Loss of morphine-induced suppression of NK cell activity and T-cell functions in [mu]-opioid receptor knockout mice.
Subject: Morphine (Dosage and administration)
Lymphocytes (Research)
Immunological research
Authors: Weber, Richard J.
Gomez-Flores, Ricardo
Sora, Ichiro
Uhl, George R.
Pub Date: 04/01/2006
Publication: Name: American Journal of Immunology Publisher: Science Publications Audience: Professional Format: Magazine/Journal Subject: Biological sciences Copyright: COPYRIGHT 2006 Science Publications ISSN: 1553-619X
Issue: Date: April, 2006 Source Volume: 2 Source Issue: 2
Topic: Event Code: 310 Science & research
Product: Product Code: 2834232 Morphine (Unit Dose) NAICS Code: 325412 Pharmaceutical Preparation Manufacturing SIC Code: 2834 Pharmaceutical preparations
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 156363908
Full Text: Abstract: In vivo administration of [mu]-opioid receptor selective agonists to various species is known to suppress lymphocyte, NK cell, and macrophage functions, in addition to mediate pain relief and euphoria. Using a mouse model in which the [mu]-opioid receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in natural killer (NK) cell activity and T lymphocyte function. Following peripheral morphine administration, NK cell activity was not affected in homozygous [mu]-opioid receptor knockout mice, heterozygous animals marginally responded to the immunosuppressive effects of the drug, while wild-type animals were significantly suppressed. In addition, splenic T-cell proliferative responses to concanavalin A, phytohemaglutinin and an antibody to T-cell [receptor.sup.[alpha][beta]] (TCR) plus interleukin-2 were not affected by morphine treatment in [mu]-opioid receptor knockout homozygous and heterozygous mice, whereas morphine significantly suppressed T-cell proliferation in wild-type mice. Taken together, these results suggest a role of the [mu]-opioid receptor in immunoregulation.

Keywords: Rodent, NK cells, lymphocytes, neuroimmunology, transgenic/knockout.

INTRODUCTION

Opioid agonist activities depend on binding to high-affinity receptors in the brain and on cells of the immune system, named [mu], [delta], and [kappa]which prototypic ligands are morphine, the enkephalins, and the dynorphins respectively [1-3]. Analgesic effects of opioids can be mediated by these types of opioid receptors [4]. However, in vivo administration of [mu]-opioid receptor selective agonists to various species is known to suppress a number of immune cell functions including natural killer (NK) cell cytotoxic activity, lymphocyte proliferation and production of IFN-[gamma], T-cell mediated cytotoxicity, antibody formation, and production of TNF-[alpha] and nitric oxide, and phagocytosis by macrophages [5-16]. It is recognized of the indirect effects opiate agonists elicit on leukocyte modulation through central pathways [5,9,10-15]. However, evidence for the direct modulation of opioids on the cells of the immune system has accumulated enormously in recent years [14].

The relevance of opioid receptors within the confines of immunophysiology (autocrine and paracrine regulation) has not been fully elucidated. One recent study using [mu]-opioid receptor (MOR) knockout mice showed that chronic administration of morphine induced lymphoid organ atrophy, and decreased thymic CD4+CD8+ cell ratio and NK cell activity in wild-type mice, suggesting that MOR gene product represents a molecular target for morphine action on the immune system [17]. In addition, the MOR has been involved in regulating hematopoiesis [18]. The present study was conducted to further investigate the immunomodulatory effects of acute morphine action in the MOR knockout mouse.

MATERIALS AND METHODS

Reagents and culture media: Penicillin-streptomycin solution, and RPMI 1640 and AIM-V media were obtained from Life Technologies (Grand Island, NY). Morphine sulfate was a gift from Dr. Andrew Ho from our Department. Concanavalin A (Con A), phytohemaglutinin (PHA), sodium dodecyl sulfate, and HCl were purchased from Sigma Chemical Co. (St. Louis, MO). Ig[G.sub.1] monoclonal antibodies to T-cell receptor[??] (TCR) were obtained from Harlan Bioproducts for Science (Indianapolis, IN).

