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(Journal of Leukocyte Biology. 2001;70:527-536.)
© 2001 by Society for Leukocyte Biology

Morphine modulates lymph node-derived T lymphocyte function: role of caspase-3, -8, and nitric oxide

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

* 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
 
The major objective of this paper is to characterize the mechanism by which morphine modulates lymphocyte function and if these effects are mediated through the µ-opioid receptor. We evaluated the in vitro effects of morphine on lymphocytes that were freshly isolated from lymph nodes from wild type (WT) and µ-opioid receptor knock-out (MORKO) mice. Results show that morphine inhibits Con A-induced lymph node T-cell proliferation and IL-2 and IFN-{gamma} synthesis in a dose-dependent manner. This effect was abolished in lymph node cells isolated from MORKO mice. The inhibition of T-cell function with low-dose morphine was associated with an increase in caspase-3- and caspase-8-mediated apoptosis. The inhibition of T-cell function with high-dose morphine was associated with an increase in the inducible NO synthase mRNA expression. NG-nitro-L-arginine methyl ester (L-NAME) antagonized the apoptosis induced by high-dose morphine. Our results suggest that low-dose morphine, through the µ-opioid receptor, can induce lymph node lymphocyte apoptosis through the cleavage activity of caspase-3 and caspase-8. Morphine at high doses induces NO release. This effect of morphine is also mediated through the µ-opioid receptor present on the surface of macrophages.

Key Words: µ-opioid receptor knock-out • wild type • apoptosis • induced nitric oxide synthase


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INTRODUCTION
 
Chronic morphine has been shown to alter many immune parameters. In addition to a decrease in thymic and splenic weight, these include the lymphocyte proliferative response to mitogen [1 ], T-cell rosette formation [2 ], and the total number of circulating lymphocytes [3 ]. In animal models, morphine treatment has been found to increase mortality rates in experimentally infected mice [4 5 6 ]. Furthermore, lymphocyte-proliferative responses [7 ], natural killer (NK) cell cytotoxicity activity [8 ] antibody and serum hemolysin formation [9 ], macrophage phagocytosis [10 ], and interleukin-2 (IL-2) synthesis [11 ] are all attenuated after in vivo chronic morphine exposure to animals. Although the immunomodulatory effects of morphine are well-accepted, the mechanisms responsible for morphine-induced changes in the immune system still remain controversial. Evidence exists that morphine may be mediating its effect directly through opiate receptors present on lymphocytes [2 , 12 13 14 15 16 17 ] or indirectly through opiate receptors present in the central nervous system (CNS) [18 19 20 21 ]. Several laboratories have sought to detect µ-opioid receptors (MOR) in rat peritoneal macrophages [22 ], activated lymphocytes [23 ], monocyte/macrophages [24 ], and granulocytes [25 ]. More recently, it has been shown that morphine treatment results in the up-regulation of MOR in human and monkey lymphocytes [26 ]. These observations suggest that immune cells may respond directly to opioids via cell-surface receptors.

The present experiment was designed to assess the direct effect of morphine on lymph node lymphocytes from wild type (WT) and µ-opioid receptor gene knock-out (MORKO) mice. Lymph nodes are the sites where T-cell responses to lymph-borne antigens are initiated. The anatomic organization of lymph nodes provides multiple sites for interaction between accessory cells and lymphocytes. Most antigens enter the body through the skin and the mucosal epithelia of the gastrointestinal and respiratory tracts. From these sites, samples of the antigens are initially transported to and concentrated in regional lymphoid organs. Therefore, the functional response of lymph node immune cells is essential for the initial effector phase of an immune response [27 ]. However, there are very few studies aimed at investigating the effect of chronic morphine treatment on immune cells derived from lymph nodes.

The purpose of the present study was to provide an extensive assessment of immunomodulatory effects of morphine on lymph node lymphocytes. The effect of morphine on T-cell proliferation and apoptosis was investigated. The role of inducible nitric oxide synthase (iNOS) and caspase-3 and caspase-8 in morphine-mediated effects was evaluated. The results strongly suggest that lymph node macrophage-derived NO serves as a molecular mediator of the immunosuppressive effect only with high-dose morphine. Apoptosis observed with low-dose morphine may be mediated through a NO-independent pathway involving caspase-3 and caspase-8.


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MATERIALS AND METHODS
 
Experiment animal
MORKO mice (Balb/cxC57BL/6 genetic background) were produced as previously described by Loh and co-workers [28 ]. 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 Jackson Laboratory (Bar Harbor, ME). A maximum 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, constant temperature (72±1[°F]), and 50% humidity.

