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(Journal of Leukocyte Biology. 2002;71:782-790.)
© 2002 by Society for Leukocyte Biology

The immunosuppressive effects of chronic morphine treatment are partially dependent on corticosterone and mediated by the µ-opioid receptor

Jinghua Wang*, Richard Charboneau{dagger}, Sudha Balasubramanian*, Roderick A. Barke{dagger}, Horace H. Loh* and Sabita Roy*,{dagger}

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

Correspondence: Dr. Sabita Roy, Veterans Affairs Medical Center, Research RT 151, Room 3N 107, One Veterans Drive, Minneapolis, MN 55417. E-mail: royxx002{at}tc.umn.edu.


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ABSTRACT
 
Wild-type and µ-opioid receptor knockout (MORKO) mice were used to investigate the role of corticosterone (CORT) and the µ-opioid receptor (MOR) in chronic morphine-mediated immunosuppression. We found that although plasma CORT concentrations in CORT infusion (10 mg/kg/day) and morphine-pellet implantation (75 mg) mice were similar (400–450 ng/ml), chronic morphine treatment resulted in a significantly higher (two- to threefold) inhibition of thymic, splenic, and lymph node cellularity; inhibition of thymic-lymphocyte proliferation; inhibition of IL-2 synthesis; and activation of macrophage nitric oxide (NO) production when compared with CORT infusion. In addition, results show that the inhibition of IFN-{gamma} synthesis and splenic- and lymph node-lymphocyte proliferation and activation of macrophage TNF-{alpha} and IL-1ß synthesis occurred only with chronic morphine treatment but not with CORT infusion. These morphine effects were abolished in MORKO mice. The role of the sympathetic nervous system on morphine-mediated effects was investigated by using the ganglionic blocker chlorisondamine. Our results show that chlorisondamine was able to only partially reverse morphine’s inhibitory effects. The results clearly show that morphine-induced immunosuppression is mediated by the MOR and that although some functions are amplified in the presence of CORT or sympathetic activation, the inhibition of IFN-{gamma} synthesis and activation of macrophage-cytokine synthesis is CORT-independent and only partially dependent on sympathetic activation.

Key Words: glucocorticoids • sympathetic nervous system • hypothalamic pituitary adrenal axis • neuroimmunomodulation


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INTRODUCTION
 
That chronic morphine use or abuse results in immunosuppression is well accepted [1 , 2 ], but the mechanisms by which morphine mediates its immunosuppressive effects still remain controversial. Three major mechanisms have been proposed. The first mechanism speculates that morphine binds to the µ-opioid receptor (MOR) present on cells of the immune system [3 4 5 6 ] and modulates their function directly [7 8 9 10 11 ]. The second mechanism hypothesizes that morphine binds to opioid receptors present in the central nervous system (CNS), leading to an increase in circulating levels of corticosterone (CORT) and hypothalamic pituitary adrenal axis (HPAA) activity. This increase in CORT has been implicated to be indirectly responsible for the immunomodulatory effects of morphine [12 13 14 15 16 ]. The third mechanism speculates that morphine activates the sympathetic nervous system (SNS) and causes increased circulating levels of epinephrine from the adrenal medulla and norepinephrine from sympathetic nerve terminals [17 , 18 ]. Increased catecholamine release has been linked to suppression of natural killer (NK) cell function and altered lymphocyte function [19 ]. One of the effector mechanisms involved in this process has been shown to be an increased synthesis of nitric oxide (NO) by macrophages in splenocyte cultures, which then inhibits concanavalin A (Con A)-stimulated proliferation of splenic lymphocytes [20 ].