Animals: C57BL/6J female [mu]-opiate receptor knockout mice (20-25g) were obtained from Dr. Udi Shavit from Department of Psychology, The Hebrew University, Mount Scopus, Jerusalem, Israel. They were given water and food ad libitum.

Drug preparation and administration: Morphine sulfate was dissolved in pyrogen-free saline at a concentration of 30 mg/kg. Two hundred microliters of morphine (3 mg/ml) or vehicle (saline) was then administered intraperitoneally. After 3 hours following morphine or saline injection, mice were killed by cervical dislocation.

Cell preparation and culture: The spleen was immediately removed after animal death. A single-cell suspension was then prepared by disrupting the spleen in RPMI 1640 medium. Cell suspension was washed three times in this medium, and suspended and adjusted at appropriate densities with AIM-V medium (containing 0.5% penicillin-streptomycin solution). The culture medium was changed at this step to the serum-free medium AIM-V which has been observed to support cell culture [19].

NK-cell assay: NK-cell cytotoxic activity was assessed by the chromium release assay using [[sup.51]Cr]-labeled YAC-1 murine lymphoma cell line as reported elsewhere [3]. YAC-1 cells were labeled by incubating 107 cells with 200 [micro]Ci sodium [sup.51]chromate (NEN Research Products, Boston, MA) for 2 h at 37[degrees]C, and then washed three times with RPMI 1640 medium and suspended in this medium to a density of 5x[10.sup.4] cell/ml. YAC-1 cells were added to round-bottomed 96-well plates (Becton Dickinson, Lincoln Park, NJ) containing splenic cells at various concentrations to give effector/target ratios ranging from 25:1 to 400:1. Spontaneous and maximal [sup.51]chromium release were obtained by incubating [[sup.51]Cr]-labeled YAC-1 cells in AIM-V medium alone or medium containing 2% sodium dodecyl sulfate plus 0.1N HCl, respectively. After 4 h of incubation, supernatants were harvested and [sup.51]Cr release was measured in a gamma counter (Packard, Downers Grove, IL).

T-cell proliferation assay: T cell proliferation was determined by [[sup.3]H]-thymidine uptake as previously reported [12]. Immediately after rat death, single-cell spleen suspensions were prepared as described above and adjusted to 5 x [10.sup.6] cells/ml. Cell suspensions (100 [micro]l) were added to round-bottomed 96-well plates (Becton Dickinson) containing triplicate cultures (100 [micro]l) of AIM-V medium (unstimulated control) or Con A (1 [micro]g/ml), and PHA (50 [micro]g/ml) and antiTCR (3 [micro]g/ml) for 48 h. After incubation for 44 h at 37[degrees]C with 5% C[O.sub.2], [[sup.3]H]-methylthymidine (6.7 Ci/mmol, ICN Pharmaceuticals Inc., Costa Mesa, CA) was added (1 [micro]Ci/10 [micro]l/well), and cultures were incubated for 4 h. Cell cultures were then harvested with a semiautomatic cell harvester (Tomtec, Orange, CT), and cellincorporated radioactivity was determined by liquid scintillation spectrophotometry using a Microbeta Plus liquid scintillation counter (model 1450, Wallac Oy, Turku, Finland) with a counting efficiency for tritium of 35%.

Statistical analysis: The results were expressed as mean [+ or -] SEM of 5 separate rat tissue's responses to each treatment (different effector:target ratios and mitogen concentrations) (3-4 replicate determinations per treatment) per experimental group (homozygous, heterozygous, and wild-type), from a representative experiment. All experiments were repeated at least three times with similar results. Level of significance was assessed by one-way analysis of variance.

