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0022-3565/99/2882-0635$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics JPET 288:635–642, 1999 Vol. 288, No. 2 Printed in U.S.A. The Effects of Morphine, Nicotine and Epibatidine on Lymphocyte Activity and Hypothalamic-Pituitary-Adrenal Axis Responses R. DANIEL MELLON and BARBARA M. BAYER Department of Pharmacology, Georgetown University Medical Center, Washington, DC Accepted for publication September 1, 1998 This paper is available online at http://www.jpet.org Evidence implicating the central nervous system (CNS) as a key regulator of immune function has been presented by many laboratories (Madden and Felten, 1995). Both the autonomic nervous system (ANS) and the neuroendocrine system have been suggested to be the primary mechanisms mediating CNS-induced immunomodulation (Madden and Felten, 1995). Recent studies have demonstrated that selective drugs of abuse modulate both immune cell activities and the hypothalamic-pituitary-adrenal (HPA) axis. In particular, this and other laboratories have demonstrated that opioid drugs such as morphine suppress the immune system via activation of central opioid receptors (Mellon and Bayer, 1998a). Similar immunomodulatory effects have also been reported after nicotine treatment (Caggiula et al., 1992; McAllister et al., 1994); however, the exact site of action and mechanism remains to be determined. The similarity of the Received for publication May 26, 1998. 1 This work was supported by National Institute on Drug Abuse Grants R01 DA04358 (B.M.B.) and F31 DA05779 (R.D.M.). Some preliminary observations in this paper have been reported previously (Mellon et al., 1996). glionic antagonist chlorisondamine (0.5 mg/kg, i.p.), completely blocked the effect of epibatidine on blood lymphocytes without altering the elevation of corticosterone levels. Although naltrexone (10 mg/kg, s.c.) blocked all effects of morphine, the effects of epibatidine were not blocked by the opioid receptor antagonist. Furthermore, in contrast to morphine (Hernandez et al., 1993), central injection of neither nicotine (30 or 240 nmol) nor epibatidine (5, 50, or 500 ng) altered blood lymphocyte responses. These results suggest that, like morphine, nicotinic agonists decrease blood lymphocyte proliferation responses, apparently independent of elevated corticosterone. However, unlike morphine, nicotinic agonists appear to act predominantly at peripheral receptors, suggesting that nicotinic receptors are downstream of opioid receptors in a centrally mediated opioidinduced immunomodulatory pathway. findings reported with nicotine to those produced by morphine suggest a possible relationship between nicotinic and opioid receptor-mediated immunomodulation. To examine the potential overlap between opioid and nicotinic receptors in a neuroimmunomodulatory pathway, we compared the effects of morphine with those of nicotine, and also examined the effects of epibatidine, a highly selective and potent nicotinic receptor agonist. Epibatidine, an alkaloid isolated from skin extracts of the Ecuadoran poison frog, Epipedobates tricolor, was first described by Daly and colleagues in 1992 (Spande et al., 1992). Initial interest in this compound was elicited due to its ability to produce both a Straub tail reaction (typical of opioid receptor activation) and potent antinociception at doses 200- to 500-fold lower than morphine. In subsequent studies, epibatidine was shown to bind with high affinity to the nicotinic receptor and demonstrated little, if any, activity at any other receptor (Badio and Daly, 1994; Houghtling et al., 1994; Houghtling et al., 1995). Recent studies suggest that this compound may bind with high affinity to the subtype of neuronal nicotinic receptor ABBREVIATIONS: CNS, central nervous system; ANS, autonomic nervous system; DMPP, 1,1-dimethyl-4-phenylpiperazinium; HPA, hypothalamic-pituitary-adrenal; ConA, concanavalin A. 635 Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 ABSTRACT Acute administration of morphine alters various neuroendocrine and immune parameters via opioid receptors located within the central nervous system. Similar effects have been reported after systemic nicotine treatment. To examine the possible relationship between opioid and nicotinic receptor activation on the immune system, we compared the effects of morphine with both nicotine and the highly selective nicotinic agonist, epibatidine. Male Sprague-Dawley rats were treated with either morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. 