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, or 
Opioid Receptors
From the *Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom; Department of Pharmacology, Uniformed Services University, Bethesda, Maryland; and Department of Health Sciences, Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester, United Kingdom.
Address correspondence and reprint requests to David G. Lambert, PhD, Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester, LE1 5WW, UK. Address e-mail to DGL3{at}le.ac.uk.
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Abstract |
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and KOP/kappa/
) and nonclassical NOP (nociceptin/orphanin FQ; N/OFQ). METHODS: In this volunteer study we probed for the expression of both classical and nonclassical opioid receptors using 1) radioligand binding, 2) specific antibody binding, and 3) polymerase chain reaction-based experimental paradigms.
RESULTS: Membranes prepared from PBMC from healthy volunteers did not bind either [3H]diprenorphine (a nonselective radioligand for classical opioid receptors) or [3H]N/OFQ. There was significant concentration-dependent binding of each radioligand to control tissues expressing recombinant MOP and NOP. In addition, using fluorescence-activated cell sorting paradigms, there was no binding of fluorescent naloxone or either of two MOP antibodies to whole PBMC, though fluorescent naloxone did bind to recombinant MOP (as a positive control). Using primers specific for classical and nonclassical opioid receptors, and RNA extracted from the PBMC of 10 healthy volunteers, we were also unable to detect MOP, DOP, and KOP transcripts. In contrast, NOP was detected in all samples.
CONCLUSIONS: Despite using several complementary experimental strategies, we failed to demonstrate protein for classical or nonclassical opioid receptors on PBMC from healthy volunteers. We detected NOP mRNA, suggesting low-density NOP expression on these immunocytes. It is possible that N/OFQ, produced by the PBMC itself, may be involved in the control of immune function.
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Introduction |
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Four opioid receptor subtypes are currently recognized: the classical naloxone-sensitive MOP (mu:µ), DOP (delta:) and KOP (kappa:) and the nonclassical nociceptin/orphanin FQ receptor (NOP). Activation of all four types produces antinociception in several animal models of pain; however, most opioids in clinical practice are predominantly MOP agonists.
Opiates have immunomodulatory properties (5–11), and endogenous opiate delivery is, at least partially, controlled by immunocompetent cells (12–15). An appreciation of the link between pain and the immune system has been realized, in particular by the work of Stein et al. (12,16,17) and others (18,19). However, opinion over the existence of opiate receptors and MOP, in particular on peripheral immune cells, is divided (19–24). The presence of the other classical opiate receptors and NOP is also equally controversial.
In this study we used 1) radioligand binding, 2) flow cytometry (FACS), and 3) polymerase chain reaction techniques (PCR) to make a systematic and detailed examination of the expression of the three classical (MOP/DOP/KOP) and the nonclassical NOP opioid receptor in peripheral blood mononuclear cells (PBMC) from healthy volunteers.
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METHODS |
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Chinese hamster ovary cells (CHO) expressing human MOP, DOP, KOP or NOP, and SH-SY5Y human neuroblastoma cells expressing MOP and NOP receptors were used as positive controls as described below. CHOhMOP/DOP/KOP stocks were grown in Hams-F12 supplemented with 10% FCS, penicillin (100 IU/mL), streptomycin (100 µg/mL), and fungizone (2.5 µg/mL). CHOhNOP cells were grown in Dulbecco's MEM/ HAMS F12 (50/50) supplemented with 5% FCS, penicillin (100 IU/mL), streptomycin (100 µg/mL), and fungizone (2.5 µg/mL). Stock cultures were further supplemented with geneticin (G418) (400 µg/mL MOP/DOP/KOP or 200 µg/mL NOP) and hygromycin B (200 µg/mL NOP only). SH-SY5Y cells were grown in MEM supplemented with 10% FCS, 2 mM l-Glutamine, penicillin (100 IU/mL), streptomycin (100 µg/mL), and fungizone (2.5 µg/mL). Cells were used at approximately 80% confluence.
Radioligand Binding
PBMC were harvested from 27 mL of venous blood for radioligand binding studies. PBMC and CHO controls were suspended in buffer consisting of 50 mM Tris-HCl and 5 mM MgSO4 then homogenized using an Ultra Turrax homogenizer. Membranes were collected under centrifugation; 13,500 rpm/10 min/4°C and protein mass assessed according to Lowry et al. (25).