RESULS AND DISCUSSION

Effect of morphine on NK-cell cytotoxic activity: In wild-type homozygous (+/+) mice, morphine significantly (P < 0.001) induced 40 [+ or -] 4, 47 [+ or -] 2, and 49 [+ or -] 1 percent of reduction of NK-cell activity at effector:target ratios of 50:1, 100:1, and 200:1 respectively (Fig. 1a), which represents 30 [+ or -] 1 percent reduction of lytic units (102570 [+ or -] 3693 and 71485 [+ or -] 2573 lytic units of saline and morphine treatment, respectively); in knockout heterozygous mice (+/-), morphine significantly (P < 0.01) induced 16 [+ or -] 0.1, 23 [+ or -] 0.1, and 8 [+ or -] 0.03 percent of reduction of NK-cell activity at effector:target ratios of 50:1, 100:1, and 200:1 respectively (Fig. 1b), which represents 5.7 [+ or -] 0.08 percent reduction of lytic units (99483 [+ or -] 3581 and 93799 [+ or -] 1376 lytic units of saline and morphine treatment, respectively); and in knockout homozygous mice (-/-), morphine induced (P > 0.1) 2.5 [+ or -] 0.08, 0, and 3.5 [+ or -] 0.2 percent of reduction of NK-cell activity at effector:target ratios of 50:1, 100:1, and 200:1 respectively (Fig. 1c), which represents 1 [+ or -] 0.04 percent reduction of lytic units (113136 [+ or -] 4072 and 111882 [+ or -] 4027 lytic units of saline and morphine treatment, respectively), as compared with response of cells from saline-injected control.

[FIGURE 1 OMITTED]

Effect of morphine on splenic lymphocyte

proliferation: Morphine caused significant (P < 0.001) 83 [+ or -] 10, 70 [+ or -] 14, and 65 [+ or -] 3 percent reduction of splenic lymphocyte proliferation induced by Con A, PHA, and antiTCR respectively, in wild-type homozygous (+/+) mice, compared with the response of cells from saline-injected control animals (Fig. 2). However, morphine did not significantly alter the proliferative response of splenic cells from knockout heterozygous (+/-) and homozygous (-/-) mice to these stimuli, compared with control (Fig. 2).

[FIGURE 2 OMITTED]

Lymphocytes, NK cells, and macrophages are very sensitive to opioid action. The role of opioid on regulating immune responses has become more significant because of the implications of drug abuse on immunity against infectious diseases and cancer. [mu]-Opioid receptor mediation of morphine-induced analgesia [20] and immunosuppression [21-24] has been previously suggested. The production of mu-opiate receptor heterozygous and homozygous knockout mice displaying approximately 54% and 0% of wild-type levels of [mu]-opioid receptor expression, respectively, has been of significant importance to unravel the role of [mu]-opioid receptors in analgesia and immunoregulation [17].

Elimination of [mu]-opioid receptors in -/- animals has been previously shown to abolished morphine's effects on nociceptive responses in hot plate and tail flick tests, whereas mu receptor +/- mice were shown to display right and downward shifts in morphine dose-effect relationships, which was consistent with lower morphine potencies and efficacies in tests of both spinal and supraspinal analgesia [17]. It was also reported that mice lacking the [mu]-opioid receptor gene did not respond to the immunosuppressive effects of morphine including lymphoid organ atrophy, decrease in the ratio CD4(+)/CD8(+) cells in the thymus, and NK-cell cytotoxic activity [17]. In addition, it has been observed that mice lacking the [mu]-opioid receptor gene are unresponsive to morphine [17] and heroin-induced analgesia18. Interestingly, it was reported that [mu]-opioid receptor knockout mice under chronic 12-h daily restraint stress for 2 days exhibited no effect in splenic lymphocyte proliferation, and IL-2, IFN-gamma and corticosterone production which were altered in wild-type mice [25].

In the present study, we have evidence confirming that homozygous [mu]-opioid receptor knockout mice were not affected in their NK-cell activity by morphine, heterozygous animals marginally responded to the immunosuppressive effects of the drug, while wild-type animals were significantly suppressed (Fig. 1), as previously reported by others [17]. In addition, we showed that splenic T-cell proliferative responses to various stimuli of [mu]-opioid receptor knockout homozygous and heterozygous mice were not affected by morphine treatment, whereas morphine significantly suppressed T-cell proliferation in wild-type homozygous animals (Fig. 2). In conclusion, we showed that [mu]-opioid receptor deficient mice were not immunosuppressed by acute action of morphine.