5 1 mg/kg freebase), or epibatidine (5 mg/kg, s.c.) and sacrificed 2 hours later. Each drug increased plasma corticosterone levels and decreased the magnitude of the peripheral blood lymphocyte proliferation response to the T cell mitogen concanavalin A. None of the treatments had a significant effect on splenic or thymic lymphocyte responses. The effects of nicotine treatment were dose-dependent. Pretreatment with the quaternary gan- 636 Mellon and Bayer predominating at the autonomic ganglia (Houghtling et al., 1995; Flores CM et al., 1996) and thus promises to be a powerful pharmacological tool for the study of autonomic functions. The present studies compare the effects of morphine with those of both nicotine and epibatidine on the proliferation responses of T lymphocytes and activation of the HPA axis. To determine the site of action of nicotinic drugs, the effects of systemic and central administration of nicotine and epibatidine were measured. In addition, the quaternary ganglionic antagonist chlorisondamine was used to determine the relative contribution of peripheral and central nicotinic receptors in the modulation of lymphocyte activity. A preliminary report of some of the observations in this paper has been presented previously (Mellon et al., 1996). Materials and Methods drug dispersal into the ventricle. Flow of the drug solution was monitored via tracking a small air bubble created in the polyethylene tubing that separated the drug solution and distilled water. The animal was returned to the home cage for the duration of the treatment period. Nociception was measured as described 30 min after either morphine administration or 3 min after either nicotine or epibatidine treatment. Animals were sacrificed either 1 or 2 h later, as indicated in the figure legends. Whole-Blood Lymphocyte Proliferation Assays. Animals were sacrificed via decapitation and trunk blood was collected in 50-ml polypropylene tubes containing 0.2 ml of heparin (1000 U/ml) and immediately placed on ice until processed. Whole blood was diluted 1:5 with cold RPMI 1640 cell culture media (Gibco, Grand Island, NY) containing 1% fetal bovine serum and gentamicin (20 mg/ml). Triplicate samples of blood (0.1 ml) were plated into 96-well microtiter plates containing 0.1 ml/well of the T cell-specific mitogen concanavalin A (ConA; 0, 2, 4, and 6 mg/well) and incubated for 72 h at 37°C with 8% CO2. Cells were pulsed with 0.5 mCi of [methyl3 H]thymidine (6.7 Ci/mmol; New England Nuclear, Boston, MA) in a 20 ml volume and incubated for an additional 24 h. Samples were lysed with distilled water and harvested onto glass fiber filters using a 96-well cell harvester (Brandel, Gaithersburg, MD). The amount of labeled DNA was determined via liquid scintillation spectrophotometry (Beta Plate; L.K.B. Pharmacia). Spleen and Thymus Lymphocyte Proliferation Assays. After decapitation, spleen and thymus were removed via sterile forceps and each placed in cold RPMI 1640 cell culture media (10 ml) containing 1% fetal bovine serum and gentamicin (20 mg/ml). The tissues were teased apart with sterile forceps, and the resulting cell suspension was washed twice in cold RPMI 1640 media and adjusted to a concentration of 2 3 106 cells/ml for spleen and 5 3 106 cells/ml for thymus. Cell preparations (100 ml) were then cultured with increasing concentrations of ConA (0, 0.0625, 0.125, 0.25, and/or 0.5 mg/well) as described for the blood proliferation assay to provide both optimal and suboptimal concentrations of mitogen. Antinociception. Antinociception was measured by the radiant heat tail-flick method (D’Amour and Smith, 1941), as described previously (Hernandez et al., 1993). All animals were acclimated to handling and the tail-flick device for 2 to 3 days before experimental manipulations. Light intensity was controlled to provide a predrug latency between 2 and 3 s. A cut-off of 8 s was used to prevent damage to tail tissue. Data were expressed as the percent maximum possible effect (% MPE) as defined below: % MPE 5 Postdrug latency (s) 2 Predrug latency (s) Cutoff (8 s) 2 Predrug latency (s) 3 100 Plasma Corticosterone Assay. Plasma samples were maintained at 220°C until assayed via solid phase 125I radioimmunoassay kits purchased from ICN Biochemicals, Inc. (Costa Mesa, CA). Because baseline levels of plasma corticosterone were found to vary depending on the nature of the experimental manipulations involved in the assays, matched treatment controls were included in all studies. Histological Analysis. Brains of centrally treated animals were removed postmortem and stored in 10% formaldehyde followed by 20% sucrose solution. All brains from drug-treated animals were cut into 40-mm sections on a freezing microtome (HistoSTAT Cryostat Microtome, model 975c; Reichert, Buffalo, NY) and placed on gelatincoated slides for microscopic verification of cannula placement by comparison to the atlas of Paxinos and Watson (1997). Statistical Analysis. Only animals whose cannula were determined to be accurately placed in the ventricle were used for data analysis. For proliferation assays, the median of triplicate samples was determined for each concentration of mitogen and the resulting dose-response curves generated. To compare the ConA dose-response curves, nonlinear regression analysis was completed to generate the best-fit curve using GraphPad Prism software (San Diego, CA). For blood proliferation data, regression analysis indicated that the Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 