The Journal of Immunology Human White Blood Cells Synthesize Morphine: CYP2D6 Modulation1 Wei Zhu,* Patrick Cadet,* Geert Baggerman,† Kirk J. Mantione,* and George B. Stefano2* Human plasma contains low, but physiologically significant, concentrations of morphine that can increase following trauma or exercise. We now demonstrate that normal, human white blood cells (WBC), specifically polymorphonuclear cells, contain and have the ability to synthesize morphine. We also show that WBC express CYP2D6, an enzyme capable of synthesizing morphine from tyramine, norlaudanosoline, and codeine. Significantly, we also show that morphine can be synthesized by another pathway via L-3,4-dihydroxyphenylalanine (L-DOPA). Finally, we show that WBC release morphine into their environment. These studies provide evidence that 1) the synthesis of morphine by various animal tissues is more widespread than previously thought and now includes human immune cells. 2) Moreover, another pathway for morphine synthesis exists, via L-DOPA, demonstrating an intersection between dopamine and morphine pathways. 3) WBC can release morphine into the environment to regulate them- selves and other cells, suggesting involvement in autocrine signaling since these cells express the ␮3 opiate receptor subtype. The Journal of Immunology, 2005, 175: 7357–7362. he immune-regulatory effects of opioid chemical messen- of plasma morphine include the adrenal gland and/or brain. An- gers are well established (1, 2). Exogenously adminis- other important potential source is white blood cells (WBC;3 leu- T tered opiates are immunosuppressive, inhibiting both cel- kocytes) since morphine is found in plasma constitutively (4). Al- lular and humoral responses via their cytokine-like effects in both though the presence of morphine in human plasma has been the CNS and the periphery (2). In vitro exposure of human mono- demonstrated, the source and biosynthetic pathways have not been cytes and granulocytes to morphine results in immunosuppression, identified. We therefore undertook this study to 1) determine as evidenced by a marked reduction in chemotaxis, phagocytosis, whether human WBC have the capacity to synthesize morphine; 2) and responsiveness of cells to signal molecules (3). Furthermore, identify the biosynthetic pathways for morphine synthesis in these receptors for many of the opioid peptides as well as morphine have cells; and 3) determine whether such synthesis can result in mod- been shown to be expressed outside the CNS (2). In addition, en- ulation of immune activity in a physiologically relevant manner. dogenous morphine-like compounds have been identified in di- verse organisms, including humans, and are released in response to Materials and Methods by guest on October 1, 2021. Copyright 2005 Pageant Media Ltd. surgical, pathogenic, and psychological stress (4–6). In this re- Human blood was obtained from the Long Island Blood Services (Melville, gard, because endogenous morphine is constitutively expressed NY). Human heparinized whole blood was immediately separated using and overexpressed following trauma, we have surmised that this 1-Step Polymorphs (Accurate Chemical and Scientific) gradient medium. opiate alkaloid normally down-regulates immune, vascular, and Five milliliters of heparinized blood was layered over 5 ml of Polymorphs ϫ neural responsiveness on a basal level. In addition, it also limits in a 14-ml round-bottom tube and then centrifuged for 35 min at 500 g in a swinging bucket rotor at 18°C. After centrifugation, the lower band microenvironmental noise and postinflammatory immune respon- consisting of polymorphonuclear cells (PMN) were harvested in 14-ml siveness (7–9). tubes and then washed with 10 ml of PBS (Invitrogen Life Technologies) Our laboratory recently demonstrated that human monocytes by centrifugation for 10 min at 400 ϫ g. In addition, residual RBC were and granulocytes express a novel ␮ opiate receptor, ␮3, which is lysed using ACK lysing buffer (0.15 M NH4Cl2, 1 mM KHCO3, 0.1 mM Na2EDTA (ph 7.2)) (Current Protocols in Immunology). http://classic.jimmunol.org morphine selective but opiate peptide insensitive (10). This G pro- A two-way ANOVA was used for statistical analysis after precursor tein-coupled receptor, an alternatively spliced variant of the ␮ re- exposure to the cells. Each experiment was performed four times. The ceptor gene, mediates downstream signaling via constitutive NO mean value was combined with the mean value taken from four other synthase-derived NO release (10). replicates. The SEM represents the variation of the mean of the means. All Human plasma contains low concentrations of morphine, which drugs were purchased from Sigma-Aldrich, except bufuralol, which was purchased from BD Biosciences