Twenty µg of CHO transfects and up to 1 mg of PBMC homogenate were incubated with [3H]diprenorphine (classical opioid antagonist) or [3H]N/OFQ. [3H]diprenorphine binding was performed in 0.5 mL of 50 mM Tris-HCl, pH 7.4 (KOH) supplemented with 0.5% BSA, varying concentrations of [3H]diprenorphine (approximately 3 nM–30 pM) and NSB were measured with 10 µM naloxone. [3H]N/OFQ binding was performed in 0.5 mL of 50 mM Tris-HCl, 5 mM MgSO4, pH 7.4 (KOH) supplemented with 0.5% BSA, a cocktail of 10 µM peptidase inhibitors (amastatin, bestatin, captopril, and phosphoramidon), varying concentrations of [3H]N/ OFQ (approximately 2 nM–3 pM) and NSB was measured with 1 µM unlabeled N/OFQ.
In some experiments using CHOhMOP, the binding affinity (Ki) of unlabeled fluorescent and nonfluorescent naloxone for classical opioid receptors was calculated from the concentration of unlabeled ligand producing 50% competition (IC50) using the Cheng–Prusoff equation [Ki = IC50/1 + (diprenorphine)/KD for diprenorphine] (26). Competition binding assays were performed in 0.5 mL of binding buffer containing approximately 20 µg of membrane protein, [3H]-diprenorphine (approximately 0.5 nM), and increasing concentration of naloxone/fluorescent naloxone for 1 h at room temperature. All binding reactions were terminated by vacuum filtration, using a Brandell harvester, through Whatman G/F-B filters treated with 0.5% polyethylenimine to reduce NSB. Filter bound radioactivity was quantified by liquid scintillation spectroscopy.
Fluorescent Staining for Flow Cytometry
In flow cytometry experiments designed to quantify MOP receptors, two different anti-MOP opioid receptor primary antibodies directed at extracellular portions of the receptor were used: AB1, an anti-MOP opioid receptor polyclonal antibody directed against the third extracellular loop of the receptor (raised against amino acids 301–316 (Chemicon International, CA), and AB2, an affinity purified antibody directed at the extracellular N-terminus of the receptor [raised against the amino acids 10–70 by one of the authors TC (27)]. Anti-rabbit IgG-FITC conjugate (Sigma, Poole, UK) was used as the secondary antibody in both cases.
Immunofluorescent staining for flow cytometry required that two staining steps be performed on unfixed cells in the dark, both at room temperature. Similar concentrations of primary and secondary antibody were used (1:200 primary, 1:250 secondary), with 30 min allowed for staining. Nonspecific staining was determined by omitting the primary antibody. Additional control flow cytometry was performed with normal rabbit serum for AB1 or affinity purified IgG for AB2 in the place of the primary antibody.
Fluorescent naloxone (a fluorescent conjugate of naloxone allowing investigation of classical opioid receptors) was also used for direct labeling of cells and subsequent flow cytometric analysis. Labeling with fluorescent naloxone was performed on ice in the dark for 30 min, followed by washing in PBS. Nonspecific binding was determined by the addition of excess unlabeled naloxone.
In all protocols cell suspensions were passed through the flow cytometer for analysis. In the case of fluorescent naloxone, ice-cold PBS was used as sheath fluid to limit the possibility of ligand–receptor dissociation. Mean fluorescent intensity (MFI) shift was taken as a measure of both total and nonspecific binding in all cases. Gates were set such that only 5% of MFI fell within the M1 gate at rest with no fluorescent staining. MFI shift into this gate was then calculated as a percentage of counts after fluorescent staining.
PCR
RNA was extracted from PBMC using a modification of the technique described by Chomczynski and Sacchi (28). Briefly 1 mL of TRI BD (phenol/ chloroform, Sigma, Bole, UK) was added to the PBMC pellet from 8 mL of blood, thoroughly mixed, and allowed to stand at room temperature for 5 min. After this 200 µL of chloroform was added, mixed and allowed to stand for a further 5 min before centrifugation (12,000g/4°C/15 min). From the resultant mixture the upper aqueous layer was removed and the above procedure repeated. Then the upper aqueous layer was added to 500 µL of isopropanol and allowed to stand at room temperature for 10 min, before centrifuging (12,000g/4°C/10 min). The resultant RNA pellet was washed in ethanol 75% and air-dried before resuspension in 40 µL of RNAse-free water. RNA was analyzed by biophotometry and electrophoresis on an RNA denaturing gel for integrity and quantity. (A260/280 >1.7 in all cases, with clear ribosomal RNA bands on gel analysis).
Reverse transcription kits from Applied Biosystems (Foster City, CA) were used to convert RNA into cDNA for use in PCR to investigate expression of the genes encoding for the four opiate receptors. Primer pairs were chosen, which sat on different exons allowing differentiation of genomic from copied DNA Table 1 and Figure 1. Primer pairs of Chuang et al. (29) were used to probe for DOP whereas primer pairs of Peluso et al. (30) were used to probe for NOP. Primers for the KOP and MOP receptors were designed in-house (Table 1).
