A Non-Peptide Substance P Antagonist(CP-96,345) Inhibits Morphine-InducedNF-�B Promoter Activation in HumanNT2-N Neurons

Xu Wang,1 Steven D. Douglas,1 Kathryn G. Commons,3 David E. Pleasure,2

Jianping Lai,1 Chun Ho,1 Peter Bannerman,2 Marge Williams,2 and Wenzhe Ho1*1Division of Immunologic and Infectious Diseases, Joseph Stokes Jr. Research Institute of The Children’sHospital of Philadelphia, Department of Pediatrics, University of Pennsylvania School of Medicine,Philadelphia, Pennsylvania2Division of Neurology, Joseph Stokes Jr. Research Institute of The Children’s Hospital of Philadelphia,Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania3Division of Anesthesiology, Joseph Stokes Jr. Research Institute of The Children’s Hospital of Philadelphia,Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania

Opioids and the neuropeptide substance P (SP) modulatethe expression of inflammatory cytokines and chemokines,which are under the control of nuclear factor �B (NF-�B).We investigated whether the neurokinin-1 receptor (SP re-ceptor) pathway is biologically involved in morphine-mediated modulation of NF-�B promoter activation in ahuman neuronal cell line (NT2-N) that expresses both themu-opioid receptor (MOR) and the SP receptor. Morphinesignificantly enhanced NF-�B promoter-directed luciferaseactivity in NT2-N neurons. DAMGO, a selective mu-opioidreceptor agonist, also induced NF-�B promoter activation.The induced activation of NF-�B promoter by morphine orDAMGO was abolished not only by naltrexone (a opioidreceptor antagonist) and CTAP (a selective, competitivemu-opioid receptor antagonist), but also by CP-96,345, anon-peptide SP receptor antagonist. Investigation of themechanism responsible for morphine-induced activation ofNF-�B promoter in NT2-N neurons demonstrated that mor-phine activates the SP promoter and induces SP expres-sion in these cells. We also observed that SP activatedNF-�B promoter and that CP-96,345 downregulated theexpression of endogenous SP. Furthermore, dual immuno-fluorescent labeling revealed that there is co-expression ofNK-1R and MOR in the processes of NT-2N neurons.These results suggest that morphine, by activating MOR,engages a positive feedback loop between NK-1R and SP.Activation of NK-1R could then impact NF-�B expressionand therefore may be an important participant in the effectof morphine on immune responses in the central nervoussystem. © 2003 Wiley-Liss, Inc.

Key words: NK-1R; mu-opioid receptor; DAMGO; SPpromoter

An important component that controls the synthesisof many cytokines and other proinflammatory gene prod-ucts is the transcriptional activator, nuclear factor �B (NF-

�B) (Baeuerle and Henkel, 1994). NF-�B has a centralrole in the overall immune response through its ability toactivate gene coding for a wide variety of cytokines andchemokines (Ghosh and Karin, 2002). NF-�B binds tospecific consensus DNA elements present on promoters oftumor necrosis factor-� (TNF-�) and interleukin-6 (IL-6)genes, initiating transcription of these genes (Collart et al.,1990; Kuprash et al., 1995; Galien et al., 1996). Cytokinesand chemokines have a dynamic role in the communica-tion between the immune system and the central nervoussystem (CNS). NF-�B activation-induced production ofcytokines and chemokines stimulates migration and mat-uration of lymphocytes. In addition, NF-�B activates genecoding for regulators of apoptosis and cell proliferation(Karin and Lin, 2002). Several NF-�B activation-inducedcytokines are involved in CNS inflammatory disease.Thus, modulation of NF-�B activity has an importantimplication for immune response and inflammation in theCNS.

Both opioids such as morphine and the neuropeptidesubstance P (SP), a potent neurotransmitter and modulatorof neuroimmunoregulation, are involved in the modula-tion of expression of inflammatory cytokines and chemo-kines under the control of NF-�B. Opioids exert immu-

Contract grant sponsor: National Institutes of Health; Contract grant num-ber: DA12815, MH49981; Contract grant sponsor: National MultipleSclerosis Society.