ACKNOWLEDGMENTS

This study was supported by NIH grants DA/AI08988, DA12095, and F32-DA05865. We thank Mary E. Riley for technical assistance.

REFERENCES

[1.] Belkowski, S.M., J. Zhu, L.Y. Liu-Chen, T.K. Eisenstein, M.W. Adler and T.J. Rogers, 1995. Sequence of kappa-opioid receptor cDNA in the R1.1 thymoma cell line. J. Neuroimmunol. 62: 113-117.

[2.] Sedqi, M., S. Roy, S. Ramakrishnan, R. Elde and H.H. Loh, 1995. Complementary DNA cloning of a mu-opioid receptor from rat peritoneal macrophages. Biochem. Biophys. Res. Commun. 209: 563-574.

[3.] Weber, R.J. and C.B. Pert, 1984. Opiatergic Modulation of the Immune System. In: Central and Peripheral Endorphins: Basic and Clinical Aspects (eds E.E. Muller and A.R. Genazzani) pp. 35-42. Raven Press, New York.

[4.] Pasternak, G.W., 1993. Pharmacological mechanisms of opioid analgesics. Clin. Neuropharmacol. 16: 1-18.

[5.] Band, L.C., A. Pert, W. Williams, B.R. de Costa, K.C. Rice and R.J Weber, 1992. Central [mu]-opioid receptors mediate suppression of natural killer activity in vivo. Prog. Neuroendocrinimmunol. 5: 95-101.

[6.] Bussiere, J.L., M.W. Adler, T.J. Rogers and T.K. Eisenstein, 1992. Differential effects of morphine and naltrexone on the antibody response in various mouse strains. Immunopharmacol. Immunotoxicol. 14: 657-673.

[7.] Carr, D.J., 1991. The role of endogenous opioids and their receptors in the immune system. Proc. Soc. Exp. Biol. Med. 198: 710-720.

[8.] Chuang, L.F., T.K. Chuang, K.F.J. Killam, A.J. Chuang, H.F. Kung, L. Yu and R.Y. Chuang, 1994. Delta opioid receptor gene expression in lymphocytes. Biochem. Biophys. Res. Commun. 202: 1291-1299.

[9.] Fecho, K., K.A. Maslonek, L.A. Dykstra and D.T. Lysle, 1996. Evidence for sympathetic and adrenal involvement in the immunomodulatory effects of acute morphine treatment in rats. J. Pharmacol. Exp. Ther. 277: 633-645.

[10.] Freier, D.O. and B.A. Fuchs, 1994. A mechanism of action for morphine-induced immunosuppression: corticosterone mediates morphine-induced suppression of natural killer cell activity. J. Pharmacol. Exp. Ther. 270: 1127-1133.

[11.] Gomez-Flores, R., J.L. Suo and R.J. Weber, 1999. Suppression of splenic macrophage functions following acute morphine action in the rat mesencephalon periaqueductal gray. Brain Behav. Immun. 13: 212-224.

[12.] Gomez-Flores, R. and R.J. Weber, 1999a. Inhibition of interleukin-2 production and downregulation of IL-2 and transferrin receptors on rat splenic lymphocytes following PAG morphine administration: a role in natural killer and T cell suppression. J. Interferon Cytokine Res. 19: 625-630.

[13.] Gomez-Flores, R. and R.J. Weber, 1999b. Opioids, opioid receptors, and immune function. In: Cytokines: Stress and Immunity (eds N. Plotnikoff, R. Faith, A. Murgo and R. Good) pp. 281-314. CRC Press, Inc., New York.