Recently, we have shown that the immunomodulatory effects of chronic morphine treatment are attenuated significantly in MOR knockout (MORKO) mice. Experiments with these animals also showed that chronic morphine treatment does not result in an increase in plasma CORT levels [21 ]. Elevated corticosteroids have been associated with the modulation of a number of immune parameters [22 , 23 ], but the duration and concentrations required to bring about changes in specific immune cells are still unknown. It is well established that the in vitro effects of glucocorticoids (GCs) are generally anti-inflammatory [24 ] and immunosuppressive, but it is becoming increasingly evident that the in vivo effects of glucorticoids are frequently different from in vitro treatment or treatment with synthetic GCs such as dexamethasone (DEX) [25 ]. In general, low concentrations of GCs have been shown to decrease blood lymphocyte numbers [26 ], but higher doses and prolonged exposure have been associated with decreases in lymphocyte numbers in the spleen and thymus [27 28 29 ]. In humans, prolonged exposure to elevated GC levels, as in Cushing’s syndrome, has been associated with decreased thymus size and decreased CD4-to-CD8 ratios [30 , 31 ]. The effect of GCs on cytokines expression and T-cell proliferation is dependent on the nature of the experiment and the dose and time applied [32 ]. Studies have shown that GCs suppressed cytokine production [33 , 34 ] but also induced or enhanced cytokine activity [35 , 36 ]. More recently, gene-profiling studies show that GCs have differential effects on proinflammatory cytokine synthesis [37 ].

In this study, we hypothesize that the immunosupressive effects of morphine are mediated by the MOR and that all three mechanisms, i.e., HPAA, SNS, and a direct effect through MOR, play a synergistic role in modulating the immunosuppressive effects of morphine. To test our hypothesis, wild-type (WT) and MORKO mice were implanted with alzet pumps delivering CORT at a rate consistent with plasma levels seen with morphine-treated animals. Immune parameters were tested and compared between morphine and CORT-treated animals. To test the role of catecholamines, animals were pretreated with chlorisondamine (a ganglionic blocker) and then treated with morphine. Our results showed that morphine induced a decrease in splenocyte proliferation and interferon-{gamma} (IFN-{gamma}) synthesis and an increase in macrophage interleukin (IL)-1 and tumor necrosis factor {alpha} (TNF-{alpha}) production. These effects were corticosteroid-independent. However, the effects of morphine on thymocyte proliferation and thymic cellularity are partially dependent on GCs. The contribution of the SNS on morphine mediated, but independent effects of GCs were investigated by using the ganglionic blocker chlorisondamine. Our results show that chlorisondamine was only able to reverse morphine’s inhibitory effects partially. These results suggest that these effects may be mediated by a direct effect of morphine on cells of immune system.


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MATERIALS AND METHODS
 
Animals
MORKO mice (Balb/cxC57BL/6 genetic background) were produced as described previously by Loh and coworkers [38 ]. Briefly, a XhoI/XbaI fragment, which spans the entire exons 2 and 3, was replaced with a Neor cassette followed by the ligation of a thymidine kinase expression cassette to the 3' end of this segment. WT mice (CB6F1/J, Balb/c femalexC57BL/6 male), 8 weeks of age, were obtained from The Jackson Laboratory (Bar Harbor, ME). Maximums of four mice were housed per cage. Food and tap water were available ad libitum. The animal room was maintained on a 12-h light/dark cycle with constant temperature (72±1[°F]) and 50% humidity.

Chemicals
Morphine pellets (75 mg) were generously provided by the National Institute on Drug Abuse. CORT was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in polyethylene glycol 400 (Sigma Chemical Co.). Alzet micro-osmotic pumps (Alza Corp., Palo Alto, CA) were filled with CORT or polyethylene glycol 400 vehicle. Chlorisondamine diiodide (Tocris, Ballwin, MO) was dissolved in 0.9% saline.

Animal treatment
Mice were anesthetized with methoxyflurane (Mallinckodt Veterinary, Mundelein, IL). Osmotic pumps and pellets were placed into pockets formed to the left of the dorsal midline. WT and MORKO mice were divided into four treatment groups: polyethylene glycol 400 (PEG 400) osmotic pump + placebo pellet (vehicle/placebo); CORT osmotic pump (10 mg/kg/day) + placebo pellet (CORT/placebo); PEG 400 osmotic pump + morphine (75 mg) pellet (vehicle/morphine); and CORT osmotic pump + morphine (75 mg) pellet (CORT/morphine). Animals were sacrificed 48 h after osmotic pump and pellet implantation.