*Correspondence to: Dr. Wen-Zhe Ho, Division of Immunologic andInfectious Diseases, The Children’s Hospital of Philadelphia, 34th Streetand Civic Center Boulevard, Philadelphia, PA 19104.E-mail: ho@email.chop.edu

Received 31 July 2003; Revised 13 October 2003; Accepted 17 October2003

Journal of Neuroscience Research 75:544–553 (2004)

© 2003 Wiley-Liss, Inc.

nomodulatory activity and affect regulatory actions of theCNS (Carr and Serou, 1995). Morphine induces apoptosisof human microglia and neurons (Hu et al., 2002), andNF-�B expression in human immune cells (Roy et al.,1998). DAMGO, a selective mu-opioid receptor agonist,induces NF-�B activity in rat cortical neurons (Hou et al.,1996). SP activates NF-�B and gene expression ofinterleukin-8 (IL-8), an NF-�B-regulated target gene inhuman astrocytes (Lieb et al., 1997). SP is produced byseveral cell types in the brain, including astrocytes, neu-rons, and microglia. SP release as an early event in immunecomplex-mediated injury could act as an amplifier of thesubsequent inflammatory cascade (Bozic et al., 1996). SPstimulates monocytes to produce proinflammatory cyto-kines (Wagner et al., 1987; Lotz et al., 1988; Blum et al.,1993; Lee et al., 1994), presumably through activation ofNF-�B, and induces expression of interleukin-1 (IL-1)(Martin et al., 1992), IL-6 (Gitter et al., 1994), and TNF-�(Luber-Narod et al., 1994) by neuroglia cells.

The biological functions of opioids are mediatedthrough opioid receptors. In the CNS, mu-opioid recep-tors are present not only in astrocytes and neurons (Sharpet al., 1998), but also in microglia (Chao et al., 1997).Similarly, SP exerts its biological activities upon binding toa G protein-coupled receptor of the neurokinin (NK)receptor family (Regoli et al., 1989; Gerard et al., 1991).

Opioid receptors co-localized with SP-like immu-noreactivity in dorsal root ganglion (DRG) neurons (Be-langer et al., 2002), and repeated morphine exposureincreased levels of SP in cultured DRG neurons (Ma et al.,2000). We have demonstrated recently that there is abiological interaction between morphine and SP in humanimmune cells (Li et al., 2000). Thus, it is important todetermine the role of morphine and SP in modulation ofimmune response and inflammation in the CNS. Weinvestigated whether morphine and the SP-NK 1 receptor(SP-NK-1R) pathway modulate NF-�B promoter activa-tion in human neuronal cells.

MATERIALS AND METHODS

Reagents

The non-peptide SP antagonist CP-96,345 and its inac-tive enantiomer CP-96,344 (Sindel et al., 1984) were providedgenerously by Pfizer Inc. (Groton, CT). CP-96,345 (10�3 M)and CP-96,344 (10�3 M) were dissolved in water and filteredthrough a 0.22-�m filter (Millipore, Bedford, MA). Morphinesulfate injection (15 mg/ml) was purchased from Elkins-Sinn,Inc. (Cherry Hill, NJ) with prescription. Naltrexone was ob-tained from Sigma-Aldrich (St. Louis, MO). DAMGO waspurchased from Tocris Cookson (Ballwin, MO). CTAP wasobtained from Phoenix Pharmaceuticals (Mountain View, CA).

Cell Culture

Ntera2/c1.D1 (NT2) cells were cultured and differenti-ated into neurons, as described previously (Pleasure et al., 1992;Younkin et al., 1993). Briefly, cells were plated at a density of2.3 � 106 cells per T75 flask and fed twice weekly withDulbecco’s modified Eagle medium (DMEM)-high glucose

(HG; Gibco, Grand Island, NY) with 10% fetal bovine serum(FBS; Hyclone, Logan, UT), 100 IU/ml penicillin, 100 �g/mlstreptomycin (Gibco), and 10 �g retinoic acid (Sigma) for up to6 weeks. The cells were then split (1:4) and cultured for anadditional 48 hr in identical medium without retinoic acid.Neuronal cells growing atop a monolayer of nonneuronal cellswere dislodged with trypsin and plated in 12-well plates (Falcon;Becton Dickinson, Lincoln Park, NJ) at 106 cells/well for RNAand transfection studies, and in 24-well plates (Flcon; BectonDickinson) at 0.5 � 106 cells/well for immunoreactivity assays.