Preparation of lymph node cells and treatment with variable concentrations of morphine
Mesenteric lymph nodes were removed with sterile forceps, and a single-cell suspension was prepared. The cell suspension was washed twice in cold RPMI 1640 medium, adjusted to a concentration of 2 x 106 cells/ml, and used for all experiments described in this study. Cells were treated for 2 h in media [RPMI+10% newborn calf serum (NCS)] containing vehicle (control) or variable concentrations of morphine (1 nM–10 µM; National Institute on Drug Abuse, Rockville, MD). Cells were stimulated with concanavalin A (Con A; 5 µg/ml) for varying periods of time, depending on the experiment. Three sets of experiments were performed, each in triplicate.

Depletion of adherent cells using the plastic-adherence procedure
To deplete adherent cells from lymph node cultures, 5 ml of a 106 cells/ml lymph node cell suspension was plated in the six-well cultural plates in 5% NCS RPMI 1640 medium. Nonadherent lymph node cells were removed by washing the plate 4 h after plating. The lymphocytes depleted of macrophages were adjusted with 10% NCS RPMI 1640 to a density of 5 x 106 cells/ml and used for the study of apoptosis. The purity of the lymphocytes was determined by flow cytometry and found to be devoid of macrophages.

Lymph node T-cell proliferation assay
Following treatment with vehicle (control), variable concentrations of morphine or µ-receptor antagonists lymph node cells (4x105 cells) were incubated in complete medium in 96-well plates (Costar, Cambridge, MA) in the presence of Con A (5 µg/ml). Lymphocyte proliferation was measured after 48 h. [Methyl-3H]-thymidine (1 µCi; Amersham, Pistacaway. NJ) was added 8 h before the termination of the cultures. Samples were harvested onto glass filters using an automatic 96-well cell harvester (Skatron Instrument, Norway). The amount of labeled DNA was determined using a liquid scintillation counter (model 1900CA, Packard Instrument Co., Downers Grove, IL).

Enzyme-linked immunosorbent assay (ELISA) for IL-2 and interferon-{gamma} (IFN-{gamma}) measurement
Mesenteric lymph node cells from MORKO and WT mice were adjusted to a final concentration of 2 x 106 cells/ml in 24-well plates. Cells were treated with vehicle (control) or different concentrations of morphine for 2 h and then incubated in the presence of Con A (5 µg/ml) for 24 h at 37°C in a humidified 5% CO2 incubator. Culture supernatants were assayed for cytokine secretion using a cytokine-specific sandwich ELISA Kit (R&D System, Minneapolis MN) according to the manufacturer’s instruction.

Reverse transcriptase-polymerase chain reaction (RT-PCR) for iNOS and MOR-1 mRNA levels
Total RNA was extracted from cultures of lymph node cells. Total RNA (1 µg) was 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 up-stream and down-stream primers specific for mouse MOR-1 (Oligos Etc., Wilsonville, OR), iNOS, and ß2-microglobulin (Clontech, Palo Alto, CA). The primer sequences are as follows: MOR-l, sense 5'-CATCAAAGCACTGATCACGATTCC-3', anti-sense 5'-TAGGGCAATGGAGCAGTTTCTGC-3'; iNOS, sense 5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3', anti-sense 5'-GGCTGTCAGAGCCTCGTGGCTTTGG-3'; ß2-microglobulin, sense 5'-ATGGCTCGCTCGGTGACCCTAG-3', antisense 5'-TCATGATGCTTGATCACATGTCTCG-3'. The mouse MOR-1, iNOS, and ß2-microglobulin primers amplify 305, 496, and 373 bp fragments, respectively. The first-strand cDNA reaction mixture was added to PCR buffer containing 2.5 units AmpliTaq DNA polymerase (Perkin-Elmer, Foster City, CA), 2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, and 0.1 µM each primer in a total volume of 50 µl. PCR conditions were 94°C for 45 sec (denaturation), 60°C for 45 sec (annealing), 72°C for 45 sec (extension) at 30 cycles, followed by a final extension at 72°C for 15 min. PCR products were analyzed on 1.5% agarose gel and visualized by ethidium bromide staining.

Determinations of apoptosis
Apoptotic nuclear DNA fragment was determined using terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein isothiocyanate (FITC)-deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) ApoAlert® DNA Fragmentation Assay Kit (Clontech) according to the manufacturer’s instruction. Briefly, 5 x 105/sample was fixed in 1% formaldehyde/phosphate-buffered saline (PBS) for 20 min at 4°C. Cells were then permeablized with 70% ice-cold ethanol at least for 4 h at -20°C. Each sample was then washed twice with PBS and incubated for 1 h at 37°C with terminal deoxynucleotidyl transferase enzyme and FITC-dUTP in a reaction buffer. Cells were washed with PBS, resuspended in 0.5 ml propidium iodide (PI)/RNase/PBS, and then incubated for 30 min at room temperature. The frequency of apoptotic cells was detected by fragment nuclear DNA and measured by flow cytometry, and the data were analyzed using CellQuest.