Animals. Pathogen-free male Sprague-Dawley rats (200 –225 g upon receipt) were obtained from Taconic Laboratories (Germantown, NY). Animals were group-housed three per cage with microisolator tops and were provided food (Purina rat chow, Ralston Purina Co., St. Louis, MO) and water ad libitum. The light cycle was regulated automatically (lights on at 6 AM, off at 6 PM) and temperature was maintained at 23 6 1°C. All animals were allowed to acclimate to this environment for 1 week before use in an experiment or surgical implantation of a guide cannula into the ventricular system. All experiments were conducted between 7 and 11 AM. Drugs. Morphine sulfate was generously provided by the National Institute on Drug Abuse (Research Triangle Park, NC). Epibatidine dihydrochloride was purchased from Research Biochemicals International (Natick, MA). Nicotine hydrogen tartrate and naltrexone hydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). Chlorisondamine chloride (Ecolid) was kindly provided by Ciba-Geigy Corporation (Summit, NJ). All drugs were dissolved in sterile isotonic saline, which also served as the control treatment in these studies. The injection volume for all systemic studies was 1 ml/kg and the route of administration was as indicated in the figure legends. The doses of all drugs reported in these studies are based on the molecular weight of the salt and not that of the freebase; however, the freebase doses of nicotine are also provided in parentheses following that of the salt. Cannulation and Microinjection Procedures. Animals were anaesthetized with Equithesin (3 ml/kg, i.p.). Equithesin was prepared by dissolving chloral hydrate (4.25 g) and magnesium sulfate (2.13 g) in a solution consisting of deionized water (43 ml), propylene glycol (26.6 ml), ethyl alcohol (12.45 ml), and pentobarbital (18 ml, 50 mg/ml). Surgical implantation of a guide cannula into either the right lateral ventricle (final coordinates relative to bregma: anteriorposterior 5 23.6; medial-lateral 5 24.6, dorsal-ventral 5 27.5) or the dorsal third ventricle (final coordinates relative to bregma: anterior-posterior 5 24.2; medial-lateral 5 0; dorsal-ventral 5 23.4) was completed based on the atlas of Paxinos and Watson (1997). After surgery, a stylet was inserted into the guide cannula to maintain sterility and patency. All animals were given gentamicin sulfate (4 mg, s.c.) and allowed to recover for approximately 1 week before experimentation. Microinjection of drugs was accomplished in freely moving awake animals as described previously (Hernandez et al., 1993). Briefly, test animals were removed from the home cage, the protective stylet was removed, and the internal cannula, which extended 1 mm beyond the end of the guide cannula, was inserted. The animal was placed into a separate cage for the injection procedure. The injection volume for all centrally administered drugs was 2 ml, administered over 45 s using a Sage Infusion pump set to dispense 2.7 ml/min. The internal cannula remained in place for an additional 75 s to ensure Vol. 288 1999 Opioid/Nicotinic Immunomodulation Results Comparison of the Effects of Morphine with Nicotinic Agonists. The antinociception, lymphocyte suppression, and activation of the HPA axis induced by morphine were compared to those of the prototypical ganglionic agonist nicotine and the potent nicotinic agonist epibatidine. The rationale for the dose of each drug was based on the results of previously published studies in our and other investigators’ laboratories (Tripathi et al., 1982; Bayer et al., 1992; Caggiula et al., 1992; Qian et al., 1993; McAllister et al., 1994). A representative study comparing the antinociceptive effects of these compounds as measured by tail-flick latency is shown in Fig. 1. Both nicotine (2.85 mg/kg, s.c. 5 1.0 mg/kg freebase) and epibatidine (5 mg/kg, s.c.) treatment produced approximately 60% of the maximum possible effect 3 min after administration, whereas the response to morphine (10 mg/kg, s.c.) was not significant at this time point. However, when tested 30 min after drug treatment, the antinociceptive Fig. 1. Effect of morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. 5 1.0 mg/kg freebase), and epibatidine (5 mg/kg, s.c.) on tail-flick latency to radiant heat. Animals (n 5 7 per group) were treated with the indicated drug and tail-flick latency was measured 3 min later. Morphine-treated animals were again tested 30 min after the injection. Data are expressed as the maximum possible effect as described in Materials and Methods. **P , .01 compared with saline and morphine treatment at 3 min, ***P , .001 compared with saline and morphine treatment at 3 min, and P , .05 compared with both nicotine and epibatidine treatment (one-way ANOVA, Newman-Keuls). effect of morphine was maximal (Fig. 1), whereas by this time, nicotine and epibatidine were only marginally effective (data not shown). In addition to their antinociceptive effects, treatment with morphine, nicotine or epibatidine significantly