of June 21, 2018.This information is current as

Suppression of HIV-1 Expression T Cells and DOR-Dependent+Blood CD4

Receptors (DOR) on Human Peripheral OpioidδImmunofluorescence Detection of

Shahabi and Phillip K. PetersonBurt M. Sharp, Kathy McAllen, Genya Gekker, Nahid A.

http://www.jimmunol.org/content/167/2/1097doi: 10.4049/jimmunol.167.2.1097

2001; 167:1097-1102; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/167/2/1097.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Immunofluorescence Detection ofd Opioid Receptors(DOR) on Human Peripheral Blood CD41 T Cells andDOR-Dependent Suppression of HIV-1 Expression1

Burt M. Sharp, 2* Kathy McAllen,* Genya Gekker, † Nahid A. Shahabi,* and Phillip K. Peterson†

The d opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover,in DOR-transfected Jurkat cells, d opioids have been shown to suppress HIV-1 p24 Ag expression. These observations led us tocharacterize the expression of DORs by human peripheral blood T cells and to determine whether a specific DOR agonist,benzamide,4-{[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]-N,2,{2S[1(S*),2a,5b]}-(9Cl) (SNC-80), cansuppress p24 Ag expression by HIV-1-infected CD41 T cells obtained from normal donors. By immunofluorescence flow cytom-etry, PHA stimulated the expression of DOR from 1.946 0.70 (mean6 SEM) to 20.706 1.88% of the PBMC population by 48 h(p < 0.0001). DOR expression was;40% of both the PHA-stimulated CD41 and CD81 T cell subsets, and virtually all DORs werefound on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA,and then CD41 T cells were purified, pretreated with SNC-80, and infected with HIV-1. In a concentration-dependent manner,SNC-80 inhibited production of p24 Ag. SNC-80 10210 M maximally suppressed (;50%) both lymphocytotropic (HIV-1 MN) andmonocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished theinhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection withHIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD41 T cells significantly suppressedthe expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive tostandard antiviral approaches to HIV-1 infection. The Journal of Immunology,2001, 167: 1097–1102.

T he d opioid agonists modulate the immune function oflymphocytes located in solid lymphoid organs and theperipheral circulation. Some of these effects of syntheticd

opioids appear to emulate the actions of the endogenous opioidpeptides produced by lymphocytes. Indeed, enkephalin peptides(e.g., methionine enkephalin) have been identified in splenic ex-tracts obtained from naive rats, and Con A induced the expressionof preproenkephalin A mRNA by CD41 murine thymocytes invitro (1, 2). Acting through DORs,3 endogenous enkephalins ap-pear to modulate Con A-stimulated murine thymocyte prolifera-tion (3). Pharmacological studies also have shown that syntheticDOR agonists modulate proliferation and IL-2 production byhighly purified murine splenic CD41 and CD81 T cells stimulatedthrough the TCR complex in vitro (4).

DOR ligands have been shown to affect lymphocyte intracellularsignaling. Thus, methionine enkephalin exerted biphasic effects oncAMP levels in human PBL andb-endorphin (antagonized byDOR-selective naltrindole (NTI)), or (D-Ala(2), D-Leu(5)) en-

kephalin (DADLE) enhanced the Con A-induced mobilization ofintracellular free calcium by murine splenic T cells (5, 6). Recentstudies also have shown that DADLE inhibited the anti-CD3-induced phosphorylation of the mitogen-activated protein ki-nases, extracellular signal-related kinases (ERKs) 1 and 2, in mu-rine splenocytes (7).

DOR transcripts have been found in mononuclear cells fromseveral species. To detect DOR mRNA in simian PBMC, humanPBL, and murine splenocytes, several laboratories have used RT-PCR techniques (8–11). DOR transcripts were identified in freshlyobtained simian mononuclear cells and murine splenocytes (8, 11),and expression in murine splenic T cells was enhanced by cellculture in the absence of mitogens, by Con A, and by cross-linkingthe TCR with anti-CD3-e (10–12). Apparently, mitogenic stimu-lation with PHA was required to detect DOR transcripts in humanPBL (9). Thus, low levels of DOR mRNA are present in lympho-cytes found in the systemic circulation of several species, and sub-stantial induction occurs with lymphocyte stimulation. The induc-ible expression of DOR transcripts is consistent with recentobservations on the detection of immunofluorescent DOR proteinon murine splenocytes and T cells obtained after in vivo stimula-tion with a single injection of the superantigen, staphylococcalenterotoxin B (7).