Apoptosis was also determined using gel electrophoresis. Lymph node cells were pretreated with vehicle (control) or variable concentrations of morphine (1 nM–10 µM) for 2 h and then incubated in the presence of Con A (5 µg/ml) for 24 h. At the end of the incubation period, lymph node cells were lysed in DNA lysis buffer. The extracted DNA was run on 1.8% agarose gel.

Apoptosis was also determined using the Hoechst (H)-33342 and PI-staining method. Hoechst (H)-33342 (Sigma Chemical Co., St. Louis, MO) stains the nuclei of live cells and identifies apoptotic cells by increased fluorescence, whereas PI (Sigma Chemical Co.) costains the necrosed cells. Double-staining enables us to obtain the percentage of live, apoptotic, and necrosed cells.

Caspase-3 and -8 activity colorimetric assay
Caspase activity was evaluated by measuring proteolytic cleavage of chromogenic substrate using the colorimetric assay kit (R&D Systems) according to the manufacturer’s instruction. The cleavage of synthetic caspase-3 substrate DEVD-pNA and caspase-8 substrate IETD-pNA was detected spectrophotometrically by the formation of pNA. Briefly, 1 x 106 cells were washed twice with PBS and resuspended in 60 µl lysis buffer (100 mM HEPES, pH 7.4, 140 mM NaCl, 1% protease inhibitor cocktail). Cells underwent three cycles of freeze/thawing. Cellular debris was removed by centrifugation at 13,000 g at 4°C for 10 min. Supernatant (50 µl) was incubated with 200 µM DEVD-pNA or IETD-pNA and 5 mM dithiothreitol (DTT) in 50 µl assay buffer [100 mM HEPES, pH 7.4, 2% glycerol, 0.5 mM EDTA, 1% bovine serum albumin (BSA)] in 96-well plates at 37°C for 2 h. The absorbance of enzymatically released pNA was measured at 405 nm in a microplate reader (Packard).

Western blot for caspase-3 and –8
Cells were lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, and 1 mM sodium vanadate, supplemented with protease inhibitor cocktail) for 15 min on ice. Lysates were centrifuged at 10,000 rpm at 4°C for 5 min, and protein concentration was determined by Bradford assay using BSA as the standard. Total protein (5 µg) was loaded on 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto nitrocellulose membrane. Nonspecific binding was blocked by incubating the membrane in 1 x Tris-buffer saline-Tween 20 (TBST) containing 5% enhanced chemiluminescence (ECL)-blocking agent (Amersham) for 1 h. Membranes were incubated overnight at 4°C in primary antibody [anti-caspase-3 polyclonal antibody (H-227); anti-caspase-8 polyclonal antibody (H-134); Santa Cruz Biotechnology, Santa Cruz, CA]. The blots were washed three times with TBST buffer and then incubated for 1 h at room temperature with anti-rabbit secondary antibody conjugated with horseradish peroxidase. Western blot analysis was conducted according to standard procedures using ECL detection (Amersham).

Statistical analysis
For comparison of mean values between two groups, the unpaired t-test was used. To compare values among multiple groups, analysis of variance (ANOVA) was applied. All values are mean ± SE except where otherwise indicated. Statistical significance was accepted at P < 0.05.


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RESULTS
 
Morphine inhibits Con A-induced lymph node T-cell proliferation and IL-2 and IFN-{gamma} synthesis through a MOR
The effect of morphine on lymph node function was evaluated at a pharmacological concentration (1 nM–1 µM). However, because the plasma concentration of morphine may be much higher in drug addicts, morphine effects were also evaluated at a higher morphine concentration (10 µM). Our result show that morphine, in concentrations of 10 nM–10 µM, inhibits Con A-induced T-cell proliferation in a dose-dependent manner in the WT animals. In contrast, this inhibition of low-dose morphine treatment on T-cell proliferation was completely abolished in cells isolated from the MORKO animals (Fig. 1 ). At doses greater than 10 µM, however, a 20% decrease in T-cell proliferation was also observed in the MORKO animals (unpublished results). Morphine treatment of lymph node cells from WT animals resulted in a dose-dependent decrease in IL-2 and IFN-{gamma} synthesis. In contrast, morphine treatment of lymph node cells from MORKO animals resulted in no significant inhibition (Fig. 2A and B ).



