1998.. In contrast, the effects of exogenous morphine on immune function have been frequently demonstrated to be centrally-mediated ŽHernandez et al., 1993; Fecho et al., 1996a; Mellon and Bayer, 1998., acting predominantly through m opioid receptors expressed in the periacqueductal gray of the mesencephalon. Evidence exists for either peripheral b-adrenergic ŽFecho et al., 1993; Fecho et al., 1996b. andror glucocorticoid mechanisms in morphine-induced changes in immune function ŽFreier and Fuchs, 1994; Fecho et al., 1996b.. Morphine injection has been associated with an increase in glucocorticoids ŽLockwood et al., 1996; Mellon and Bayer, 1998.; however, the glucocorticoid increase does not always appear to correlate with decreased immune responses ŽLockwood et al., 1996.. Of relevance to our study are the findings of Sutton et al. Ž1994. who showed that opioidmediated analgesia induced by acute inescapable tail shock in rats was attenuated by adrenalectomy ŽADX.. Basal corticosterone ŽCort. replacement in the drinking water of the ADX rats restored the opioid-induced analgesia; therefore, basal Cort was permissive for the shock-induced analgesia, but shock-induced increases in Cort were not necessary. This is of interest because previous studies from our laboratory indicated that some of the immunological effects of SOE Ži.e., increased IL-4 production. could be abrogated by pretreatment of mice with the glucocorticoid receptor antagonist RU486 ŽMoynihan et al., 1994. even though glucocorticoids were not significantly elevated in the SOE paradigm Žunpublished data.. In conclusion, our studies demonstrate that the balance between Th1 and Th2 cytokines is a potential target for neuroendocrine influences on immunity. Future studies will be directed at determining the consequence of immune deviation due to neuroendocrine perturbations for disease pathogenesis and in clinical populations.
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Acknowledgements The authors wish to thank Tracey Smith for her excellent technical assistance. This research was supported by research grants from the PHS ŽMH-45681. and The Fetzer Institute.
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