Immunofluorescence Staining of Mu-Opioid Receptorsin NT2-N Neurons

Undifferentiated NT2 cells were cultured on glass cover-slips (12 mm) in a 24-well plate. After being cultured for6 weeks in vitro, the differentiated NT2-N neurons were rinsedtwice in 0.1 M phosphate-buffered saline (PBS) and fixed in 4%paraformaldehyde in PBS for 20 min. After additional rinses,cells were incubated in either 0.2% bovine serum albumin (BSA)in PBS (PBS-BSA) or primary antisera diluted in PBS-BSAovernight at 4°C. Primary antisera were raised in rabbits forNK-1R (1:200; Novus Biologicals, Littleton, CO) or in guineapigs for MOR (1:500; Chemicon, Temecula, CA). For dualimmunolabeling, a combination of the antisera for NK-1R andMOR was used. Secondary antisera raised in donkey (1:200;Jackson Immunoresearch, West Grove, PA) were conjugated toeither Cy2 or Cy3 fluorophores. The coverslips were washedwith 1� PBS and then mounted in 90% glycerol in PBS. Thesamples were examined and imaged using a conventional fluo-rescence microscope. Digital images of each fluorophore werecaptured in black and white, then pseudocolored and mergedusing Openlab software (Improvision, Inc. Coventry, UK). Fig-ures were assembled using Photoshop 5.5 and adjusted for bright-ness and contrast.

RNA Extraction and Reverse Transcription

Total cellular RNA was isolated from NT2-N neuronsusing Tri-Reagent (Molecular Research Center, Cincinnati,OH). In brief, total RNA was extracted by a single step, gua-nidium thiocyanate-phenol-chloroform extraction. After cen-trifugation at 13,000 � g for 15 min at 4°C, the RNA-containing aqueous phase was precipitated in isopropanol. RNAprecipitates were washed once in 75% ethanol and resuspendedin 30 �l of RNase-free water. Total RNA (1 �g) was subjectedto reverse transcription using the Reverse Transcription System(Promega, Madison, WI) with specific antisense primers, de-scribed below for SP or mu-opioid receptor for 1 hr at 42°C.The reaction was terminated by incubating the reaction mixtureat 99°C for 5 min. One-tenth of the resulting cDNA was usedas a template for PCR amplification.

Real-Time PCR: TaqMan Detection

We have developed recently a highly sensitive and specificreal-time RT-PCR assay to measure quantitatively SP mRNA(Lai et al., 2002a). PCR primers and the probe (molecularbeacon; MB) used for SP mRNA qualification were designedwith Primer Express software (PE Applied Biosystems) and weresynthesized by Integrated DNA Technologies (Coralville,Iowa). The HSP4-HSP3 primer pair (HSP4, 5�-CGACCA-

CP96,345, Morphine and NF-�B Promoter 545

GATCAAGGAGGAACTG-3�; HSP3, 5�-CAGCATCCC-GTTTGCCCATT-3�), which is specific for a 121-base pair(bp) fragment of the SP transcript, was described previously (Laiet al., 1999), but was modified by the addition of four nucleo-tides at the 3� end of primer HSP3 (underlined) due to thedesign obtained from the Primer Express software. Sequences forthe primers and the MB were selected from the sequences ofexons 2 and 3 of the preprotachykinin-A (PPT-A) gene (Lai etal., 1998; 2002a) so that they amplify a single cDNA fragmentthat represents all four SP isoforms of PPT-A mRNA transcript(Lai et al., 1999). The primer pair specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is as follows: 5�-GGTG-GTCTCCTCTGACTTCAACA-3� (sense) and 5�-GTTGCT-GTAGCCAAATTCGTTGT-3� (antisense). The sequences ofthe MB specific for SP and GAPDH were designed to becomplementary to the target sequence in exon 3 of the SP geneand to be complementary to the sequence between the sequencesof each primer in the primer pair for GAPDH, respectively. Thefollowing are the sequences of the two MBs: SP, 5�-FAM(6-carboxyfluorescein)-GCGAGCAGAATCGCCCGGAGACC-CAAGCGCTCGCDABCYL (4-benzoic acid, 4�dimethyl-aminophenylaso)-3�; GAPDH, 5�-FAMGCGAGCCTGGCA-TTGCCCTCAACGACCACGCTCGCDABCYL-3�. Stemsequences were selected such that they would not complementsequences within the loop region. The lengths of the MBs weredesigned such that the annealing temperature was slightly higherthan those of the PCR primers. The MB was labeled at the 5�end with FAM and the quencher DABCYL at the 3� end. Theprimers and MBs were resuspended in Tris-EDTA buffer andstored at �30°C.

Real-Time PCR: SYBR Green Detection

Real-time PCR for the quantification of mu-opioid re-ceptor mRNA was carried out with the ABI PRISM 7000-Sequence Detection System using the Brilliant SYBR GreenQPCP Master Mix (Stratagene, La Jolla, CA) as recommendedby the manufacturer. The primer pair specific for mu-opioidreceptor was 5�-ACCAACATCTACATTTTCAACCTT-3�(sense) and 5�-CAGTACCAGGTTGGATGAGAG-3� (anti-sense) as described previously (Madden et al., 2001).