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  		Figure 1. Morphine inhibits lymph node T-cell proliferation to Con A via MOR-1. Morphine in concentrations of 10 nM–10 µM inhibited lymph node cell proliferation to Con A in WT mice but not in MORKO mice. *, P < 0.05; **, P < 0.001 compared with vehicle control.



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  		Figure 2. Morphine inhibits IL-2 and IFN-{gamma} synthesis in a dose-dependent manner in WT mice. Morphine inhibited IL-2 synthesis at doses of 10 nM–10 µM (A) and IFN-{gamma} synthesis from doses of 100 nM–10 µM (B) in WT mice. *, P < 0.05; **, P < 0.001 compared with vehicle control.

We have previously shown the existence of a naloxone-insensitive morphine-binding site on T cells and macrophages [35 ]. To eliminate the possibility that morphine might exhibit its effects observed in this study by acting through these receptors, we investigated the effect of naloxone on morphine-induced inhibition in lymph node cells isolated from WT mice. Cell proliferation was assayed using lymph node cells in 96-well plates, pretreated with naloxone (10 nM–100 µM) 45 min before the addition of morphine (1 nM–10 µM). Naloxone attenuated morphine-mediated inhibition of lymph node T-cell proliferation to Con A (Fig. 3 ). Naloxone also attenuated the effect of morphine on IL-2 and IFN-{gamma} synthesis (unpublished results). Naloxone alone, at doses up to 10 µM, had no effects on the functions tested. However, naloxone treatment at 100 µM resulted in a decrease in T-cell proliferation and cytokine synthesis. These results suggest that morphine-mediated inhibition of Con A induced lymph node proliferation and IL-2, and IFN-{gamma} synthesis is mediated via the classical MOR.



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  		Figure 3. Naloxone attenuates morphine-mediated inhibition of lymph node T-cell proliferation to Con A in WT mice. Cell proliferation was assayed using 2 x 106 cells/ml of lymph node cells from WT mice in 96-microwell plates containing naloxone (10 nM–100 µM) 45 min before adding morphine (1 nM–10 µM). Data are presented as mean ± SE of triplicate determination from three sets of experiments. *, P < 0.05; **, P < 0.001 compared with vehicle control.

Determination of MOR-1 mRNA in lymphocytes and macrophages from lymph node using RT-PCR
To identify if MOR-1 are expressed on lymph node-derived lymphocytes and macrophages, lymphocytes and macrophages were purified from lymph nodes as described in Materials and Methods. RT-PCR analysis showed that the MOR-1 was expressed in lymph node-derived lymphocytes and macrophages harvested from WT animals. As expected, MOR-1 mRNA was not expressed in lymphocytes and macrophages isolated from MORKO animals (Fig. 4 ).



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  		Figure 4. RT-PCR of MOR mRNA in lymphocytes and macrophages. Lane 1, lymphocytes from WT mice; 2, lymphocytes from WT mice, DNase-1-treated; 3, macrophages from WT mice; 4, macrophages from WT mice, DNase-1-treated; 5, lymphocytes from MORKO mice; 6, macrophages from MORKO mice.

Morphine-enhanced lymph node cell apoptosis
To investigate if the decrease in T-cell proliferation was a result of clonal deletion of T cell by apoptosis, DNA fragmentation of lymph node cells following morphine treatment was carried out using the TUNEL assay. Our results show that there was a significant increase in the number of apoptotic cells in lymph node cells harvested from WT mice (Fig. 5 ). This effect was dose-dependent. To determine if this effect was mediated through the MOR-1, similar studies were done with MORKO mice. Our results show that morphine-mediated apoptosis was completely obliterated in the MORKO mice, suggesting that the morphine’s effect shown in the WT mice must be mediated by the MOR-1. To further verify that the effects of morphine were mediated by the MOR-1, cells were pretreated with the opioid antagonist naloxone (10 µM) or the MOR antagonist CTOP (10 µM; Fig. 6 ). Our results show that naloxone and CTOP completely antagonized the effect of morphine (1 µM)-induced lymph node cell apoptosis. These results provide additional evidence supporting the role of MOR in morphine-mediated effects.



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  		Figure 5. Morphine promotes lymph node cell apoptosis. The DNA fragmentation of lymph node cells by morphine treatment was compared using TUNEL assay. A significantly increased proportion of TUNEL+ cell was observed in lymph node cells from WT mice following morphine treated for 2 h and incubated in the presence of Con A (5 µg/ml) for 24 h. Lymphocytes from WT animals (•); lymphocytes harvested from MORKO animals ({circ}). *, P < 0.05; **, P < 0.001 compared with vehicle control.