To determine if the autonomic nervous system (ANS) is involved in these morphine-mediated inhibitory effects, initial experiments were performed in WT mice treated with chlorisondamine, an autonomic nervous system ganglion blocker. Mice received an intraperitoneal (i.p.) injection with saline or the autonomic ganglionic blocker, chlorisondamine diiodide (5 mg/kg), 1 h before implantation of morphine or placebo pellet. On day 2 of experiment, the same mice received another i.p. injection of chlorisondamine (5 mg/kg). Based on the studies by Abdel-Rahman [39 ], animals were treated with 5 mg/kg chlorisondamine to produce ganglionic blockade in mice. Chlorisondamine diiodide is an exceptionally long-lasting nicotinic antagonist; blockade of the central nicotinic responses induced by chlorisondamine diiodide can persist for several weeks [40 ].

CORT radioimmunoassay
Animals were sacrificed at 10:00 AM, and plasma samples were stored at -70°C until time of analysis. Plasma concentrations of CORT were determined using a [125-I]-coupled, double-antibody radioimmunoassay (ICN Biochemicals, Costa Mesa, CA). CORT concentrations were expressed as nanograms per milliliter.

Thymic-, splenic-, and lymph node-lymphocyte proliferation assays
Thymus, spleen, and peritoneal mesenteric lymph nodes were removed with sterile forceps and placed on a metal sieve (Sigma Chemical Co.) and were submerged in cold 10% newborn calf serum (NCS) RPMI 1640. The cell suspension was prepared by forcing the tissues through the sieve with a sterile plunger of syringe. The resulting cell suspension was washed in cold RPMI-1640 medium and adjusted to a concentration of 2 x 106 cells/ml for splenocytes and lymph node cells and 5 x 106 cells/ml for thymocytes. Triplicate samples were plated onto 96-well plates containing 5 µg/ml Con A (Sigma Chemical Co.) and incubated for 48 h (lymph node cells) or 72 h (splenocytes and thymocytes) at 37°C 5% CO2. Cells were pulsed with 0.2 µCi [methyl-3H]-thymidine (Amersham, Piscataway, NJ) in a 20 µl volume and incubated for 8 h. Samples were lysed with distilled water and harvested onto glass filters using an automatic 96-well cell harvester (Skayron Instrument, Norway). The amount of labeled DNA was determined with a 1900CA liquid scintillation counter (Packard, Downers Grove, IL). The results were expressed as the relative activity, where the activity of the vehicle/placebo group was taken as 100%. That of other groups was then calculated as percentage thereof.

NO determination
Macrophages were obtained by peritoneal lavage with ice-cold RPMI-1640 medium from WT- and MORKO-implanted mice. Peritoneal macrophages were washed twice and resuspended in 5% NCS RPMI 1640. Macrophages were purified by incubating them in 6-well plates overnight at 37°C 5% CO2 and then removing nonadherent cells by washing three times with Click’s medium (Sigma Chemical Co.). The adherent cells were cultured in triplicate in 96-well plates at 2 x 105 cells/well in 10% NCS RPMI 1640 with a final concentration of lipopolysaccharide (LPS; Sigma Chemical Co.) at 10 µg/ml. Plates were incubated for 24 h at 37°C 5% CO2 and were then centrifuged. A volume of 100 µl resultant supernatant was pipetted onto a 96-well plate, and an equal volume of modified Griess reagent (Sigma Chemical Co.) was added. Plates were read at wavelength 550 nm using a spectracount plate reader (Packard) after 15 min. The detection limit of the assay was approximately 0.43 µM. Using sodium nitrite (Sigma Chemical Co.) as a standard, a standard curve was obtained. Sample NO concentrations were expressed as mean of the triplicate µM concentrations of nitrite.

Enzyme-linked immunosorbent assay (ELISA) for IL-2, IFN-{gamma}, IL-1, and TNF-{alpha}
Splenocytes and peritoneal macrophages from each mouse were adjusted to a final concentration of 2 x 106 cells/ml in 24-well plates and stimulated with Con A (5 µg/ml) for splenocytes and LPS (10 µg/ml) for macrophage activation. The microtiter plates were then incubated for 24 h at 37°C in a humidified 5% CO2 incubator. Culture supernatant was analyzed using cytokine-specific ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