NF-�B and SP Promoter Activation Assay

The pNF-�B-luc (NF-�B promoter containing plasmid)multimer-driven luciferase construct was generated by Dr. D.Petrak (Petrak et al., 1994). Two copies of the mouse � lightchain enhancer (Pierce et al., 1988) were cloned into pBLCAT3vector (Luckow and Schutz, 1987), and then the construct wasmodified by replacing the CAT reporter gene with the luciferasegene obtained from pGEM-luc (Petrak et al., 1994). The pPPT/NO-luc (SP promoter containing plasmid) multimer-driven lu-ciferase construct was generated (Qian et al., 2001) and kindlyprovided by Dr. P. Rameshwar (University of Medicine andDentistry of New Jersey, New Jersey Medical School). ThepPPT/NO was ligated in pGL3-basic, upstream of the luciferasereporter. The pNF-�B-luc and pPPT/NO-luc plasmids wereprepared by miniprep techniques (Wizard plus Minipreps; Pro-mega) according to the manufacturer’s instructions and used intransfection experiments. For transfection experiments, NT2-Nneurons were seeded in a 12-well tissue culture plate

(106 cells/well). Cells were transfected with the pNF-�B-luc orpPPT/NO-luc plasmids using Fugene 6 Transfection Reagent(Roche Molecular Biochemicals, Indianapolis, IN) with a ratioof Fugene 6:plasmid 6:1 (�l:�g), and 24 hr after transfection,cells were treated with SP (10�8 to 10�6 M) for 24 hr or withmorphine (10�12 to 10�8 M) for 48 hr. In some experiments,pNF-�B-luc-transfected NT2-N neurons were also incubatedwith naltrexone, CTAP, or CP-96,345 for 1 hr before theaddition of morphine or SP. At the termination of the experi-ments, cells were harvested and washed twice with PBS bycentrifugation at 3,300 � g for 3 min at room temperature. Cellpellets were lysed by adding 0.25 ml of 1 � Reporter LysisBuffer (Promega) and a cycle of freezing and thawing in dry ice.Cell-free lysates were obtained by centrifugation at 10,000 � gfor 30 sec at room temperature. The effects of morphine or SPon activation of the SP or NF-�B promoter were determined bymeasurement of luciferase activity.

Statistical Analysis

Each variable was measured in triplicate, and experimentswere repeated at least three times. Variability between triplicatewells was less than 15%. One-way analysis of variance(ANOVA) was used to test for differences in means and post-hoc t-test was used for comparison. Differences were consideredsignificant if P � 0.05.

RESULTSExpression of NK-1R and MOR in NT2-NNeurons

Because biological activities of morphine and SP aremediated through their receptors, it is important to deter-mine whether NT2-N neurons express these receptors.We have demonstrated recently that human NT-2N neu-rons express functional NK-1R (Li et al., 2003). Becausethe biological functions of morphine are mediated mainlythrough the MOR, we investigated whether terminallydifferentiated NT2-N neurons contain MOR mRNA andprotein. MOR mRNA was detected in NT2-N neuronsby real-time PCR (Table I). We also determined whethercell differentiation affects MOR mRNA expression inNT2-N neurons. We extracted cellular RNA at fourdifferent time points (Day 2, 14, 28, and 42) during the

TABLE I. Mu-Opioid Receptor mRNA Expression During NT2Differentiation*

Days post retinoic acidtreatment

Relative mRNA (fold increase)

Exp 1 Exp 2

2 (control) 1.0 1.014 1.9 2.028 11.1 11.942 10.9 13.2

*The cellular RNA was extracted at four different time points as indicatedduring the course of NT2 differentiation into NT2-N neurons. Themu-opioid receptor mRNA levels are determined by real-time RT-PCRand expressed as fold of control (Day 2), which is defined as 1.0. Datashown are means of duplicate cultures.

546 Wang et al.

course of NT2 differentiation into NT2-N neurons.There was a consistent increase of MOR mRNA expres-sion during cell differentiation over time in cultures asdemonstrated by the real-time (RT) PCR assay (Table I).Because the cell membrane expression of MOR andNK-1R is related directly to their biological functions, weexamined whether NT2-N neurons expressed membraneproteins for MOR and NK-1R and demonstrated, byimmunofluorescence staining using specific antibodies forMOR and NK-1R, that NT2-N neurons express bothMOR (Fig. 1A) and NK-1R protein (Fig. 1B). Dualimmunofluorescent labeling with antibodies to NK-1Rand MOR revealed that there was co-expression of bothNK-1R and MOR in processes of NT-2N neurons (Fig.1C).