Previous studies have shown that morphine enhanced HIV-1propagation in acutely infected PBMC, apparently throughm-likeopioid receptors (13). Several lines of evidence suggest that DORsmay modulate the expression of HIV-1 by normal human CD41 Tcells. These include the modulatory effects of DORs on T cellfunction, the induction of DOR transcripts in T cells from severalspecies, the recent report of DOR immunofluorescence on murineT cells, and our previous study showing that DOR ligands sup-pressed HIV-1 p24 production by Jurkat cells stably transfectedwith a DOR cDNA (DOR-Ju.1 cells) (14, 15). In the latter study,

*Department of Pharmacology, Health Science Center, University of Tennessee,Memphis, TN 38120; and†Department of Medicine, Hennepin County Medical Cen-ter and University of Minnesota, Minneapolis, MN 55415

Received for publication November 10, 2000. Accepted for publication May 3, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by U.S. Public Health Service Grants DA-04196 (B.M.S.)and DA-09224 (P.K.P.).2 Address correspondence and reprint requests to Dr. Burt M. Sharp, Department ofPharmacology, Health Science Center, University of Tennessee, 874 Union Avenue,Memphis, TN 38163. E-mail address: bsharp@utmem.edu3 Abbreviations used in this paper: DOR,d opioid receptor; ERK, extracellular signal-related kinase; NRS, normal rabbit serum; NTI, naltrindole; SNC-80, benzamide,4-{[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl}-N,2,{2S[1(S*),2a,5b]}-(9Cl).

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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two DOR agonists, deltorphin and benzamide,4-{[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl}-N,2, [2S-[1(S*),2a,5b]}-(9Cl) (SNC-80), concentration dependently inhib-ited the production of p24 Ag, an index of HIV-1 expression;maximal suppression was observed with 10213–1029 M SNC-80(.60% reduction) or 10215–10211 M deltorphin (.50%reduction).

The objectives of the present study were to characterize theexpression of DOR immunofluorescence by human T cells and todetermine whether DOR agonists suppress p24 Ag production bynormal peripheral blood CD41 T cells that have been acutely in-fected with HIV-1. Immunofluorescence flow cytometry was usedto measure DOR expression by resting and mitogen-stimulatedCD41 vs CD81 T cells and to determine whether memory and/ornaive cells express DOR. To evaluate whether activated DORsaffect HIV-1 propagation, normal PBMC were PHA stimulated,and CD41 T cells were purified and then pretreated with SNC-80before infection with either of two strains of HIV-1. Cells werecultured for 3 days, and p24 Ag production was measured in cul-ture supernatants. These studies demonstrated that PHA inducedthe expression of DORs on;40% of both CD41 and CD81 Tcells. Signaling through DORs substantially reduced the propaga-tion of both lymphocytotropic and monocytotropic strains ofHIV-1 in normal human peripheral blood CD41 T cells.

Materials and MethodsCell collection and separation of lymphocytes for DOR detection

Human PBMC were isolated from units of whole blood randomly obtainedfrom healthy male donors at Life Blood (Memphis, TN). Whole blood wasdiluted 1/2 with PBS, layered onto lymphocyte separation medium (ICN,Aurora, OH), and centrifuged (4003 g) for 25 min at 24°C. The middlelayer was removed and again spun, the pellet was resuspended, and theRBC were lysed with alkaline lysis buffer (0.15 M ammonium chloride,0.01 M potassium carbonate, 1 mM Na EDTA). After centrifugation, thepellet was resuspended at 23 106 cells/ml in culture media within smallflasks (RPMI 1640, 5% FBS with penicillin, streptomycin, and glutamine),and either vehicle or PHA, 5mg/ml (Sigma, St. Louis, MO), was added for48 or 96 h of culture.