Reverse transcriptase-polymerase chain reaction (RT-PCR) for MOR-1 mRNA levels
To identify if the MOR is expressed on lymphocytes and macrophages, as well as on hypothalamus and pituitary cells, lymphocytes were purified from thymus, spleen, and lymph nodes with mouse CD3+ T-cell-enrichment columns (R&D Systems), according to the manufacturer’s instructions. Peritoneal macrophages were harvested and isolated as described above. Total RNA was extracted from these cells or tissues and reverse-transcribed to synthesize the first-strand cDNA (42°C, 30 min) using random hexamers (2.5 µM), Moloney murine leukemia virus RT (2.5 units), and 1 mM each dATP, dCTP, dGTP, and dTTP in a final reaction volume of 40 µl. Following first-strand synthesis, the reaction mixture was heated (95°C, 5 min) to inactivate the RT. Amplification was performed using upstream and downstream primers specific for mouse MOR-1 (Oligos Etc., Wilsonville, OR) and ß2-microglobulin (Clontech, Palo Alto, CA). The primer sequences are as follows: MOR-l, sense 5'-CATCAAAGCACTGATCACGATTCC-3', antisense 5'-TAGGGCAATGGAGCAGTTTCTGC-3'; ß2-microglobulin, sense 5'-ATGGCTCGCTCGGTGACCCTAG-3', antisense 5'-TCATGATGCTTGATCACATGTCTCG-3'. The mouse MOR-1 and ß2-microglobulin primers amplify 305 and 373 bp fragments, respectively. The first-strand cDNA reaction mixture was added to PCR buffer containing 2.5 units AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA), 2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, and 0.1 µM of each primer in a total volume of 50 µl. PCR conditions were 94°C for 45 s (denaturation), 60°C for 45 s (annealing), 72°C for 45 s (extension) at 30 cycles, followed by a final extension at 72°C for 15 min. PCR products were analyzed on a 1.5% agarose gel and visualized by ethidium-bromide staining.

Statistical analysis
Data were analyzed for statistically significant differences by two-way analysis of variance between animals. Individual group comparisons were made by the two-tailed Student’s t-test. Statistical significance was accepted at P < 0.05.


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RESULTS
 
Effect of continuous CORT infusion and morphine pellet implantation on plasma CORT levels
Initially, experiments were conducted to achieve plasma levels of CORT, following continuous CORT infusion through an Alzet osmotic pump that were equivalent to levels observed following morphine pellet implantation. Previous studies have shown that implantation of 75 mg morphine pellets results in plasma CORT levels in the 400–450 ng/ml range [21 ]. In the present experiment, we show that infusion of CORT at a rate of 10 mg/kg/day elevated plasma CORT levels to 420.7 ± 23.4 ng/ml in WT mice and 448.8 ± 36.0 ng/ml in MORKO mice after 48 h of continuous CORT infusion (Fig. 1A ). To prove that these two methods produce the same kinetic profile of CORT levels throughout the 48-h treatment period, plasma CORT levels were measured at various times points (4, 8, 20, 36, and 48 h) after CORT pumps or morphine pellet implantation in WT mice. The elevated levels in plasma CORT were not significantly different between morphine-pelleted animals and CORT pump-implanted mice, except at the 4-h time point. Results show that the two methods of drug delivery are comparable pharmacological treatment profiles (Fig. 1B) .



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  		Figure 1. (A) Effect of CORT infusion, 2 days continuous, and morphine-pellet implantation on plasma CORT concentration in WT and MORKO mice. (B) Effect of CORT infusion or morphine-pellet implantation on pharmacokinetic profiles of plasma CORT at various times point (4, 8, 20, 36, and 48 h). Data are expressed as mean ± SEM with six animals in each group. *, Significance at level P < 0.05; **, significance at level P < 0.01.

Implantation of morphine pellets (75 mg) elevated serum CORT level to 414.6 ± 29.2 ng/ml when measured at 48 h in WT mice. This increase in plasma CORT level was not observed in the MORKO mice following morphine-pellet implantation. In WT mice that received a morphine-pellet and CORT infusion, the plasma CORT levels increased to 607.7 ± 46.0 ng/ml. In MORKO animals, there was no significant increase in plasma CORT levels between animals that received CORT alone and animals that received CORT infusion and a morphine pellet. These results suggest that morphine-induced increases in plasma CORT levels are a function of the MOR. Furthermore, these results support the use of the MORKO mouse model to delineate the role of CORT in morphine-induced immunosuppression.