Morphine Activates the NF-�B PromoterTo determine whether morphine activates the

NF-�B promoter, we transfected NT2-N neurons withNF-�B promoter-containing plasmid (pNF-�B-luc) andincubated cells in the presence or absence of morphine(10�12 to 10�8 M) for 48 hr. Morphine significantlyenhanced NF-�B promoter activity in a concentration-dependent manner (2.1-, 2.9-, and 3.6-fold of control at10�12, 10�10, and 10�8 M, respectively) in NT2-N neu-rons (Fig. 2A). We then examined whether morphine,through opioid receptors, activated the NF-�B promoter.We incubated NF-�B promoter plasmid-transfectedNT2-N neurons with or without morphine (10�8 M) ornaltraxone (10�8 M). Naltrexone abrogated morphine-induced NF-�B promoter activation (Fig. 2B). Becausenaltraxone is a pan-opioid receptor antagonist, we furtherexamined whether morphine, specifically through themu-opioid receptor, exerts its effect on the NF-�B pro-moter. For this purpose, we determined whether CTAP,a selective, competitive antagonist specific to the mu-

opioid receptor, blocks morphine action (Kramer et al.,1989; Xu et al., 1999). The addition of CTAP to thecultures also abrogated morphine-induced NF-�B pro-moter activity, whereas CTAP alone had no effect onactivation of the NF-�B promoter (Fig. 2B).

DAMGO Activates the NF-�B PromoterMorphine has a high affinity and sensitivity to the

mu-opioid receptor (Herz, 1997; Peterson et al., 1998,1999). To determine further whether the effect of mor-phine was mediated through the mu-opioid receptorrather than other opioid receptors, we examined whetherDAMGO, a selective mu receptor agonist, activatesNF-�B promoter in NT2-N neurons. We incubatedNF-�B promoter plasmid-transfected NT2-N neuronswith DAMGO at different concentrations or withoutDAMGO. DAMGO significantly enhanced NF-�Bpromoter-directed luciferase activity in a concentration-dependent fashion (Fig. 3A). DAMGO-induced activationof NF-�B promoter was abolished by the addition ofCTAP, a specific mu-opioid receptor antagonist (Fig. 3B).

CP-96,345 Inhibits Morphine- or DAMGO-Induced NF-�B Promoter Activation

Biological interaction of morphine with the SP-NK-1R pathway in the CNS may play a critical role ininflammatory processes related to neurological disorders.Because both morphine and SP are involved in the mod-ulation of expression of inflammatory cytokines and che-mokines that are under the control of NF-�B, we hypoth-esized that SP and SP receptors (NK-1R) may be involvedin morphine or DAMGO-mediated activation of theNF-�B promoter. To test this hypothesis, we first exam-ined whether a non-peptide SP antagonist (CP-96,345)interferes with the effects of morphine or DAMGO on theNF-�B promoter in NT2-N neurons. CP-96,345 abro-

Fig. 1. NT2-N neurons express both MOR and NK-1R. A: MOR immunofluorescence staining(red) of processes and fascicles (arrows)of processes of NT2-N neurons. B: Single labeling (green) forNK-1R is localized to fascicles (arrows) of processes radiating from NT-2N cell bodies. C: Double-immunofluorescence staining (yellow) of NT2-N neurons. Note that co-localization of NK-1R andMOR is common throughout processes (arrows). Scale bars � 50 �m.

CP96,345, Morphine and NF-�B Promoter 547

gated the effects of morphine on the activation of NF-�Bpromoter in NT2-N neurons (Fig. 4A). This effect ofCP-96,345 is specific because CP-96,344, an inactiveenantiomer of CP-96,345, had no impact on morphineaction (Fig. 4A). In addition, CP-96,345 abolished theenhancing effect of DAMGO on the NF-�B promoter,whereas CP-96,344 did not affect DAMGO action on theNF-�B promoter (Fig. 4B). The addition of either CP-

96,345 or CP-96,344 alone had no effect on NF-�Bpromoter activation (Fig. 4A,B).