decreased the magnitude (Emax) of the peripheral blood lymphocyte proliferation response to the T cell mitogen ConA when compared with responses of saline-treated control animals (Fig. 2). No significant alteration in the sensitivity (EC50) of lymphocytes to ConA was noted after any drug treatment. In contrast to the effect of these agents on peripheral blood lymphocytes, no significant effects were detected in lymphocytes isolated from either the spleen or the thymus under these conditions (data not shown). Since activation of the HPA axis has been reported to produce altered immune parameters (Parrillo and Fauci, 1979), plasma levels of corticosterone were also measured in these studies. Figure 3 represents the combined results from two studies where animals were sacrificed at either 1 or 2 h after drug administration. As depicted, all drug treatments produced significantly elevated plasma levels of corticosterone compared with saline treatment when measured at 1 h. However, only morphine treatment significantly continued to sustain elevated corticosterone levels for 2 h (Fig. 3). Dose Dependence of Nicotine-Induced Alterations in Peripheral Blood Lymphocyte Proliferation. Unlike epibatidine or morphine, the dose of nicotine used in these studies (2.85 mg/kg, s.c. 5 1.0 mg/kg freebase) produced some seizure activity. Because this could complicate the interpretation of the results, experiments were carried out to determine whether the effects of nicotine on lymphocyte responses could be observed at lower doses. Figure 4 represents the combined results from two studies testing nicotine at four Fig. 2. Effect of morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. 5 1.0 mg/kg freebase), and epibatidine (5 mg/kg, s.c.) on blood lymphocyte proliferation responses to ConA. Animals (n 5 6 –7 per group) were sacrificed 2 h after drug treatment. All drug treatments significantly decreased the magnitude (Emax) of the blood lymphocyte proliferation responses compared with saline-treated controls (***P , .001, one-way ANOVA, Newman-Keuls). The EC50 for ConA treatment was unaltered by any drug treatment (P . .05, one-way ANOVA, Newman-Keuls). Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 curves best fit a sigmoidal dose-response curve with the following equation: Y 5 Emin 1 (Emax 2 Emin)/(1 1 10LogEC50 2 X), where Y is the proliferation response measured in cpm, Emin is the calculated minimum proliferation response (i.e., absence of mitogen), Emax is the calculated maximal proliferation response, EC50 is the concentration of mitogen producing 50% of the maximal response, and X is the logarithm of concentration. From the resulting equations the values for maximal response (Emax) and EC50 were obtained and compared. Significant differences in Emax were interpreted as alterations in the magnitude of the response of lymphocytes to ConA treatment, whereas alterations in EC50 values were interpreted as alterations in the sensitivity of the lymphocytes to ConA treatment. For all analyses, Student’s t test was used to compare results from experiments involving only two treatment groups, whereas a oneway analysis of variance (ANOVA) with Newman-Keuls post hoc analysis was conducted for comparison of three or more groups. When no significant difference between basal proliferation in the control groups occurred, the results obtained in individual experiments were combined directly and proliferation data were expressed as the mean cpm 6 S.E.M. For all parameters, any value greater than 2 SDs from the mean of the treatment group was omitted. 637 638 Mellon and Bayer Fig. 3. Effect of morphine (10 mg/kg, s.c.), nicotine (2.85 mg/kg, s.c. 5 1.0 mg/kg freebase), and epibatidine (5 mg/kg, s.c.) on plasma levels of corticosterone. Animals (6 –7 per group) were treated with the indicated drug and sacrificed either 1 or 2 h later. Plasma corticosterone was determined as described in Materials and Methods. *P , .05 compared with salinetreated control value for indicated time (one-way ANOVA, NewmanKeuls). different doses. ANOVA indicated that nicotine at a dose of either 2.0 mg/kg (0.7 mg/kg freebase) or 2.85 mg/kg (1.0 mg/kg freebase) significantly decreased the magnitude of blood lymphocyte proliferation responses compared with saline-treated controls and doses of nicotine at either 0.5 mg/kg (0.175 mg/kg freebase) or 1 mg/kg (0.35 mg/kg freebase). Although not significant, a dose of 1 mg/kg (0.35 mg/kg freebase) slightly decreased peripheral blood lymphocyte responses, whereas the lowest dose of nicotine (0.5 mg/kg, s.c. 5 0.175 mg/kg freebase) slightly elevated the response. The sensitivity of lymphocytes to ConA as measured by EC50 values was not significantly altered. In these studies, nicotine at doses of 2 mg/kg (0.7 mg/kg freebase) or 2.85 mg/kg, s.c. (1.0 mg/kg freebase) also significantly increased latency to tail-flick, whereas lower doses were without any detectable effect (data not shown). The Role of Central Nicotinic Receptor Activation on Peripheral Lymphocyte Activity. As