Immunofluorescence flow cytometry

After cell culture, human PBMC were fixed with 4% paraformaldehyde for10 min at 4°C, washed three times with TBS containing 1% donkey serum(50 mM Tris-HCl, 150 mM NaCl, pH 7.4), and then incubated overnight at4°C with blocking buffer (5% donkey serum in TBS). Cells were incubatedwith both rabbit anti-DOR antisera (1/400 dilution; raised against the N-terminal peptide of DOR (aa 3–17 of the murine receptor); Chemicon In-ternational, Temecula, CA) and mouse anti-human-CD4 or anti-human-CD8 (BD PharMingen, San Diego, CA) for 2 h at 22°C, and then washedand incubated with biotinylated donkey anti-rabbit IgG for 60 min at 22°C.Thereafter, cells were washed extensively, incubated for 10 min at 4°Cwith fluorescein avidin defined calf serum (Vector Labs, Burlingame, CA),again washed, and then resuspended in TBS. To evaluate the expression ofDORs by naive vs memory cells, CyChrome anti-human CD45RA orCyChrome anti-human CD45RO (PharMingen, San Diago, CA) and theirisotype (CyChrome mouse IgG1,k) were used. Cytofluorometric analyses(104 cells per run) were performed using an EPICS XL flow cytometer(Coulter, Palo Alto, CA) equipped with an argon laser, and filtered forexcitation at 488 nm and emission at 526 and 682 nm. For backgroundcontrol, normal rabbit serum (NRS) substituted for the primary antiseraagainst DOR, CyChrome mouse IgG1,k (isotype) for anti-CD4, anti-CD8,anti-CD45RA, or anti-CD45RO. Mean background immunofluorescencelevels at 48 and 96 h ranged from 1.1 to 2.1% (NRS) or 1.7 to 2% (isotype)in unstimulated cells, and from 6.1 to 4.5% (NRS) or 2 to 3.7% (isotype)in PHA-stimulated cells. The background cytofluorometric signal, deter-mined for each quadrant in each experiment, was subtracted from the totalsignal in that quadrant. Immunoneutralization with the N-terminal DOR Agreduced PHA-stimulated anti-DOR immunofluorescence from 20.561.5% of total PBMC to 3.36 0.4% of the population.

Lymphocyte activation and purification before HIV-1 infection

Four healthy, HIV-1-seronegative laboratory personnel served as donors ofvenous blood. From heparinized blood, PBMC were obtained by Ficoll-Hypaque centrifugation using lymphocyte separation medium. PBMCwere then activated for 3 days with 4mg/ml PHA in RPMI supplementedwith 10% heat-inactivated FBS, 5 U/ml IL-2, 2 mM-L-glutamine, 100 U/mlpenicillin, and 100mg/ml streptomycin. CD41 lymphocytes were then iso-lated from the activated PBMC using Dynabeads (Dynal, Lake Success,NY), according to directions supplied by the manufacturer. Briefly, mag-netic polystyrene beads coated with primary mAb to CD4 were incubatedwith PBMC for 45 min at 4°C on an orbital rotator at cell-bead ratio of 1:4.The lymphocytes bound to the beads were separated using a magnet (DynalMPC) and washed four times with PBS containing 2% FBS. After isola-tion, DETACHaBEAD was used to remove the isolated CD41 cells fromDynabeads (1mm/100-ml cell suspension was used to detach positivelyselected lymphocytes from the magnetic beads using a Dynal-MPC mag-net). Isolated CD41 lymphocytes were$98% pure by FACScan analysisand were$98% viable by trypan blue dye exclusion criteria.

Human immunodeficiency virus-1

The HIV-1 MN isolate used in this study was originally recovered from theperipheral blood of an asymptomatic HIV-1-infected patient and preparedas previously described (13). This viral isolate has characteristics mostsuggestive of a T-tropic strain, i.e., it replicates readily in the T cell line H9and in primary activated CD41 lymphocytes, but is not expressed in cul-tures of human microglial cells, which are primary brain macrophages thatare productively infected by M-tropic, but not by T-tropic HIV-1 strains. Inaddition, a monocytotropic HIV-1 isolate, SF162, was tested. This wasprovided by the National Institutes of Health AIDS Research and Refer-ence Reagent Program (National Institute of Allergy and Infectious Dis-eases, Rockville, MD).

Drug treatment and HIV-1 infection of CD4 lymphocytes

Purified activated CD41 lymphocytes were incubated with SNC-80, aDOR-specific agonist, and/or NTI, a DOR-specific antagonist, at varyingconcentrations for indicated time periods before or postinfection with ei-ther strain of HIV-1 at a multiplicity of infection of 0.02. NTI and SNC-80were provided by Drs. P. Portoghese (University of Minnesota) and K.Rice (National Institutes of Health), respectively. After 2 h of incubationwith HIV-1 at 37°C, CD41 lymphocytes were washed three times withPBS and resuspended in culture medium (RPMI 1640, 10% FBS, penicil-lin/streptomycin, 2mg/ml PHA, and 5 U/ml IL-2) containing SNC-80.Three days postinfection, culture supernatants were collected in duplicatefor HIV-1 p24 Ag assay.