Morphine Activates the SP Promoter andEnhances SP Expression

We demonstrated previously that morphine en-hances SP expression in mononuclear phagocytes andlymphocytes (Li et al., 2000). We hypothesized that mor-

Fig. 2. Effect of morphine on the activation of NF-�B promoter inNT2-N neurons. A: NT2-N neurons transfected with pNF-�B-lucconstruct were incubated with or without morphine at the concentra-tions indicated for 48 hr. The effect of morphine on the activation ofNF-�B promoter was determined by NF-�B-directed luciferase activ-ity. B: NT2-N neurons transfected with pNF-�B-luc construct wereincubated with or without morphine (10�8 M) or naltrexone (10�8 M)or CTAP (10�8 M) for 48 hr. The data are presented as relative lightunit (RLU) per �g protein. The data shown are the mean SD ofthree independent experiments (**P � 0.01, *P � 0.05, morphine vs.control).

Fig. 3. Effect of CTAP on DAMGO-induced activation of NF-�Bpromoter in NT2-N neurons. A: NT2-N neurons transfected withpNF-�B-luc construct were incubated with or without DAMGO atthe indicated concentrations for 48 hr. The effect of DAMGO on theactivation of NF-�B promoter was determined by NF-�B-directedluciferase activity. B: NT2-N neurons transfected with pNF-�B-lucconstruct were incubated with or without DAMGO (10�8 M) orCTAP (10�8 M) for 48 hr. The data are presented as relative light unit(RLU) per �g protein. The data shown are the mean SD of threeindependent experiments (**P � 0.01, *P � 0.05, DAMGO vs.control).

548 Wang et al.

phine upregulates SP expression in NT2-N neurons.Thus, we first examined whether morphine activates theSP promoter. We transfected NT2-N neurons with an SPpromoter containing plasmid (pPPT/NO-luc) and in-cubated the cells with or without morphine (10�12 to10�8 M) for 24 hr. Morphine significantly enhanced SPpromoter activation (1.8-, 2.0-, and 3.0-fold of control at10�12, 10�10, and 10�8 M, respectively) in NT2-N neu-

rons (Fig. 5A). The enhancing effect of morphine on SPpromoter activity prompted us to investigate whethermorphine modulates SP gene expression in NT2-N neu-rons. We demonstrated that morphine increased SP geneexpression in a concentration-dependent fashion (Fig. 5B).

SP-NK-1R Pathway May Account for Morphine-Mediated NF-�B Promoter Activation

To determine the role of the SP-NK-1R pathway inmorphine-mediated NF-�B promoter activation in

Fig. 4. Effects of CP-96,345 on morphine- or DAMGO-inducedNF-�B promoter activation in NT2-N neurons. A: NT2-N neuronstransfected with pNF-�B-luc construct were incubated with or with-out morphine (10�8 M) or CP-96,345 (10�8 M), CP96,344 (10�8 M)for 48 hr. B: NT2-N neurons transfected with pNF-�B-luc constructwere incubated with or without DAMGO (10�8 M) or CP-96,345(10�8 M), or CP96,344 (10�8 M) for 48 hr. NF-�B promoter-directedluciferase activity in cell-free lysates were quantitated using a luciferaseassay system and a luminometer. The data are presented as relative lightunit (RLU) per �g protein and are the mean SD of three indepen-dent experiments (**P � 0.01, *P � 0.05, morphine or DAMGO vs.control).

Fig. 5. Effect of morphine on the activation of SP promoter (A) and SPmRNA expression (B) in NT2-N neurons. A: NT2-N neurons trans-fected with pPPT/NO-luc construct were incubated with or withoutmorphine at the concentrations as indicated for 24 hr. The effect ofmorphine on the activation of SP promoter was analyzed by luciferaseactivity. The data are presented as relative light unit (RLU) per �gprotein. B: NT2-N neurons were incubated with or without morphinefor 3 hr. SP real-time RT-PCR was carried out to measure SP mRNAcopy numbers. Data shown are the mean SD of three independentexperiments (**P � 0.01, *P � 0.05, morphine vs. control).

CP96,345, Morphine and NF-�B Promoter 549

NT2-N neurons, we first studied whether SP has theability to enhance NF-�B promoter activation in NT2-Nneurons. SP, when added to the cultures of NT2-Nneurons transfected with the NF-�B promoter-containingplasmid (pNF-�B-luc), activated NF-�B promoter-directed luciferase activity (2.2-, 2.4-, and 1.7-fold ofcontrol at 10�8, 10�7, and 10�6 M, respectively) in thesecells (Fig. 6A). CP-96,345 abolished the SP-induced ac-

tivation of NF-�B promoter (Fig. 6B). We also examinedthe effect of CP-96,345 on SP mRNA expression inNT2-N neurons, and found that CP-96,345 inhibited SPmRNA expression in NT2-N neurons in a concentration-dependent fashion (Fig. 7).