nicotine and epibatidine readily enter the CNS, the role of central nicotinic receptors in the effect of these drugs on peripheral blood lymphocyte proliferation was examined by administering these compounds directly into the ventricular system of freely moving conscious rats. Figure 5 shows the effects of administration of nicotine at doses of 30 and 240 nmol into the third ventricle. Neither of these doses of nicotine significantly altered either the magnitude (Emax) or sensitivity (EC50) of blood lymphocytes to ConA treatment. To maximize the central tissues potentially exposed to injected drug, the higher dose of nicotine (240 nmol) was administered into the lateral ventricle. As shown in the inset in Fig. 5, nicotine treatment was still without effect on blood lymphocyte proliferation. No significant alterations in spleen lymphocyte proliferation were noted after microinjection into either the third or the lateral ventricle under these conditions (data not shown). To evaluate whether epibatidine may have centrally mediated effects on lymphocyte activity, the drug was also administered directly into the lateral ventricle of the rat. The doses of epibatidine used (5, 50 and 500 ng) were based on our previous experience with central administration of morphine (Hernandez et al., 1993), which detected significant effects on blood lymphocyte proliferation and antinociceptive responses after microinjection of doses 1000-fold lower than those used systemically. After the central administration of epibatidine, the highest dose tested (500 ng) produced a slight but insignificant decrease in the magnitude of the peripheral blood lymphocyte proliferation response (Fig. 6). Nicotinic Antagonist Studies. To further examine whether the effects of epibatidine were mediated predominantly by peripheral nicotinic receptors, chlorisondamine (0.5 mg/kg, i.p.), the quaternary nicotinic antagonist, was administered 30 min before epibatidine (5 mg/kg, s.c.) treatment. Animals were sacrificed 1 h later, at a time when epibatidine significantly elevates corticosterone levels (Fig. 3). The rationale for the dose of chlorisondamine was based on a review of the published literature. Although higher doses of chlorisondamine have been reported in the literature (Irwin et al., 1988; Britton and Indyk, 1989; Nikolarakis et al., 1989; Saito et al., 1991), we chose a lower dose to decrease Fig. 5. Effect of central (3V) nicotine on the magnitude of the peripheral blood lymphocyte proliferation response. Animals (n 5 5–7) were treated with the indicated dose of nicotine directly into the third ventricle and sacrificed 2 h later. Blood lymphocyte proliferation responses were measured as described in Materials and Methods. No significant differences in either Emax or EC50 between treatment groups were detected (P . .05, one-way ANOVA, Newman-Keuls). Inset, nicotine (240 nmol, n 5 11–14) was administered directly into the lateral ventricle and animals were sacrificed as above. No significant differences in either Emax or EC50 between treatment groups were detected (P . .05, Student’s t test). Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 Fig. 4. Dose-dependent effects of nicotine on the magnitude of blood lymphocyte proliferation response. Animals were treated with either saline (n 5 13) or the indicated dose of nicotine (n 5 6 –7 per group) and sacrificed 2 h later. The proliferation response of blood lymphocytes was determined as described in Materials and Methods. Calculated Emax values (cpm): saline, 40,770 6 2,506; 0.5 mg/kg, 47,780 6 5,574; 1.0 mg/kg, 33,040 6 3,738; 2.0 mg/kg, 23,650 6 2,890**; 2.85 mg/kg, 17,900 6 1751***. **P , .01 compared with saline and nicotine 0.5 mg/kg (0.175 mg/kg freebase), ***P , .001 compared with saline, nicotine 0.5 mg/kg (0.175 mg/kg freebase), and P , .05 compared with nicotine 1 mg/kg, s.c. (0.351 mg/kg freebase) (one-way ANOVA, Newman-Keuls). The EC50 for ConA treatment was unaltered by any drug treatment (P . .05, one-way ANOVA, Newman-Keuls). Vol. 288 1999 the potential for this quaternary drug to gain access to the CNS (Clarke, 1984). As previously shown at 2 h (Fig. 2), epibatidine treatment also significantly suppressed the magnitude of the blood lymphocyte proliferation response within 1 h (Fig. 7). Furthermore, pretreatment with chlorisondamine completely antagonized the suppressive effects of systemic epibatidine on the blood lymphocyte proliferation response. To examine the possibility that this dose of chlorisondamine may be blocking central nicotinic receptors, plasma levels of corticosterone were measured. As shown in Fig. 8, epibatidine significantly elevated corticosterone 1 h after treatment; however, pretreatment with chlorisondamine did not antagonize this effect. Thus, in contrast to the effects of chlorisondamine on blood lymphocyte proliferation, chlorisondamine did not antagonize the effects of epibatidine on plasma levels of corticosterone (Fig. 8). Naltrexone Antagonist Studies. To determine whether the effects of epibatidine