Expression of MOR-1 in macrophage and lymphocyte derived from thymus, spleen, lymph node, hypothalamus, and pituitary in WT mice
To identify if the MOR is expressed on lymphocytes, macrophages, and on hypothalamus- and pituitary-derived cells, lymphocytes were purified from thymus, spleen, and lymph nodes using mouse CD3+ T-cell-enrichment columns. Peritoneal macrophages were harvested and isolated as described in Materials and Methods. Total RNA isolated from these cells was subjected to RT-PCR analysis. These results show that the MOR-1 was expressed in lymphocytes derived from thymus, spleen, and lymph node, peritoneal macrophages, as well as in the hypothalamus and pituitary harvested from WT animals. MOR-1 was not expressed in any of these cells or tissues isolated from MORKO animals (Fig. 2 ).



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  		Figure 2. MOR-1 mRNA was expressed in lymphocytes derived from thymus, spleen, and lymph node, peritoneal macrophages, as well as in the hypothalamus and pituitary harvested from WT mice but not in any of these cells or tissues isolated from MORKO mice.

Role of morphine and exogenous CORT on immune organ cellularity
We conducted experiments to investigate the role of CORT in morphine-induced changes in thymic-, splenic-, and lymph node-lymphocyte and macrophage cellularity. After 48 h of continuous CORT infusion, there was a 19% reduction in thymic cellularity in the WT and a 13.2% decrease in the MORKO mice when compared with the vehicle/placebo-treated control. These decreases were significantly different from the vehicle/placebo-treated group (P<0.05). Treatment with morphine pellets, however, resulted in a 54% decrease in thymic cellularity in the WT animals compared with the vehicle/placebo-treated animals (P<0.01). In contrast, morphine treatment of the MORKO animals did not result in any significant decrease in thymic cellularity compared with the vehicle/placebo-treated animals. In WT animals that were treated with morphine and CORT, the decrease in thymic cellularity was similar to that of animals treated with morphine alone. In MORKO animals treated with CORT and morphine, thymic cellulary was similar to animals that were treated with CORT alone. Comparing the effects of morphine on splenic and lymph node cellularity between WT and MORKO animals, it was seen that morphine treatment resulted in a significant decrease (P<0.01) in splenic and lymph node cellularity. Morphine treatment did not result in any significant decrease in these parameters in the MORKO animals. CORT treatment of WT and MORKO, however, resulted in the same amount of decrease in splenic and lymph node cellularity when compared with placebo/vehicle-treated animals. It should be noted that the effect of morphine on WT animals was significantly different from the effect of CORT in MORKO animals (P<0.01), even though the CORT levels were similar in both groups. It was interesting to observe that although morphine treatment alone or CORT treatment alone in WT animals did not result in a significant decrease in the number of peritoneal macrophages, a combination of morphine and CORT resulted in a 27% decrease in peritoneal macrophage cell number. However, this effect was not observed in MORKO animals, suggesting the MOR plays a role in this function (Table 1 ).


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  		Table 1. Cellular Number of Thymus, Spleen, and Lymph Nodes in Each Group

Effect of morphine and CORT on thymic-, splenic-, and lymph node-lymphocyte proliferation
Next, we investigated the role of CORT in a morphine-induced decrease in thymic-, splenic-, and lymph node-lymphocyte proliferation. Our results show that although the plasma CORT concentrations in CORT infusion and morphine pellet implantation were similar, CORT infusion resulted in a significant decrease in only Con A-induced, thymic-lymphocyte proliferation (Fig. 3A ). In contrast, morphine treatment resulted in significant decreases in Con A-induced proliferation of T lymphocyte derived from all three immune organs. When Con A-induced, thymic-lymphocyte proliferation was compared between animals treated with morphine or CORT, it was seen that the effect was more pronounced in animals treated with morphine (P<0.05). The inhibitory effect of morphine was abolished completely in the MORKO mice. These results suggest that the chronic in vivo effects of morphine on thymocyte proliferation are mediated by the MOR and partially dependent on CORT. However, the decrease in splenocyte and lymph node-derived T-lymphocyte proliferation is clearly a function of the MOR and is CORT-independent (Fig. 3B and 3C) .