DISCUSSIONThere are biological interactions between opioids

and SP. SP participates in the pathogenesis of opiate with-drawal (Tiong et al., 1992), and SP levels are alteredduring opiate dependence and after withdrawal. SP levelsin the brain are elevated during long-term morphine treat-ment, and this is attenuated after an injection of naloxone(Morley et al., 1980). These findings were confirmed bythe study of Bergstrom et al. (1984), in which it wasdemonstrated that elevated SP levels could also be reversedby passive withdrawal. Endogenous opioid systems may beinvolved in the activity-induced expression of spinaltachykinin peptides and neurokinin receptor (McCarsonand Krause, 1995). Blockade of the SP receptor induces adecrease in the expression of naloxone-precipitated mor-phine withdrawal syndrome in rats, supporting the partic-ipation of endogenous SP in the opiate withdrawal re-sponse (Maldonado et al., 1993). The SP receptorantagonist CP-96,345 inhibited SP production and mor-phine withdrawal response in guinea pigs (Chahl andJohnston, 1993), and it has been reported that there is aloss of the rewarding properties of morphine in mice witha genetic disruption of the SP receptor (Murtra et al.,2000). We documented recently that morphine upregu-lates SP and NK-1R expression in human immune cells(Li et al., 2000). Suarez-Roca and Maixner (1992) re-

Fig. 6. Effect of SP or CP96,345 on the activation of NF-�B promoterin NT2-N neurons. A: NT2-N neurons transfected with pNF-�B-lucconstruct were treated with SP at the concentrations as indicated for24 hr. The effect of SP on the activation of NF-�B was determined byNF-�B promoter-directed luciferase activity. B: NT2-N neuronstransfected with pNF-�B-luc construct were incubated with or with-out SP (10�7 M) or CP-96,345 (10�8 M) for 24 hr. The data arepresented as relative light unit (RLU) per �g protein and are themean SD of three independent experiments (*P � 0.05, SP vs.control).

Fig. 7. Effect of CP-96,345 on SP production in NT2-N neurons.NT2-N neurons were incubated with or without CP-96,345 at indi-cated concentrations for 3 hr. SP mRNA levels were measured byreal-time RT-PCR. Untreated NT2-N neurons were used as thebaseline control, which is defined as 100% (*P � 0.05, CP-96,345 vs.control).

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ported that morphine, through activation of different opi-oid receptor subtypes, has a multiplicity effect on SPrelease from trigeminal nucleus caudalis slices. Repeatedexposure to morphine enhanced SP expression in DRGneurons (Ma et al., 2000). These data strongly support ourhypothesis that the SP-NK-1R pathway is involved bio-logically in morphine-mediated neuroimmunoregulationin the CNS.

NT2-N neurons have morphologic features similarto primary human neurons, and have processes that dif-ferentiate into axons and dendrites (Andrews, 1984).NT2-N neurons also express cytoskeletal proteins, secre-tory markers, and surface markers, which are characteristicof neurons. They also express functional neuropeptides(Guillemain et al., 2000) and functional N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors(Younkin et al., 1993). Undifferentiated human NT2 cellsgrafted into mouse brain differentiated into neuronal andglial cells (Ferrari et al., 2000). NT2-N neurons expressfunctional SP and SP receptors, which are coupled with-chemokine expression (Li et al., 2003). Multiple celltypes (neurons, astrocytes, and oligodendrocytes) in thebrain express NK-1R and may therefore be potential sitesfor the action of SP antagonists. The SP receptor (NK-1R)is highly expressed in areas of the brain implicated inmultiple sclerosis. NT2-N neurons also express mu-opioidreceptors (Table I, Fig. 1A), which is in agreement withthe report showing that NT2-N neurons express inducibledelta- and mu-opioid receptors (Beczkowska et al., 1997).The mu-opioid receptors expressed on NT2-N neuronsare functional biologically, because both naltrexone andCTAP, a selective, competitive mu-opioid receptor an-tagonist, abolished morphine-induced NF-�B promoteractivation (Fig. 2B). Most importantly, we demonstratedthat there is co-expression of NK-1R and MOR inNT-2N neurons (Fig. 1C). Thus, the NT-2N cell line isan appropriate in vitro model for investigation of cellularand molecular interactions between opioids and SP.