were due to interaction with opioid Fig. 7. Effect of chlorisondamine pretreatment on magnitude of epibatidine-induced suppression of peripheral blood lymphocyte proliferation response. Animals (n 5 5– 6 per group) were pretreated with either saline or chlorisondamine (0.5 mg/kg, i.p.) 30 min before a 1-h systemic treatment of epibatidine (5 mg/kg, s.c.). Blood lymphocyte proliferation response was measured as described in Materials and Methods. Calculated Emax values (cpm): saline saline, 49,240 6 4,452; saline epibatidine, 21,240 6 3,042 ***; chlorisondamine saline, 55,810 6 5,153; chlorisondamine epibatidine, 45,580 6 3,148. *** P , .001 compared with all other treatment groups (one-way ANOVA, Newman-Keuls). No significant differences in EC50 were detected (P . .05, one-way ANOVA, NewmanKeuls). 639 Fig. 8. Effect of chlorisondamine pretreatment on epibatidine-induced elevation of plasma corticosterone. Animals were treated as described in the legend to Fig. 7. Plasma corticosterone was determined as described in Materials and Methods. **P , .01 compared with saline saline- and chlorisondamine saline-treated groups (one-way ANOVA, NewmanKeuls). receptors, animals were pretreated with the opioid receptor antagonist naltrexone (10 mg/kg, s.c.) 30 min before epibatidine treatment (5 mg/kg, s.c.). Animals were sacrificed 1 h after epibatidine treatment. As shown in Fig. 9, epibatidine treatment significantly suppressed the blood lymphocyte proliferation response. In contrast to the effects of the nicotinic antagonist chlorisondamine, the opioid antagonist naltrexone did not antagonize the effect of epibatidine. Likewise, epibatidine treatment significantly elevated plasma levels of corticosterone in an opioid-independent manner (Fig. 10). Additional experiments were carried out under identical experimental conditions to confirm that the effects of morphine were sensitive to antagonism by naltrexone. Morphine treatment (10 mg/kg, s.c.) significantly decreased blood lymphocyte proliferation compared with all other treatment groups (Fig. 11). However, in contrast to the lack of an effect of naltrexone in epibatidine-treated animals, naltrexone (10 mg/kg, s.c.) pretreatment completely antagonized the effect of morphine on the blood lymphocyte proliferation response Fig. 9. Effect of naltrexone pretreatment on epibatidine-induced suppression of peripheral blood lymphocyte proliferation response. Animals (n 5 6 per group) were pretreated for 30 min with either naltrexone (10 mg/kg, s.c.) or saline, and then treated with either saline or epibatidine (5 mg/kg, s.c.) and sacrificed 1 h later. Blood lymphocyte proliferation response to ConA was determined as described in Materials and Methods. Calculated Emax values (cpm): saline saline, 38,710 6 5,197; saline epibatidine, 14,000 6 1,235 ***; naltrexone saline, 44,290 6 2,370; naltrexone epibatidine, 17,710 6 2,306 **. **P , .01 and ***P , .001 compared with both saline saline- and naltrexone saline-treated animals (one-way ANOVA, Newman-Keuls). No significant differences in EC50 were detected (P . .05, one-way ANOVA, Newman-Keuls). Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 Fig. 6. Effect of central epibatidine (LV) on peripheral blood lymphocyte proliferation response. Animals were treated with either saline (n 5 14) or epibatidine at a dose of 5 ng (n 5 9), 50 ng (n 5 4), or 500 ng (n 5 6) directly into the lateral ventricle and sacrificed 2 h later. Blood lymphocyte proliferation responses were measured as described in Materials and Methods. No significant differences in either Emax or EC50 were detected (P . .05, one-way ANOVA, Newman-Keuls). Opioid/Nicotinic Immunomodulation 640 Mellon and Bayer Fig. 10. Effect of naltrexone pretreatment on epibatidine-induced elevation of plasma corticosterone. Animals were treated as described in the legend to Fig. 9. Plasma corticosterone was determined as described in Materials and Methods. **P , .01 compared with saline saline- and naltrexone saline-treated groups (one-way ANOVA, Newman-Keuls). (Fig. 11). Likewise, naltrexone antagonized the effect of morphine on the HPA axis (Fig. 12). Discussion The close similarity of the reported findings with nicotine on immune function (Caggiula et al., 1992; McAllister et al., 1994) with the results of the studies conducted in our laboratory with morphine led us to further explore the relationship between nicotinic and opioid-induced alterations in immune function. The results (Figs. 1–3) with acute systemic morphine, nicotine and epibatidine treatment presented here demonstrated that each of these compounds produce: 1) antinociception, 2) decreased magnitude of peripheral blood lymphocyte proliferation responses to mitogen without altering the sensitivity of the lymphocytes, 3) no alteration of either splenic or thymic proliferation responses, and 4) an elevation of circulating corticosterone levels. Collectively, these results indicate that the effects of systemic morphine are largely mimicked by both nicotine and epibatidine