[14.] McDonough, R.J., J.J. Madden, A. Falek, D.A. Shafer, M. Pline, D. Gordon, P. Bokos, J.C. Kuehnle and J. Mendelson, 1980. Alteration of T and null lymphocyte frequencies in the peripheral blood of human opiate addicts: in vivo evidence for opiate receptor sites on T lymphocytes. J. Immunol. 125: 2539-2543.

[15.] Roy, S. and H.H. Loh, 1996. Effects of opioids on the immune system. Neurochem. Res. 21: 1375-1386.

[16.] Weber, R.J. and A. Pert, 1989. The periaqueductal gray matter mediates opiate-induced immunosuppression. Science 245: 188-190.

[17.] Gaveriaux-Ruff, C., H.W. Matthes, J. Peluso and B.L. Kieffer, 1998. Abolition of morphineimmunosuppression in mice lacking the mu-opioid receptor gene. Proc. Natl. Acad. Sci. U.S.A. 95: 6326-6330.

[18.] Tian, M., H.E. Broxmeyer, Y. Fan, Z. Lai, S. Zhang, S. Aronica, S. Cooper, R.M. Bigsby, R. Steinmetz, S.J. Engle, A. Mestek, J.D. Pollock, M.N. Lehman, H.T. Jansen, M. Ying, P.J. Stambrook, J.A. Tischfield and L. Yu, 1997. Altered hematopoiesis, behavior, and sexual function in mu opioid receptor-deficient mice. J. Exp. Med. 185: 1517-1522.

[19.] Kaldjian, E.P., G.H. Chen and K.B. Cease, 1992. Enhancement of lymphocyte proliferation assays by use of serum-free medium. J. Immunol. Methods 147: 189-195.

[20.] Sora, I., N. Takahashi, M. Funada, H. Ujike, R.S. Revay, D.M. Donovan, L.L. Miner and G.R. Uhl, 1997. Opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia. Proc. Natl. Acad. Sci. U.S.A. 94: 1544-1549.

[21.] Bayer, B.M., S. Daussin, M. Hernandez and L. Irvin, 1990. Morphine inhibition of lymphocyte activity is mediated by an opioid dependent mechanism. Neuropharmacology 29: 369-374.

[22.] Hamra, J.G. and T.L. Yaksh, 1996. Equianalgesic doses of subcutaneous but not intrathecal morphine alter phenotypic expression of cell surface markers and mitogen-induced proliferation in rat lymphocytes. Anesthesiology 85: 355-365

[23.] Tomassini, N., F.L. Renaud, S. Roy and H.H. Loh, 2003. Mu and delta receptors mediate morphine effects on phagocytosis by murine peritoneal macrophages. J. Neuroimmunol. 136 (1-2): 9-16.

(24.] Roy, S., J.H. Wang, S. Balasubramanian, R. Charboneau, R. Barke and H.H. Loh, 2001. Role of hypothalamic-pituitary axis in morphine-induced alteration in thymic cell distribution using mu-opioid receptor knockout mice. J. Neuroimmunol. 116 (2): 147-155.

(25.] Wang, J., R. Charboneau, R.A. Barke, H.H. Loh and S. Roy, 2002. Mu-opioid receptor mediates chronic restraint stress-induced lymphocyte apoptosis. J. Immunol. 169 (7): 3630-3636.

(1) Richard J. Weber, (1,2) Ricardo Gomez-Flores, (3) Ichiro Sora, (3) George R. Uhl

(1) Section of Medical Sciences, Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, Illinois 61656, USA

(2) Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Departamento de Microbiologia e Inmunologia, San Nicolas de los Garza, Nuevo Leon, Mexico

(3) Laboratory of Molecular Neurobiology, Addiction Research Center, National Institute on Drug Abuse, Baltimore, Maryland 20892, USA

Corresponding Author: Ricardo Gomez-Flores, Universidad Autonoma de Nuevo Leon, Facultad de Ciencias Biologicas, Departamento de Microbiologia e Inmunologia, San Nicolas de los Garza, Nuevo Leon, Mexico
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