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A standard PCR reaction was used with an initial 95°C, 3 min denaturing step followed by 40 cycles of 95°C (30 s), X°C (30 s), 72°C (30 s), then 10 min at 72°C followed by 4°C. The annealing temperature X was primer pair-dependent. All experiments were performed using an Eppendorf (Combs, UK) Mastercycler. PCR products were run on a 3% agarose gel stained with ethidium bromide for 45 min at 100 V and imaged under ultraviolet illumination.
Further quantitative real-time PCR (QPCR) reactions were run using commercially available TaqMan® Gene expression assays from Applied Biosystems for all four opiate receptors (Hs00538331_m1, Hs00175127_m1, Hs00168570_m1 and Hs00173471_m1) on PBMC samples from healthy volunteers and various CHO/SH-SY5Y positive controls. These reactions had a thermal profile of 95°C (10 min), 40 cycles of 95°C (15 s) 60°C (1 min). QPCR reactions were performed using a Stratagene Mx4000 machine and inbuilt software. These assays were capable of differentiating gDNA from cDNA by virtue of primers being located on different exons. A deflection from the baseline before the 35th cycle of amplification was considered significant for QPCR.
Analysis of Data
All data are expressed as mean ± sem, from n experiments performed as single points or as duplicates. All curve fitting was performed using GraphPad PRISM V3.0 (San Diego). Where appropriate differences in binding affinity were compared using Student's t-test (GraphPad PRISM V3.0) with P < 0.05 being significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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FACS Analysis
In flow cytometric analysis, both AB1 and AB2 failed to bind to MOP in CHOhMOP cells. These antibodies were therefore not used in PBMC. With AB1 in CHOhMOP cells there was a shift into the M1 gate that was also produced by normal rabbit serum as a control (data not shown). As measured by FACS, the binding (Mean Fluoresence Intensity shift) of fluorescent naloxone to CHOhMOP cells (Figs. 3A and B) was concentration-dependent and saturable with an apparent pKD of 7.69 ± 0.38, Figure 3C. This value did not differ from that obtained in radioligand competition experiments. There was no binding of fluorescent naloxone to PBMC, Figure 3C.
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PCR
From 8 mL of blood 18.03 ± 14.25 µg of RNA was extracted with 1.35 ± 0.73 µg added to the standard 40 µL reaction. Using end product PCR amplification with gel-based imaging we failed to demonstrate cDNA for MOP, DOP or KOP, Figure 4. In a separate gel (data not shown) using SH-SY5Y cells an amplicon of approximately 200 base pairs was observed using MOP primer sets. In Figure 4 controls for CHODOP and CHOKOP are included and produced amplicons of the predicted approximately 350 and 200 base pairs respectively. Using NOP-specific primer sets, amplicons of 5–600 bp corresponding to cDNA encoding for the NOP receptor was identified in all 10 volunteers. QPCR techniques using TaqMan primer/probe combinations supported these findings i.e., presence of NOP transcripts. A typical "growth curve" from MOP/DOP/KOP/NOP is shown in Figure 4. In PBMC the cycle threshold for NOP was 32–39 (n = 10). SH-SY5Y and CHOhNOP cells used as very crude controls for low and high expression respectively produced cycle thresholds consistent with this pattern.
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Radioligand binding experiments with [3H]diprenorphine as a nonselective opioid ligand showed binding to CHOMOP cells and [3H]N/OFQ showed binding to CHOhNOP. These types of assays, using relatively low specific activity ligands, do not enable very low expression to be accurately quantified. At face value, the lack of [3H]N/OFQ binding in PBMC is not consistent with clear identification of NOP mRNA in PCR. However, we feel that expression is likely very low (see below).
In immunofluorescent and direct staining experiments using flow cytometry, both anti-MOP receptor antibodies proved to be ineffective at specific staining of the MOP receptor in CHOhMOP cells when adequate negative controls were used. AB1 is a commercially prepared polyclonal directed against a 15 amino acid sequence located on the third extracellular loop of rMOP which, allowing for a shift in alignment, displays 93% homology with hMOP. In contrast, AB2 (TC) is directed against a longer 60 amino acid C-terminal sequence of rMOP which, allowing for a shift in alignment, displays 75%–80% homology with hMOP. These primary antibodies were not used to attempt to stain PBMC. Beck et al. (20) and Caldiroli et al. (21) used immunofluorescent and direct fluorescent staining with primary anti-MOP receptor antibodies and fluorescent opioid receptor antagonists respectively to study MOP receptors on peripheral blood mononuclear cells taken from volunteers or rodent models. A third fluorescent probe used to image classical opiate receptors was fluorescent naloxone. Radioligand competition binding experiments showed reduced affinity, but this was still sufficiently high for use in FACS studies. This antagonist bound to CHOhMOP cells with an affinity (approximately 20 nM) close to that in competition binding experiments (approximately 6 nM). This probe did not bind to PBMC.