treatment. Although considerable evidence supports the involvement of a central site of action for systemic morphine on blood Fig. 12. Effect of naltrexone pretreatment on morphine-induced elevation of plasma corticosterone. Animals were treated as described in the legend to Fig. 11. Plasma corticosterone was determined as described in Materials and Methods. ** P , .01 compared with all other groups (one-way ANOVA, Newman-Keuls). lymphocyte responses (Hernandez et al., 1993; Mellon and Bayer, 1998), the site(s) of action for nicotine-induced immunomodulatory effects has not been well characterized. Based on several observations in this report, the CNS does not appear to be the predominant site of action of nicotine- and epibatidine-induced alterations in peripheral blood lymphocyte responses. First, central administration of either nicotine or epibatidine failed to significantly alter blood lymphocyte proliferation (Figs. 5 and 6). Although the highest dose of epibatidine tested did produced a slight trend toward a decrease in blood lymphocyte proliferation, this dose was only 10-fold lower than the dose used for systemic administration. Second, chlorisondamine, a quaternary nicotinic antagonist, completely antagonized the effects of systemic epibatidine on blood lymphocyte proliferation responses. High doses of chlorisondamine (10 mg/kg, s.c.) have been reported to produce prolonged blockade of central nicotinic receptors (Clarke, 1984; Clarke et al., 1994), suggesting that some of the drug gained access to the central compartment under these conditions. However, the lower doses of chlorisondamine used in the present study (0.5 mg/kg, i.p.) had no effect on the stimulation of the HPA axis by epibatidine (Fig. 8). Because nicotinic receptor-mediated activation of the HPA axis appears to be due to central nicotinic receptors (Cam et al., 1979; Matta et al., 1987), these data suggest that it is unlikely that chlorisondamine gained access to the CNS. The conclusion that this dose of chlorisondamine does not gain access to the CNS is further supported by studies examining nicotine-induced antinociception (Caggiula et al., 1995). These observations also suggest that systemic administration of epibatidine decreases peripheral blood lymphocyte responses by stimulating peripheral rather than central nicotinic receptors. Similar to the findings reported here, Caggiula et al. (1992) observed that systemic nicotine administration to the Sprague-Dawley rat decreased peripheral blood lymphocyte proliferation responses in a dose-dependent manner. Additional studies by this group also demonstrated that acute systemic nicotine treatment in the Sprague-Dawley rat had no effect on splenic lymphocyte proliferation responses to ConA, suggesting that the blood lymphocytes were specifically targeted by nicotine (McAllister et al., 1994). However, in contrast to these observations, in the Lewis rat, Fecho et al. (1993) found that systemic administration of low doses of Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017 Fig. 11. Effect of naltrexone pretreatment on morphine-induced suppression of peripheral blood lymphocyte proliferation response. Animals (n 5 6 per group) were pretreated for 30 min with either naltrexone (10 mg/kg, s.c.) or saline, and then treated with either saline or morphine (10 mg/kg, s.c.) and sacrificed 1 h later. Blood lymphocyte proliferation responses to ConA were determined as described in Materials and Methods. Calculated Emax values (cpm): saline saline, 76,740 6 6,758; saline morphine, 41,870 6 5,918 ***; naltrexone saline, 95,870 6 7,589; naltrexone morphine, 94,970 6 6,706. *** P , .001 compared with all other groups (one-way ANOVA, Newman-Keuls). No significant differences in EC50 were detected (P . .05, one-way ANOVA, Newman-Keuls). Vol. 288 1999 641 munosuppressive (Keller et al., 1983; Keller et al., 1988; Bayer et al., 1990; Flores et al., 1990). Overall, the results are consistent with the conclusion that activation of the autonomic ganglia appear to lead to decreased peripheral blood lymphocyte proliferation. However, the possibility for a direct effect of nicotinic agonists on lymphocyte proliferation response has not been eliminated by these studies. Although there are a few published reports which provide evidence for nicotinic receptors on lymphocytes (Richman and Arnason, 1979; Maslinski et al., 1980; Konno et al., 1986; Menard and Rola-Pleszczynski, 1987; Toyabe et al., 1997), the pharmacology and functional significance of these receptors are not entirely clear. In summary, these studies provide the first comparison of the immune effects of nicotine with the potent nicotinic agonist epibatidine and suggest that nicotine and epibatidine treatment appear to mimic the effects of morphine on peripheral lymphocyte responses. These treatments appear to decrease the overall magnitude of the response to mitogen treatment without clearly altering the sensitivity of the cells to this potent stimuli. Although the physiological significance of this altered