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  		Figure 6. Morphine-induced lymphocyte apoptosis is antagonized by naloxone and CTOP. The DNA fragmentation of lymph node cells was measured using TUNEL assay. Cells were pretreated with naloxone (10 µM) or CTOP (10 µM) 1 h before treatment with morphine (1 µM). Solid bar represents lymphocytes from WT animals, and open bar represents lymphocytes harvested from MORKO animals. **, P < 0.001 compared with vehicle control.

The effect of morphine on lymph node cell apoptosis was also investigated using the classical DNA fragmentation method (Fig. 7A ) and morphological techniques using H-33342 and PI staining (Fig. 7B 7C 7D) . Results show that vehicle-treated lymph node cells had the typical morphology of live, healthy cells (Fig. 7B) . Morphine treatment at a low concentration (10 nM) resulted in significant apoptosis (bright fluorescence as well as fragmentation of a few cells; Fig. 7C ). Morphine treatment at a high concentration (1 µM) triggered apoptosis and some secondary necrosis in a significant number of cells (Fig. 7D) . The necrotic effect of high-dose morphine was also observed in MORKO animals (unpublished results).



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  		Figure 7. (A) Morphine treatment increases DNA ladder in lymphocytes. Gel electrophoresis of DNA isolated from lymph node cells treated with vehicle (control) or variable concentrations of morphine for 2 h and then incubated in the presence of Con A (5 µg/ml) for 24 h. M, 123-bp Marker; 1–6, lymph node cells from WT mice; 7–12, lymph node cells from MORKO mice. 1 and 7, Vehicle control; 2 and 8, 1 nM morphine; 3 and 9, 10 nM morphine; 4 and 10, 100 nM morphine; 5 and 11, 1 µM morphine; 6 and 12, 10 µM morphine. (B–D) Morphological evaluation of lymph node cell apoptosis induced by morphine. An equal number of lymph node cells were pretreated in media containing vehicle or morphine (1 nM–10 µM) for 2 h and then incubated in the presence Con A (5 µg/ml) for 24 h. At the end of the incubation period, cells were stained with Hoechst-33342 and PI and examined under fluorescent microscopy. (B) Vehicle control. (C) Morphine treatment at 10 nM. (D) Morphine treatment at 1 µM. Original magnification, x400.

Role of caspase-3 and -8 in morphine-induced lymph node lymphocyte apoptosis
To investigate if caspases play a role in lymph node lymphocyte apoptosis, the cleavage activity of caspase-3 and caspase-8 was assayed using the synthetic peptide substrate DEVD-pNA or IETD-pNA. The cleaved product was measured spectrophotometrically. An increase in caspase-3 and caspase-8 activity was seen following morphine treatment in the WT mice (Fig. 8 ). To further verify the role of caspase-3 and -8 in morphine-mediated effects, caspase-3 and -8 cleavage products were examined using Western blot analysis. The cleaved forms of caspase-8 (P31 and P20) were clearly observed in morphine-treated lymph node cells. The cleaved forms of caspase-3 (P17 and P11) were also seen to be augmented following morphine treatment in lymph node cells harvested from WT mice (Fig. 9 ). Morphine treatment increased the cleavage activity of caspase-3 and -8 in a dose-dependent manner.



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  		Figure 8. Morphine induces caspase-3 and -8 activity in lymph node cells via MOR-1. Caspase-3 activity is represented by closed circles (WT) and closed squares (MORKO). Caspase-8 is represented as open triangles (WT) and open diamonds (MORKO). Cells were treated with the concentrations of morphine indicated. *, P < 0.05; **, P < 0.001 compared with vehicle control.



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  		Figure 9. Morphine increases cleavage activity of caspase-3 and -8. Lymph node cells from WT mice were treated with different doses of morphine for 2 h. Following incubation, the proteins were extracted, and caspase-3 and -8 cleavage was examined using Western blot analysis. (A) Activation products of caspase-8 (P31 and P20) were clearly observed in morphine-treated lymph node cells. (B) Cleaved forms of caspase-3 (P17 and P11) were seen following morphine treatment in lymph node cells from WT mice. To verify equality of loading, the blots were reprobed with anti-tubulin antibody (H-300, Santa Cruz Biotechnology).