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  		Figure 3. Effect of morphine and exogenous CORT on Con A-induced proliferation of (A) thymic, (B) splenic, and (C) lymph node lymphocyte in WT and MORKO mice. The relative activity of the vehicle/placebo group was taken as 100%. That of other groups was then calculated as percentage thereof. Data are expressed as mean ± SEM with six animals per group. *, Significance at level P < 0.05; **, significance at level P < 0.01.

Effects of morphine and exogenous CORT on splenic-lymphocyte IL-2 and IFN-{gamma} synthesis
To determine if morphine-induced inhibition of T-lymphocyte cytokine synthesis is a function of increased CORT production, we compared the effect of morphine and exogenous CORT on splenic-lymphocyte IL-2 and IFN-{gamma} synthesis. Analysis of IL-2 and IFN-{gamma} levels in animals that were treated with CORT alone showed a significant inhibition of splenic-lymphocyte IL-2 production when compared with vehicle/placebo-treated animals. In the same treatment group (CORT alone), it was interesting to observe that there were no significant differences in IFN-{gamma} protein levels when compared with vehicle/placebo-treated animals. In the morphine-treated WT animals, however, production of IL-2 and IFN-{gamma} was decreased significantly. Therefore, our results show that morphine treatment resulted in a greater inhibition of IL-2 and IFN-{gamma} when compared with CORT-treated WT mice (Fig. 4A and 4B ). Morphine-induced decreases in IL-2 and IFN-{gamma} were not observed in the MORKO animals. Our studies provide novel in vivo evidence that the decrease in IFN-{gamma} following morphine treatment is MOR-dependent and not mediated by CORT.



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  		Figure 4. Effect of morphine and exogenous CORT on (A) IL-2 and (B) IFN-{gamma} synthesis of splenic lymphocytes in WT and MORKO mice. Data are expressed as mean ± SEM with six animals per group. *, Significance at level P < 0.05; **, P < 0.01.

Effect of morphine and exogenous CORT on macrophage NO production
Because NO has been demonstrated by several investigators to trigger lymphocyte and macrophage apoptosis, we tested the effect of morphine and exogenous CORT on macrophage NO production. Our results show that morphine and exogenous CORT treatment increased macrophage NO production in the WT animals. Morphine treatment in the WT animals resulted in a two- to threefold induction of macrophage NO when compared with the vehicle/placebo-treated group and a 1.5-fold induction over the CORT-treated group (Fig. 5 ). Similar to what was observed with WT, treatment of the MORKO group with CORT resulted in a 1.5-fold increase over vehicle/placebo control. Morphine treatment in the MORKO showed no significant increase in NO production compared with the vehicle/placebo-treated group. These results suggest that the decrease in T-cell proliferation may be a result of an apoptotic deletion of T cells, possibly mediated by an increase in NO synthesis.



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  		Figure 5. Concentration of NO2- in LPS-stimulated macrophage cultures from morphine-treated WT mice is significantly greater than that in cultures from CORT-treated WT mice. The data are expressed as mean ± SEM with six animals per group. *, Significance at level P < 0.05; **, P < 0.01.

Effect of morphine and exogenous CORT on macrophage TNF-{alpha} and IL-1ß production
To further evaluate the effect of morphine and exogenous CORT on macrophages, we measured the inflammatory cytokines, TNF-{alpha} and IL-1ß. As shown in Figure 6A and 6B , in WT animals, the morphine-treated group had significantly higher TNF-{alpha} (threefold) and IL-1ß (3.4-fold) levels when compared with the vehicle/placebo group. CORT treatment did not result in a significant increase in IL-1ß and TNF-{alpha} synthesis. The effects of CORT on the MORKO animals were similar to that observed in the WT animals. The effects of morphine, once again, were abolished completely in the MORKO animals.



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  		Figure 6. Effect of CORT infusion and morphine implanted on macrophage (A) TNF-{alpha} and (B) IL-1ß synthesis in WT and MORKO mice. Data are expressed as mean ± SEM with six animals per group. **, Significance at level P < 0.01.