Clontech. The medium containing the can increase following trauma or exercise (4–6). Potential sources PMN was then separated after and before precursor exposure at varying Downloaded from concentrations for 1 h. Cells were washed and the endogenous morphine content was determined. *Neuroscience Research Institute, State University of New York College at Old West- bury, Old Westbury, NY 11568; and †Laboratory of Developmental Physiology and Molecular Biology, Zoological Institute, Catholic University of Leuven, Leuven, Bel- Morphine determination gium The morphine extraction, isolation, and identification and quantification Received for publication August 9, 2005. Accepted for publication September protocols were performed as described elsewhere in great detail for the past 21, 2005. 15 years (11). Several blank HPLC purifications were performed between The costs of publication of this article were defrayed in part by the payment of page each sample to prevent residual morphine contamination remaining on the charges. This article must therefore be hereby marked advertisement in accordance column. Furthermore, invertebrate molluscan mantle tissue was run as a with 18 U.S.C. Section 1734 solely to indicate this fact. negative control, demonstrating a lack of contamination (12). All solutions, 1 This work was supported in part by grants from the National Institute of Mental Health (Grant 47392) and National Institute on Drug Abuse (Grant 09010). 2 Address correspondence and reprint requests to Dr. George B. Stefano, Neuro- 3 Abbreviations used in this paper: WBC, white blood cell; PMN, polymorphonuclear science Research Institute, State University of New York–College at Old Westbury, cell; L-DOPA, L-3,4-dihydroxyphenylalanine; FF, form factor; Q-TOF, quadrupole P.O. Box 210, Old Westbury, NY 11568. E-mail address: [email protected] time-of-flight; MS, mass spectrometry. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 7358 WBC SYNTHESIZE MORPHINE medium etc., were also examined for any presence of morphine. The results chemical adhesion molecule alterations (19–21) as well as cytokine of these tests revealed a lack of morphine contamination. production (19–21). Nanoflow electrospray quadrupole time-of-flight mass spectrometry (Q- All pharmacological agents were purchased from Research Biochemi- TOF-MS) offers an effective way of demonstrating endogenous alkaloids in cals or Sigma-Aldrich. biological tissues (11, 13, 14). Q-TOF-MS was performed on a Q-TOF hybrid system (Q-Tof2, Micromass). One microliter of acetonitrile/water/ Results formic acid (30:69:1, v/v/v) of the PMN HPLC extraction was loaded in a gold-coated borosilicate capillary (Proxeon). Authentic morphine standard In control (vehicle-exposed) WBC, morphine was identified at a was used as a control. This sample was sprayed at a flow rate of ϳ30 level of 12.33 Ϯ 5.64 pg/million cells Ϯ SEM (Fig. 1). Mass nl/min, giving extended analysis time in which MS spectra, as well as spectrometric analysis demonstrated the molecular mass of the sin- MS/MS spectra, were obtained. During MS/MS or tandem MS, fragment gle charged ion of morphine, which is identical with authentic ions are generated from a selected precursor ion by collision-induced dis- morphine (Fig. 1, top and middle). It is important to note that these sociation. The collision energy is typically varied between 20 and 35 eV so that the parent ion is fragmented into a number of different daughter ions, cells were extensively washed in serum-free RPMI 1640, limiting allowing a comparison to be made between the experimental and authentic any plasma morphine that may be found on the cells. However, it samples. is possible that the cells nonspecifically accumulated morphine RIA determination from plasma. To determine whether WBC contain morphine due to endogenous synthesis, cells were incubated with specific morphine The morphine RIA determination is a solid-phase, quantitative RIA, 125 precursors, including tyramine, previously shown to be present in wherein I-labeled morphine competes for a fixed time with morphine in whole animals (12, 16, 22) and previously shown to increase mor- the test sample for the Ab binding site. The detection limit was 0.5 ng/ml. The commercial kit used was obtained from Diagnostic Products (11–13, phine synthesis in healthy neural tissue (16, 22). Tyramine, nor- 15, 16). laudanosoline tetrahydropapaveroline (THP), reticuline, and L-3,4- dihydroxyphenylalanine (L-DOPA) significantly increase WBC CYP2D6 molecular demonstration morphine concentrations above those found in untreated cells in a Human heparinized whole blood obtained from volunteer blood donors concentration-dependent manner (ANOVA test, p Ͻ 0.001; Fig. 1, (Long Island Blood Services) was prepared