Role of iNOS and NO in morphine-induced lymph node lymphocyte apoptosis
Morphine treatment increases iNOS mRNA expression
To investigate if morphine-induced lymph node lymphocyte apoptosis was mediated by an increase in iNOS production, lymph node cells (2x106/well) were treated with vehicle (control) or different doses of morphine and then stimulated with Con A (5 µg/ml) for 24 h. iNOS mRNA accumulation was measured using RT-PCR with primers specific for iNOS. ß2-Microglobulin was used as the housekeeping gene and to control for equality of loading. Results were quantified using Image-pro plus software and presented as a ratio of iNOS/ß2-microglobulin. Our results show that iNOS mRNA expression was significantly elevated in WT lymph node cells pretreated with high-dose morphine (100 nM–10 µM; Fig. 10 ). However, when lymph node cells were depleted of adherent cells and then treated with morphine, no significant increase in iNOS production was observed by RT-PCR (unpublished results). This result suggests that the elevation of iNOS mRNA following morphine treatment may be macrophage-derived. In the MORKO animals, morphine treatment of lymph node cells at doses 100 nm–10 µM did not result in any significant increase in iNOS mRNA. However morphine treatment of lymph node cells with doses greater than 10 µM showed significant increases in iNOS activity in the MORKO animals.



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  		Figure 10. Morphine induces iNOS mRNA expression in lymph node cells. M, Marker; 1–6, lymph node cells from WT mice; 7–12, lymph node cells from MORKO mice. 1 and 7, Vehicle control; 2 and 8, 1 nM; 3 and 9, 10 nM; 4 and 10, 100 nM; 5 and 11, 1 µM; 6 and 12, 10 µM morphine. **, P < 0.001 compared with vehicle control.

Apoptosis induced by high-dose morphine is antagonized by the iNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME)
To further substantiate that NO is involved in the suppressive effects of high-dose morphine, we examined the effect of L-NAME (1 mM) on morphine-induced apoptosis (Fig. 11 ) in total (unfractionated) and adherent, cell-depleted (fractionated) lymph node cultures using flow cytometry. Vehicle treatment of fractionated (A), unfractionated (D), or L-NAME-treated, unfractionated (L-NAME control) cells (G) did not result in any significant apoptosis. Treatment of fractionated cells with 1 nM morphine (B) resulted in a similar level of apoptosis as seen with treatment of unfractionated cells with 1 nM morphine (E). However, treatment of unfractionated cells with 100 nM morphine resulted in a greater increase in the percentage of apoptotic cells (F) when compared with treatment with fractionated cells with the same concentration of morphine (C and bar graph). It was interesting to observe that L-NAME significantly decreased the percentage of apoptotic cells seen with high-dose morphine (100 nM) treatment (I and bar graph). In contrast, L-NAME pretreatment did not result in any significant decrease in the percentage of apoptotic cells (H and bar graph) with low-dose morphine (1 nM) treatment. These results are quantitatively illustrated in the bar graph (Fig. 11) . Morphine treatment results in a dose-dependent increase in apoptotic cells in fractionated and unfractionated cultures. Treatment with high-dose morphine (100 nM and 1 µM) results in a greater number of apoptotic cells in the unfractionated cultures when compared with the fractionated cultures. When cells are pretreated with L-NAME, the increase in the percentage of apoptotic cells following high-dose morphine treatment in unfractionated cultures is reversed to the same levels as fractionated cultures. L-NAME pretreatment does not reverse the percentage of apoptotic cells following high- or low-dose morphine treatment in fractionated cultures (unpublished results).



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  		Figure 11. L-NAME antagonizes morphine-induced apoptosis of lymph node cells. Pretreatment of lymph node cultures with L-NAME (10 mM), an iNOS inhibitor, partly antagonized apoptosis induced by high-dose morphine in WT mice. (A) Lymph node cells depleted of macrophage, vehicle control; (B) lymph node cells depleted of macrophage, pretreatment with 1 nM morphine; (C) lymph node cells depleted of macrophage, pretreatment with 100 nM morphine; (D) unfractionated lymph node cells, vehicle control; (E) unfractionated lymph node cells, pretreatment with 1 nM morphine; (F) unfractionated lymph node cells, pretreatment with 100 nM morphine; (G) unfractionated lymph node cells, addition of L-NAME (10 mM) to media and vehicle control; (H) unfractionated lymph node cells, addition of L-NAME (10 mM) to media and pretreatment with 1 nM morphine; (I) unfractionated lymph node cells, addition of L-NAME (10 mM) to media and pretreatment with 100 nM morphine.

From these results, it can be concluded that morphine-induced apoptosis seen with high-dose morphine may be partially mediated by NO. However, the apoptosis observed with low-dose morphine is NO-independent. Because treatment of fractionated cells with morphine does not result in an increase in NO and also because L-NAME decreases the percentage of apoptotic cells only in unfractionated cells, it can be further concluded that NO is produced by adherent cells following morphine treatment.