Effect of chlorisondamine on morphine-induced suppression of splenocyte proliferation and IFN-{gamma} synthesis
Our results demonstrate that splenic-lymphocyte proliferation and splenic IFN-{gamma} synthesis were inhibited significantly after morphine treatment. These functions were not altered significantly following continuous CORT infusion, suggesting that the inhibitory action of morphine is not mediated through HPAA and is possibly CORT-independent. To determine if the ANS is involved in these morphine-mediated inhibitory effects, experiments were performed in WT mice treated with chlorisondamine (5 mg/kg), an autonomic nervous system ganglion blocker, 1 h prior to implantation with morphine or placebo pellets. This dose of chlorisonadamine (5 mg/kg) has been shown by several authors to produce a long-lasting, irreversible, and insurmountable blockade of the ganglionic nicotinic receptors [40 , 41 ]. At the time of sacrifice, autonomic functions, such as gastrointestinal transit and ptosis of the eyelids, were also monitored to ensure complete blockade (unpublished results).

Chronic morphine administration resulted in a 70% suppression of splenic-lymphocyte proliferation and a 57% inhibition of IFN-{gamma} synthesis. Chlorisondamine treatment alone (chlorisondamine/placebo group) had no significant effect on Con A-induced splenic-lymphocyte proliferation nor IFN-{gamma} synthesis, as compared with saline-treated controls (vehicle/placebo group). It was interesting to note that ganglionic blockade with chlorisondamine did not completely antagonize the inhibitory effect of morphine on splenic-lymphocytes proliferation and IFN-{gamma} synthesis (Figs. 7 and 8 ). These results suggest that the inhibitory effects of morphine on splenic-lymphocyte proliferation and IFN-{gamma} synthesis are not mediated totally by the activation of the ANS.



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  		Figure 7. Ganglionic blockades with chlorisondamine partially antagonized the morphine-induced inhibitory activity of splenic-lymphocyte proliferation Con A. Data are expressed as mean ± SEM with six animals per group. *, Significance at level P < 0.05; **, P < 0.01.



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  		Figure 8. Effect of chlorisondamine on morphine-induced suppression of IFN-{gamma} synthesis. Data are expressed as mean ± SEM with six animals per group. *, Significance at level P < 0.05; **, P < 0.01.


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DISCUSSION
 
In these studies, we tested the hypothesis that the immunosuppressive effects of chronic morphine treatment in vivo are a function of the MOR. We also investigate the role of the HPAA and the ANS in morphine-induced immunosuppression. Our previous studies show that treatment of MORKO mice with chronic morphine does not result in an increase in plasma CORT [21 ]. This animal model is a valuable tool for delineating the mechanisms by which morphine mediates its immunosuppressive effects. To investigate the role of CORT in morphine-mediated immunosuppression, WT and MORKO mice were treated with placebo, morphine (75 mg pellet) alone, continuous infusion of CORT (10 mg/kg/day) alone, or morphine + CORT. Our results show that morphine pellet implantation in WT mice resulted in a much greater decrease in thymic, splenic, and lymph node cellularity when compared with MORKO animals infused with the same plasma concentration of CORT. These results suggest that the decrease in immune organ cellularity may be a result of a direct effect of morphine on cells of the immune system and an indirect effect mediated through CORT. These results support the work by Bryant and colleagues [12 , 13 ], who demonstrated that immune suppression in morphine-pelleted mice is mediated partially by adrenal-dependent mechanisms. The conclusion made by these authors—that morphine-induced immunosuppression is at least in part mediated by the increase in serum CORT levels after implantation of the morphine pellet—is corroborated further by our studies.

In addition, several other studies including ours have shown that splenocytes, lymph node lymphocytes, and Jurkat T cells treated in vitro with morphine result in T-cell apoptosis and decreased T-cell proliferation [7 , 9 , 10 ]. However, it is also well established that CORT and DEX are potent inducers of apoptosis in thymocytes [42 ] and splenocytes [43 ] and have been shown to decrease splenic and thymic size. Because our results show that morphine treatment results in a greater decrease in thymic, lymph node, and splenic cellularity compared with CORT treatment alone, it is reasonable to conclude that the morphine-mediated decreases cannot be attributed to CORT alone. Rather, it is more likely that morphine and CORT play an additive role in decreasing thymic, splenic, and lymph node cellularity.