This inability to detect opioid receptors on PBMC led us to use polymerase chain techniques. Specific primer design allowed for discrimination of genomic from complimentary DNA (Fig. 1). When used with CHO transfects, consistent images corresponding to the gene of interest were seen on agarose gels or in real-time PCR fluorescence plots; however PBMC from volunteers showed no such bands for the classical opiate receptor genes. These findings imply that the RNA message for DOP, KOP, and MOP receptors are not produced in these cells, and this is consistent with binding and FACS data. In contrast, both types of PCR clearly indicate that the mRNA encoding for the novel opiate receptor NOP is present.
Collectively, these experiments provide good evidence that peripheral blood mononuclear cells do not express the classical opiate receptors but do produce RNA encoding for the NOP receptor. It is worth re-emphasizing that message does not automatically mean protein at the cell surface. As early as 1983, it was reported in radioligand binding studies that PBMC and other immunocytes (separated from as little as 20 mL of human blood donated by healthy volunteers) expressed opioid receptors (31). Using PCR, Chuang et al. have described the expression of the mRNA encoding for the MOP receptor in immunocompetent cells and cell lines of humans and other primates (32,33). However, the primer pairs used in these experiments were directed against regions of the genetic code located within one exon and so raise the possibility of amplification of genomic, rather than complimentary, DNA.
There is evidence to indicate that opiate agonists can exert an effect upon a central neuroimmune axis, possibly via classical opiate receptors and the hypothalamic–pituitary–adrenal axis, with immunomodulatory consequences (7,34). This physiological axis could explain opioid immunosuppression in human and whole animal studies. However, the finding that immune cells do not display classical opiate receptors suggests this functional effect of opioid agonists is unlikely via an interaction with classical receptors on PBMC within the periphery. A number of different theories could explain how opioids influence immune cell function in the absence of these receptors. Most simply, it is possible that opioid agonists may act at PBMC via a nonclassical receptor. Cadet et al. have postulated a µ3 receptor capable of interaction with classical MOP agonists but displaying a different DNA and protein sequence; this type of receptor has not been sequenced (22,35,36) and if it exits it would probably amplify with classical MOP. A second possibility is the expression of receptors only after exposure of immune cells to a variety of cytokines and proinflammatory compounds. In our view, this is the most attractive explanation. Indeed, Kraus et al. have shown that MOP receptor expression on immune cells increases during the first 24 h after administration of tumor necrosis factor-
(TNF-) (37,38). However RNA transcripts were found in very low number, requiring multiple PCR amplification cycles to show evidence of amplification product.
N/OFQ and NOP have been classified as the fourth opioid receptor system. However, while the receptor shares a degree of homology with classical opioid receptors, it has been shown to differ significantly in its pharmacological characteristics. Despite these differences, previous studies have provided laboratory evidence supporting the view that N/OFQ, along with the classical opioid receptors, plays a role in the modulation of immune responses at the cellular level. In vitro experiments with T-lymphocytes stimulated with Staphylococcal enterotoxin B show an upregulation of the cluster differentiation markers involved in T-cell activation after the addition of N/OFQ (39). It is believed that these effects are, at least in part, modulated via changes in prostaglandin synthesis within the cell in response to the administration of N/OFQ. Further in vitro studies indicate that N/OFQ can act as a potent chemotactic agent on monocytes, with less impressive results on neutrophils, where it may promote lysozomal release (40). Additional studies have also found that N/OFQ is released by neutrophils in a time-dependent manner in response to the inflammatory mediators fMLP and cytochalasin B (18). These studies provide some evidence that N/OFQ can elicit functional immune responses at the cellular level, and that immunocompetent cells produce and respond to local N/OFQ.
SH-SY5Y human neuroblastoma cells express a very a low density of NOP [approximately 10 fmol/mg protein using the high specific activity (125I) N/OFQ, unpublished] and QPCR growth curves for PBMC lie to the right of this cell line (Fig. 4). If we assume equal starting template and comparable reactions, then it is not unreasonable to suggest that PBMC express <10 fmol/mg protein NOP. This level would be below the detection limit of [3H]binding protocols and corroborates the data in Figure 2.
In summary, we were unable to detect classical opioid receptors on PBMC from healthy volunteers. Based on PCR findings these cells probably express a very low density of the nonclassical NOP.
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Footnotes |
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Funded in part by a grant from British Journal of Anesthesia and The Royal College of Anesthetists.
Conflict of Interest: We have no Conflict of Interests to declare.
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