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DISCUSSION
 
Chronic morphine use has been associated with an increased incidence of many diseases [4 , 30 ], and animal studies have demonstrated that morphine can alter many immune parameters [12 , 31 32 33 ]. Although there is overwhelming evidence indicating that chronic morphine use or abuse results in immunosuppression, there are still some contradictory studies as to the mechanism by which morphine mediates its effects. Although the specific mechanisms responsible for morphine-induced changes in the immune system are undefined, the initial step is thought to be mediated through MOR. Several binding studies suggest that immune cells contain MOR [3 , 29 , 34 , 35 ]. However, the existence of morphine binding sites that are different from the classical neuronal MOR has also been shown [36 37 38 39 ]. In addition, Makman et al. [40 ] and Stefano and Scharrer [41 ] demonstrate the presence of yet another morphine receptor, the MOR-3, which is opiate alkaloid-sensitive and opioid peptide-insensitive. Several groups using RT-PCR technique have shown the existence of the classical (neuronal) MOR-1 in rat peritoneal macrophage and in several human T- and B-cell lines in addition to human CD4+ T cells, human monocytes/macrophages, human polymorphonuclear neutrophil (PMN), monkey peripheral blood mononuclear cells (PBMC), and monkey PMN [22 , 42 ]. In this paper, we have used MORKO mice to address the question of whether morphine’s immunosuppressive effect is mediated by the classical MOR. In such animals, morphine’s effect on immune cells can no longer be mediated by MOR. Therefore, it is possible to evaluate the role of other classical (naloxone-sensitive) opioid receptors ({delta} and {kappa}) as well as nonclassical (naloxone-insensitive) receptors in the immunosuppressive effects of morphine. The purpose of the present study was to investigate the role and mechanism by which the MOR modulate lymph node lymphocyte function. Our results show that morphine inhibits Con A-induced proliferation and IL-2 and IFN-{gamma} synthesis of lymph node cells in WT animals. In contrast, these effects of morphine were completely abolished in lymph node cells isolated from MORKO mice. These results provide novel data showing that the MOR present on lymph node lymphocytes is directly involved in morphine’s modulatory effect.

It has been shown previously that morphine promoted apoptosis of Jurkat T cells and freshly isolated human T lymphocyte [15 ]. To investigate if the decrease in T-cell proliferation following morphine treatment in lymph node-derived T cells was also a result of clonal deletion of T cells by apoptosis, DNA fragmentation of lymph node cells following morphine treatment was carried out using the TUNEL assay. Our results confirm the observation of Singhal et al. [15 ] that morphine treatment of lymph node cells harvested from WT mice results in a significant increase in the number of apoptotic cells. Recently, it has been shown that morphine at high dose (3 µM) induces the expression of the Fas protein, a receptor on the cell surface that triggers the cell’s suicide by apoptosis. This induction of Fas expression by morphine appears to prime lymphocytes for elimination by apoptosis [43 ]. It has also been shown that Jurkat T cells can be rescued from Fas-induced apoptosis through the inhibition of caspases [44 ]. The caspases are known to play a pivotal role in triggering and executing apoptosis in virtually all cell types [45 ]. Caspase-8 exists as an inactive 56-kDa precursor [46 ]. Caspase-8/FADD-like IL-1 ß converting enzyme is a third component of the DISC (death-inducing signaling complex) and is the first caspase in the cascade of ICE (IL-1 ß-converting enzyme)-like protease activated by CD95. Upon binding to FADD (Fas-associated death domain), through the DED (death-effector domain), caspase-8 is cleaved to its active subunits, which are released to the cytosol and activate a cascade of ICE-like protease [47 ]. Caspase-3 is usually thought of as the down-stream effector protease most important for the classic nuclear changes associated with apoptosis [48 , 49 ]. Caspase-3 has been implicated in sepsis-induced thymocyte apoptosis [50 ] and in Jurkat T-lymphocyte apoptosis induced by agonistic anti-Fas [15 ]. To determine whether morphine-induced lymph node lymphocyte apoptosis is mediated through activation of the caspase cascade, we evaluated the effect of morphine on the cleavage activity of caspase-3 and -8. Our results from spectrophotometric determinations and Western blot studies show that caspase-3 and caspase-8 activities were significantly augmented following morphine treatment. In this study, we provide in vitro evidence for the first time that cleavage activity of caspase-3 and caspase-8 is an essential step for only low-dose morphine-induced lymph node T-cell apoptosis.