Although CORT treatment resulted in a significant decrease in thymic, splenic, and lymph node cellularity, CORT exposure alone altered thymic-lymphocyte proliferation and did not decrease splenic and lymph node-lymphocyte proliferation significantly. These results indicate that thymic-lymphocyte proliferation is more sensitive to the effects of CORT exposure than splenic- and lymph node-lymphocyte proliferation. It was interesting to observe that although morphine treatment inhibited IL-2 and IFN-{gamma} synthesis, continuous infusion of CORT at 10 mg/kg/day only inhibited IL-2 synthesis significantly. CORT had no significant effect on IFN-{gamma} synthesis. This observation is consistent with a number of in vivo studies, where it has been demonstrated that elevated GCs had no effect or a slightly stimulatory effect on IFN-{gamma} synthesis [44 45 46 ]. However, in vitro treatment of GCs has been shown to be inhibitory. These results strongly suggest and support our recent observation that morphine-mediated inhibition of IFN-{gamma} may be a direct effect of morphine acting on cells of the immune system [7 ]. This observation is very significant, because IFN-{gamma} plays a central role in host resistance to infection, notably to viral infections, and may explain why there is a higher incidence of HIV and other viral infections in the drug-abuse population [47 , 48 ].

Because macrophage-derived NO has been implicated in morphine-mediated apoptosis, we investigated the effect of morphine treatment on NO production. CORT and morphine caused an increase in NO production. We speculate that NO-mediated apoptosis may partly account for the decrease in thymic, splenic, and lymph node cellularity. A noteworthy observation was that although morphine and CORT increased NO production, only morphine treatment resulted in a significant augmentation in TNF-{alpha} and IL-1ß secretion. Because this induction was abolished completely in the MORKO mice, it can be concluded that the increase in TNF-{alpha} and IL-1ß observed following chronic morphine treatment is CORT-independent and mediated by the MOR. These studies are consistent with our previous studies and with Eisenstein and coworkers’ findings [49 ] that in vivo morphine treatment primes macrophages for enhanced production of proinflammatory cytokines.

It has been shown that acute morphine treatment-induced suppression of lymphocyte activity is HPAA-independent and can be reversed with the ganglionic blocker chlorisondamine [50 ]. These studies suggest that the inhibitory effect of acute morphine administration on lymphocyte activity may be mediated through activation of the ANS [51 , 52 ]. Little is known about the role of the ANS in alteration of immune-cell activity induced by chronic morphine administration. Because our results show that the chronic effect of morphine on splenocyte proliferation and Con A-induced IFN-{gamma} synthesis was CORT-independent, we investigated if these effects were mediated through the ANS. Our results show that chlorisondamine pretreatment only partially blocked the inhibitory effect of morphine on splenic-lymphocyte proliferation and IFN-{gamma} synthesis (Figs. 7 and 8) . These results suggest that the inhibitory effect of morphine on splenic-lymphocyte proliferation and IFN-{gamma} synthesis is not totally dependent on activation of the ANS. A direct effect of morphine on these cells is strongly implicated to observe complete inhibition of these functions by morphine. These results support several studies, including ours, that have shown that chronic in vitro treatment of morphine results in a significant decrease in T-cell proliferation and cytokine synthesis [7 8 9 10 11 ]. These results also explain why a higher dose of morphine is required to observe the inhibitory effects of morphine in vitro. At steady state, the morphine concentration in plasma observed with 75 mg morphine pellet is approximately 200 ng/ml.

In summary, our studies demonstrate that chronic morphine-induced immunosuppression is clearly a function of the MOR. These studies also indicate that indirect and direct mechanisms may be responsible for mediating morphine’s inhibitory role. Although the indirect mechanisms through activation of HPAA and ANS play a contributing, possibly additive role, the direct effects of morphine cannot be discounted and are strongly indicated.


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ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grants R01-DA 12104 (S. R.), P50-DA 11806-01 (S. R.), and the Department of Defense/Veterans Affairs (R. A. B.).

Received November 18, 2001; revised January 4, 2001; accepted January 14, 2002.


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