HIV-1 p24 Ag assay

HIV-1 p24 Ag levels were measured using an enzyme-linked immunoassay(Abbott Laboratories, Abbott Park, IL), as previously described. A stan-dard dilution curve derived from known amounts of p24 Ag was used toquantify the Ag levels in culture supernatants. The sensitivity of this assayis 30 pg/ml.

Statistical analysis

Data are expressed as means6 SEM. For comparisons of multiple groupmeans, analysis of variance was performed and, except where noted,posthoc testing utilized Scheffe’s test. As indicated, other comparisonswere performed witht tests.

ResultsCytofluorometric analyses were performed on PBMC that hadbeen cultured with PHA or vehicle for 48 or 96 h. Fig. 1A is arepresentative experiment in which the distribution of cells ex-pressing either DOR and/or CD4 or DOR and/or CD8 is shown. By48 h, PHA increased the fraction of DOR1/CD41 and DOR1/CD81 T cells. Both groups were composed of subsets expressinghigher and lower levels of CD41 or CD81. To determine whetherthis was related to differences in cell size, the T cell subsets weregated to measure the fraction of small cells. In both the CD41 andCD81 populations, similar percentages of small cells were foundin the subsets expressing higher or lower levels of these surfacemarkers (fraction of small cells in the lower CD41 subset, 79%61.4 vs higher CD41, 81.8%6 1.9; and lower CD81, 86.5%6 0.6

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vs higher CD81, 87.2%6 0.8). Therefore, regardless of the levelof CD41 or CD81 expression, DOR was predominantly detectedon small cells.

Fig. 1B represents the quantitative data (mean6 SEM) fromfour experiments in which PBMC were stimulated with PHA for48 or 96 h, and the background cytofluorometric signal (NRS orisotypes) was subtracted. Considering the total PBMC population,

2–3% was DOR positive after 48 or 96 h of cell culture in theabsence of mitogen; PHA increased the DOR-positive fraction by10-fold at 48 h (p , 0.001). By 96 h, DOR expression remainedelevated in PHA-stimulated cells (p , 0.001 compared with salinecontrol), but the fraction of DOR1 cells was less than at 48 h (p ,0.01). The fluorescence intensity per cell was determined at 48 h inthe PHA vs vehicle-treated cells. A 3.3-fold increase in DOR flu-orescence intensity per cell was observed (mean channel fluores-cence: 0.1196 0.023 for vehicle and 0.3736 0.067 for PHA,n 54 per group;p 5 0.012).

The fraction of CD41 or CD81 T cells was slightly increased byPHA (data not shown). However, Fig. 1Bshows that PHA greatlyincreased the percentage of these subsets that expressed DOR atboth time intervals (p , 0.001). By 48 h,;40% of both the CD41

and the CD81 subsets were DOR positive, an increase from;6%in the respective vehicle-treated cultures. The fraction of cells ex-pressing DOR in the PHA-stimulated CD41 and CD81 subsetsdeclined significantly by 96 h (p , 0.05). In PHA-stimulated cellcultures, calculations showed that DOR-positive cells were eitherCD41 or CD81 cells.

In three separate experiments, CD45RA and CD45RO wereused to determine whether DOR is expressed by naive or memoryT cells. These studies were performed 48 h after stimulation byPHA, a time when the fraction of CD45RA1 and CD45RO1 cellswas unaffected by PHA (Fig. 2). In contrast, additional experi-ments showed that between 2 and 6 days of PHA diminished theCD45RA1 fraction (45 6 2% to 24 6 2%, respectively;p ,0.0001,n 5 6) and elevated the CD45RO1 fraction during thistime interval (516 1% to 616 1%, respectively;p , 0.0001,n 56). The experiments in Fig. 2 showed that PHA significantly in-creased the fraction of cells positive for both DOR and CD45RAfrom ;2% (vehicle treated) to 15% of the total PBMC. Since allDOR1 cells were T cells and;40% of total PBMC were T cells,