cellular responsiveness is not known, this profile resembles that which would be produced in the presence of a noncompetitive antagonist to immunological stimuli. Given this potential immunomodulatory role, activation of opioid or nicotinic receptors may lead to a functional, although attenuated, immune response. Furthermore, these studies provide evidence that nicotinic agonists produce their immunomodulatory effects on blood lymphocyte proliferation by acting predominantly at peripheral nicotinic receptors. In addition, nicotinic receptor mediated immunomodulation, like that produced by opioids, appear to be independent of their ability to activate the HPA axis. Collectively, the studies discussed here suggest that peripheral nicotinic receptors appear to be located downstream of central opioid receptors in an neuroimmunomodulatory pathway. Acknowledgments We thank Valerie Lewis-Morris and Monica C. Hernandez for expert technical assistance in these studies, Dr. Kenneth J. Kellar (Georgetown University) for generously providing epibatidine and nicotine for the initial studies, and Dr. John C. Pezzullo (Georgetown University, Washington, DC) and Dr. Harvey J. Morulsky (GraphPad Software, Inc.) for assistance with statistical analyses. References Badio B and Daly JW (1994) Epibatidine, a potent analgesic and nicotinic agonist. Mol Pharmacol 45:563–569. Bayer BM, Gastonguay MR and Hernandez MC (1992) Distinction between the in vitro and in vivo inhibitory effects of morphine on lymphocyte proliferation based on agonist sensitivity and naltrexone reversibility. Immunopharmacology 23:117– 124. Bayer BM, Hernandez M and Irvin L (1990) Suppression of lymphocyte activity after acute morphine administration appears to be glucocorticoid independent, in Alcohol, Immunomodulation, and AIDS (Pawloski A, Seminara D and Watson R eds) pp 273–282, Alan R. Liss, Inc., New York. Britton DR and Indyk E (1989) Effects of ganglionic blocking agents on behavioral responses to centrally administered CRF. Brain Res 478:205–210. Caggiula AR, Epstein LH, Perkins KA and Saylor S (1995) Different methods of assessing nicotine-induced antinociception may engage different neural mechanisms. Psychopharmacology 122:301–306. 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DMPP treatment suppressed the proliferation response of splenic lymphocytes, splenic natural killer cell cytolytic activity, interleukin-2 production, interferon-g production, and total white blood cell counts (Fecho et al., 1993). Explanations for the reported differential sensitivity of lymphocytes from blood and spleen compartments after nicotine and epibatidine compared with those with DMPP are not clear. Strain differences may account, in part, for some of these observations. For example, Fecho et al. (1993) used the Lewis rat model, whereas Caggiula et al. (1992), McAllister et al. (1994), and our studies used the Sprague-Dawley rat. Differences in autonomic tone have been suggested between these two animals strains (Dimitrova et al., 1995). Regardless of these compartmental differences, these studies collectively suggest that, like morphine, acute systemic nicotinic agonists produce alterations in peripheral lymphocyte activity. However, the studies presented in this report suggest that, unlike acute morphine, nicotinic agonists alter lymphocyte activity via peripheral nicotinic receptors rather than central pathways. In conjunction with our previous report (Flores LR et al., 1996), these studies collectively provide strong evidence that opioid receptors are located upstream from nicotinic receptors in a neuroimmunomodulatory pathway. This conclusion is supported by the following observations: 1) acute morphine appears to act only at central opioid receptors (Hernandez et al., 1993; Mellon and Bayer, 1998b); 2) nicotinic agonists appear to act predominantly at peripheral nicotinic receptors; 3) an opioid antagonist blocks the effect of morphine without altering those of the nicotinic agonist epibatidine; and 4) the peripherally acting nicotinic antagonist chlorisondamine antagonized both the effects of morphine (Flores LR et al., 1996) and epibatidine (Fig. 7). Therefore, these pathways appear to overlap at the level of the peripheral nicotinic receptor, presumably at the autonomic ganglia. In addition, as both opioid and nicotinic receptor activation stimulate corticosterone release via central mechanisms, the neuronal pathways mediating activation of the HPA axis may overlap. The results of the studies with naltrexone pretreatment of epibatidine, however, suggest that an opioid receptor is not downstream from the nicotinic receptor in the pathway whereby epibatidine stimulates corticosterone release. The results reported here demonstrating the effects of chlorisondamine pretreatment on epibatidine-induced responses also provide evidence that the suppressive effect of epibatidine on lymphocyte proliferation is not mediated by the increase in corticosterone levels. 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