NF-�B is a central mediator of induction of host andviral genes in response to various stimuli including cyto-kines and viral products. NF-�B is an inducible transcrip-tion factor present in the CNS (Bakalkin et al., 1993;Kaltschmidt et al., 1993, 1994). Opioids (Hou et al., 1996;Roy et al., 1998) and SP (Lieb et al., 1997; Roy et al.,1998) both have the ability to stimulate NF-�B. Weexamined the effect of morphine on NF-�B promoteractivation in human neuronal cells. Morphine significantlyenhanced NF-�B promoter-directed luciferase activity inNT2-N neurons (Fig. 2). DAMGO, a selective mu-opioidreceptor agonist, also enhanced NF-�B promoter activa-tion (Fig. 3). This increased activation of NF-�B promoterby morphine or DAMGO is abolished by naltrexone (aopioid receptor antagonist) and CTAP, a selective, com-petitive mu-opioid receptor antagonist (Fig. 2 and 3),indicating that NT2-N neurons possess functional mu-opioid receptors that are responsible for morphine-mediated action. Interestingly, CP-96,345, a non-peptideSP receptor antagonist, also inhibited morphine-induced

NF-�B promoter activity (Fig. 4), suggesting that theSP-NK-1R pathway is involved biologically in morphineaction. To investigate further the mechanism(s) responsi-ble for morphine-induced activation of NF-�B promoterin NT2-N neurons, we examined whether morphineenhances SP expression in these cells. Morphine not onlyactivated the SP promoter but also induced SP expressionin NT2-N cells (Fig. 5). The addition of SP to NT2-Nneuron cultures also induced activation of NF-�B pro-moter, which is consistent with the observation that SP atnanomolar concentrations activates NF-�B in human as-trocytes (Lieb et al., 1997). CP-96,345 downregulated SPexpression (Fig. 7) in NT2-N neurons, suggesting a pos-sible mechanism by which CP-96,345 blocked morphine-SP-mediated NF-�B promoter activation. This findingalso supports our previous observation that CP-96,345downregulates SP expression in human immune cells (Laiet al., 2002b). Our data demonstrated that CP-96,345, anon-peptide SP receptor antagonist, antagonizes the ac-tion of morphine on the NF-�B promoter, which sup-ports the notion that SP-NK-1R pathway is indeed in-volved in opioid-mediated biological function within theCNS.

The SP-preferring receptor, NK-1R, has been in-vestigated as a possible pharmaceutical target based on thehypothesis that SP receptor antagonists have potential im-plications for pain relief and depression. SP is involved inthe pathophysiology of depression and is the mechanism ofaction of antidepressant drugs. SP serum levels are elevatedin individuals with major depression (Bondy et al., 2003).Several SP antagonists have been developed, some ofwhich have been tested in human trials. These antagonistsare safe and well-tolerated and have shown some success inpatients with major depression (M. S. Kramer et al., 1998).CP-96,345 was the first non-peptide SP receptor antago-nist developed, and is highly specific to NK-1R. CP-96,345 effectively blocks SP action in a variety of systems(Garret et al., 1991; McLean et al., 1991; Snider et al.,1991; Kennedy et al., 1997). Because CP-96,345 has theability to block morphine action and downregulate ex-pression of endogenous SP, which is implicated in inflam-mation and viral infection, SP receptor antagonists mayprove useful for therapeutical intervention of CNS inflam-matory diseases related to opioids.

Taken together, our data demonstrate that the SP-NK-1R pathway has a critical role in morphine-mediatedregulation of NF-�B, an important transcriptional factorthat controls many inflammatory genes in both peripheraland central nervous systems. Our study also provides ad-ditional evidence that the interaction between opioids andSP is involved biologically in regulating functions of hu-man neuronal cells. This notion is supported strongly byour finding that there is a co-localization of receptors forboth SP and opioids in NT-2N neurons (Fig. 1C). Furtherstudies are required to elucidate the precise mechanism(s)whereby the SP-NK-1R pathway participates in themorphine-mediated inflammatory and immune responsesin the CNS. In addition, future investigations into the

CP96,345, Morphine and NF-�B Promoter 551

interaction of opioids with the SP-NK-1R pathway willprovide insight to the potential application for use of SPreceptor antagonists for therapeutic intervention of opioidabuse.

ACKNOWLEDGMENTSThis work was supported by the National Institutes

of Health (grant DA12815 to W.Z.H. and MH49981 toS.D.D.) and the National Multiple Sclerosis Society (PilotResearch Award to W.Z.H.).

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