FIGURE 1. Immunofluorescence detection of DOR on CD41 andCD81 human peripheral blood T cells. After 48 or 96 h in culture withPHA (5 mg/ml (P)) or vehicle (saline (S)), PBMC were labeled with rabbitanti-DOR and mouse anti-human CD4 or anti-human CD8; NRS andCyChrome mouse IgG1,k (isotype) were used as controls. Anti-DOR wasdetected with a fluorescein avidin-biotin anti-rabbit Ab complex, whereasanti-CD4 or anti-CD8 was detected with CyChrome (directly conjugated).Cytofluorometric analyses were performed using an EPICS XL flow cy-tometer (Coulter) equipped with an argon laser, and filtered for excitationat 488 nm and emission at 526 and 682 nm. Theupper panels(A) showrepresentative distributions of cells positive for DOR and CD4 or CD8immunofluorescence in the PBMC from one donor cultured with PHA orsaline for 48 h. The value within each quadrant is the percentage of the totalcells analyzed in the experiment that were detected within that quadrant. Inunstimulated PBMC, the fraction of cells positive for DOR/CD4 (rightupper quadrant) was greater in those labeled with primary antisera thanNRS/isotype. PHA increased the fraction of cells expressing both DOR/CD4 and DOR/CD8.B, Shows the quantitative analysis of the fraction ofPBMC positive for the indicated immunofluorescent labels in blood ob-tained from four donors (values are mean6 SEM). Fluorescence detectedin the presence of NRS/isotype was subtracted from the immunofluores-cence signal emitted by the primary Ab. Compared with unstimulated cul-tures (saline), PHA 5 (mg/ml) significantly increased both the fraction ofDOR1 cells in PBMC and the fractions of double-positive cells (i.e.,DOR1CD41) in both T cell subsets at 48 h. The effects of PHA weresustained at 96 h in both T cell subsets. Analysis of differences wasperformed with ANOVA (F5 52.5, p , 0.0001 for DOR1; F 5 51.1,p , 0.0001 for DOR1CD41; F 5 27.1, p , 0.0001 for DOR1CD81).Group comparisons of the following treatments utilized Scheffe’s test: sa-line and PHA at the same time interval (p,p , 0.001), PHA at 48 h and96 h (0,p , 0.05).

FIGURE 2. Immunofluorescence detection of DOR on CD45RA1 andCD45RO1 human peripheral blood T cells. After 48 h in culture with PHA(5 mg/ml) or vehicle (saline), PBMC were labeled with rabbit anti-DORand CyChrome anti-human CD45RA or CyChrome anti-human CD45RO.Cytofluorometric analyses were performed as previously described. Fluo-rescence levels detected in the presence of NRS/isotype were subtractedfrom the immunofluorescence (FL1) signal emitted by the primary Ab. Theresulting values (mean6 SEM) indicate the fraction of PBMC positive forthe indicated immunofluorescent labels in blood obtained from three do-nors. Compared with unstimulated cultures (saline), PHA (5mg/ml) sig-nificantly increased both the fraction of DOR1 cells in PBMC and thefractions of cells double positive for DOR and CD45RA (DOR1RA1) orCD45RO (DOR1RO1). Statistical analyses were made witht tests on eachimmunofluorescently defined treatment pair.p, p , 0.0001.

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calculations demonstrated that;37% of T cells were positive forboth DOR and CD45RA. Similarly, PHA significantly increasedthe fraction of DOR1/CD45RO1 in total PBMC from 4% in thevehicle-treated group to 16%;;41% of T cells were positive forboth Ags. Thus, PHA stimulated similar fractions of bothCD45RA- and CD45RO-positive T cells to express DOR.

Fig. 3 shows that SNC-80, a DOR-specific agonist, dose depen-dently inhibited;50% of the p24 Ag accumulated in the super-natant from CD41 T cells that were cultured for 3 days after in-fection with HIV-1. SNC-80 was effective when cells werepretreated for 1 h before infection, but not 24 h pre- or postinfec-tion. A more comprehensive dose-response study was performedwith CD41 cells from four healthy donors (Fig. 4). In theupperpanelof Fig. 4, p24 Ag production by cells infected with HIV-1MN was maximally suppressed by SNC-80 10210 M; an invertedU-shaped dose-response relationship was observed. Using bloodobtained from the same donors, thelower panelof Fig. 4 showsthat SNC-80 had a similar effect on p24 Ag production by cellsinfected with the SF162 strain of HIV-1.

Fig. 5 shows that the specific DOR antagonist, NTI, markedlyreduced the suppressive effects of SNC-80 on p24 Ag accumula-tion. As expected, in the absence of NTI, SNC-80 10210 M re-duced p24 Ag expression by;50% when supernatant from cellsinfected with either the MN (Fig. 5,upper panel) or SF162 strain(Fig. 5, lower panel) was assayed. At 1029 and 10210 M, NTIeffectively blocked the suppressive effects of SNC-80, whereasNTI alone had no effect.

