Kappa-opioid receptors on lymphocytes of a human lymphocytic cell line: morphine-induced up-regulation as evidenced by competitive RT-PCR and indirect immunofluorescence
Introduction
Immune cells have been shown to express brain-like kappa opioid receptors (KOR) both at transcriptional [1], [2], [3], [4] and translational [5], [6] levels. However, the immunological functions of KOR remain largely unknown. It has been reported that there is a decrease in κ-opioid receptor expression on maturation of mouse lymphocytes [7]. Treatment of peritoneal macrophages with κ-opioid receptor-selective agonist U50,488H suppressed the production of cytokines, including TNF-α, IL-1 and IL-6 [8]. Like mu or delta opioid receptors, KOR belongs to the G-protein-coupled receptor family with seven transmembrane domains [9]. Ligand-binding studies indicate that KOR may selectively dimerize with delta but not with mu opioid receptors to form a new functional receptor [10].
In a recent study we demonstrated that opioids, at micromolar concentrations, suppress the chemokine-mediated migration of both monkey neutrophils and monkey monocytes [11]. Using various opioid receptor agonists and antagonists in the study, we found that this inhibition of leukocyte migration by opioids is mediated by opioids binding to mu or kappa receptors; binding to delta opioid receptors was rarely observed [11].
Morphine is a potent analgesic and addictive opioid; it also elicits various immunomodulatory and immunosuppressive effects on rhesus monkeys when used chronically, including suppression of T-cell proliferation response, a transient stimulation of IL-2 release [12], inhibition of polymorphonuclear cell phagocytosis and chemotaxis [13], and alteration of the disease progression of simian immunodeficiency virus (SIV)-infected animals [14]. The immunomodulatory functions of morphine were found to involve the cellular activation and phosphorylation of the MAP kinase cascade proteins [15]. It is generally recognized that morphine induces its immunological actions through binding to its specific receptors, primarily of the mu type [16]. In mice lacking the mu opioid receptor gene, the morphine-induced immunosuppression was found to be abolished [17]. However, in another study, it was reported that several morphine-induced immune functions, including morphine reduction of splenic and thymic cell number and mitogen-induced proliferation, and morphine inhibition of IL-1 and IL-6 secretion by macrophages, were not affected in mu-opioid receptor-knockout mice, suggesting that morphine may act by a mechanism mediated by either delta or kappa opioid receptors [18]. Using in vitro assay systems, the present study was undertaken to determine whether morphine might indeed activate kappa opioid receptors of immune cells.
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Cell culture
The CEM x174 cell line, a hybrid of human B cell line 721.174 and human T cell line CEM [19], was maintained in RPMI 1640 medium supplemented with 10% heat-inactivated bovine calf serum, 2 mM l-glutamine, 25 mM HEPES, penicillin and streptomycin (pen/strep). The cells were grown at 37°C in a 5% CO2 incubator.
Treatment of cells with morphine, U50,488H, and/or opioid antagonists
Morphine sulfate, U50,488H (a KOR-selective opioid), naloxone (a fairly nonselective antagonist, Ref. [16]), nor-binaltorphimine (nor-BNI, a KOR-selective antagonist, Ref. [16]), and
Amplification and quantification of KOR transcript
It was previously reported that CEM x174 cells express KOR receptors with gene sequences identical to the published human placenta KOR sequences [1]. Fig. 1A shows that a 388-bp segment of KOR transcript spanning the second extracellular loop and the seventh transmembrane domain of the receptor was amplified from the total RNA of CEM x174 cells using an RT-PCR procedure with a set of K-1 and K-2 primers. To quantify KOR mRNA on CEM x174 cells, plasmids containing segments of KOR and plasmids
Discussion
Opioids such as morphine are potent analgesic and addictive compounds. Chronic morphine use also induces immunomodulatory and immunosuppressive effects, as especially evident in HIV-infected patients. Morphine acts on the immune cells mainly through its binding to mu opioid receptors expressed by the cells [20], [21]. Since certain morphine-induced alterations in immune functions also exist in mu opioid receptor knockout mice [18], one would surmise that in addition to the mu opioid receptors,
Acknowledgments
This work was supported by NIH research grants DA 10433, DA 05901, DA 00360 and DA 04355 from the National Institute on Drug Abuse. We thank Dr. Sabita Roy for her generous gift of U50,488H.
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