Several studies have shown that systemic administration of morphine results in alterations of splenic macrophage NO production [51 52 53 ]. These studies implicate the MOR within the CNS in the regulation of splenic NO production and decrease in Con A-stimulated splenic lymphocyte proliferation. Recently, several investigators have shown that morphine can directly affect macrophage and endothelial cell function by promoting NO formation under basal and activated states [54 55 56 57 58 ]. Our results confirm these studies and also show that high-dose morphine treatment in vitro increases NO release. In addition, our results also show that iNOS mRNA expression was significantly elevated in WT lymph node cells treated with high-dose morphine (100 nM–10 µM). These effects of morphine were abolished in the MORKO mice. It has been demonstrated by Stefano et al. [55 56 57 58 ] that morphine induced NO release in PBMCs, and endothelial cells may be a result of an induction in cNOS. These authors further demonstrate that this increase in cNOS has the potential to down-regulate iNOS. These effects were shown to be mediated by the µ-3 opioid receptor [55 56 57 58 ]. Although we speculate that the increase in NO seen with morphine treatment is iNOS-derived, it is quite conceivable that morphine treatment may also increase cNOS expression as demonstrated by Stefano and co-workers [55 56 57 58 ]. Morphine-induced iNOS expression and NO release seen with high-dose morphine in WT mice were abolished in the MORKO, suggesting the role of the classical MOR-1 in these effects. Several studies have implicated NO as a mediator of T-cell function and an important pathogenic factor in a wide range of immunologically mediated diseases [59 , 60 ]. Normal and malignant T cells cease to proliferate and undergo apoptosis when cultured in the presence of NO donor compounds [61 ]. Furthermore, it has been shown that T cells, activated in the presence of alveolar macrophages, are unable to proliferate, and the process is reproduced by NO generators S-nitroso-N-acetyl penicillamine. These effects are inhibited by the NO synthase inhibitor N-methyl L-arginine. More recently, it has been shown that peroxynitrite, through nitration of tyrosine residue, is able to inhibit activation-induced protein tyrosine phosphorylation in purified lymphocytes and prime them to undergo apoptotic cell death after phytohemagglutinin (PHA)- or CD3-mediated activation. These authors show that peroxynitrite is produced during activation mainly by cells of the monocyte/macrophage lineage [59 ]. To determine the role of macrophage-derived NO in morphine-induced T-lymphocyte apoptosis, we examined the effect of L-NAME, an inhibitor of inducible NO synthase, on morphine-induced apoptosis. Lymph node cells in cultures were unfractionated or depleted of adherent cells (macrophages and dendritic cells) by the plastic-adherence procedure. Our results show that addition of L-NAME to unfractionated lymph node cells partially reversed high-dose morphine-induced apoptosis. L-NAME had no effect on morphine-induced apoptosis of fractionated (adherent cell depleted) lymphocytes. Similarly, depletion of adherent cells resulted in only a partial reversal of high-dose morphine-induced apoptosis of lymph node lymphocytes. In addition, our results also show that morphine treatment of lymph node cells depleted of adherent-cell population did not result in an increase in iNOS synthesis. From these results, it can be concluded that morphine-induced lymph node apoptosis is mediated by two different pathways. The first pathway involves caspase-3 and -8 and is mediated directly by morphine acting on MOR present on lymphocytes. The second pathway involves NO and is mediated indirectly by morphine acting on MOR present on macrophages and adherent cells.

The data presented in this work suggest the following model for MOR-mediated lymph node apoptosis: High- and low-dose morphine induces a rapid activation of the "activator caspase," caspase-8, through MOR present on lymph node lymphocytes. Following activation of caspase-8, downstream "effector caspase," including caspase-3, is activated, which, in turn, executes apoptosis. Clonal deletion of lymph node lymphocytes induced by morphine impairs the proliferation of T cells and inhibits IL-2 and IFN-{gamma} synthesis. In addition, treatment with high-dose morphine results in the induction of macrophage-derived NO, which can further induce apoptosis (Fig. 12 ). Because morphine-induced activation of caspase-3, -8, and NO is abolished in the MORKO animals, it can concluded that these effects are mediated through MOR.



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  		Figure 12. Flow chart showing mechanism of morphine-induced lymph node apoptosis.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from National Institute on Drug Abuse R01-DA 12104 (S. R.), P50-DA 11806-01 (S. R.), the Department of Defense/Veterans Affairs (R. A. B.), and the North Memorial Trauma Institute (Robbinsdale, MN).

Received May 8, 2001; revised June 18, 2001; accepted June 19, 2001.


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