DiscussionThe present investigations are, to our knowledge, the first to dem-onstrate the presence of DOR immunoreactivity on human periph-

eral blood T cells and to analyze DOR expression on CD41 orCD81 subsets and T cells positive for CD45RA or CD45RO (16–18). A small fraction of the CD41 and CD81 T cells expressedDOR in the absence of mitogenic stimulation and, by 48 h, PHAstimulated expression by;40% of these subsets. After PHA stim-ulation, the DOR-positive cells appeared in the T cell fraction, andsimilar fractions of both CD45RA1 and CD45RO1 cells had be-come DOR1. Subnanomolar concentrations of the DOR agonist,SNC-80, were shown to suppress the production of p24 Ag bylymphocytotropic and monocytotropic strains of HIV-1. The spec-ificity of this suppression was evident in its concentration depen-dence and reversal by the DOR antagonist, NTI.

FIGURE 3. Effect of pre- and posttreatment with SNC-80 on p24 Agexpression by human peripheral blood CD41 T cells. Purified activatedCD41 lymphocytes were incubated with SNC-80, a DOR-specific agonist,at varying concentrations for indicated time periods before or postinfectionwith the MN strain of HIV-1 at a multiplicity of infection of 0.02. After 2-hincubation with HIV-1 at 37°C, CD41 lymphocytes were cultured withPHA and the indicated concentrations of SNC-80. Three days postinfec-tion, culture supernatants were collected in duplicate for HIV-1 p24 Agassay. Four subjects were studied, and the data were expressed as the meanpercentage of inhibition of p24 Ag expression (p24 Ag levels were6682.56 1536.5 pg/ml in vehicle control) for the group. SNC-80 waseffective when a relatively brief (1-h) preincubation interval was tested.Analysis of differences was performed with ANOVA (F 5 37.7, p ,0.0001), followed by comparisons between concentrations of SNC-80 (pp,p , 0.0001 for 1 h preinfection with SNC-80 10210 M vs 1026 M; p, p ,0.05 for SNC-80 1028 M vs 1026 M) by Scheffe’s test.

FIGURE 4. Effect of SNC-80 on p24 Ag expression by human periph-eral blood CD41 T cells infected with HIV-1 MN or SF162. Purified ac-tivated CD41 lymphocytes were incubated with the designated concentra-tions of SNC-80 for 1 h before infection with the MN or SF162 strains ofHIV-1 at a multiplicity of infection of 0.02. After 2-h incubation withHIV-1 at 37°C, CD41 lymphocytes were cultured for 3 days with PHA andSNC-80. Four subjects were studied, and the data are expressed as themean percentage of inhibition of p24 Ag expression (in the vehicle controlgroups, p24 Ag levels were 6,6836 1,537 pg/ml and 16,1746 460 pg/mlfor the MN and SF162 strains, respectively) for the group. SNC-80 signifi-cantly suppressed p24 Ag expression by both strains of HIV-1. Statisticalanalyses were made by ANOVA (for HIV-1 MN and SF162, respectively:F 5 37.1,p , 0.0001;F 5 14.1,p , 0.0001) and between group com-parisons of SNC-80 concentrations were performed with Fisher’s least sig-nificant difference (pp,p , 0.0001 for SNC-80 10210 M vs 1026 or 10214

M; p, p , 0.01 for SNC-80 1028 M vs 1026 M or 10212 M vs 10214 M).

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In several species, mononuclear cells contain DOR transcripts,although very few studies have provided substantial evidence forthe detection of DOR itself (19, 20). DOR transcript levels areuniformly low in freshly obtained mononuclear cells, requiringreverse transcription with PCR and a relatively large number ofamplification cycles for detection in simian and human PBMC andin murine splenocytes (8–11). In murine splenocytes, recent stud-ies have shown that stimulation in vivo or in vitro significantlyincreased the level of DOR transcripts. This was evident after invivo treatment with a single injection of staphylococcal entero-toxin B, which produced a 2-fold increase by 8 h (7). In vitro, ConA and anti-CD3-e had similar effects, stimulating transcript ex-pression by T cells, including CD41 and CD81 subsets (10–12).T cell DOR transcripts increased from,1 copy/cell to 22 and 42copies/cell after 24 and 48 h of anti-CD3-e (12). Moreover, ex-periments with actinomycin D implied that transcriptional activa-tion mediates the anti-CD3-e-driven increase (12).

By 48 h in culture, PHA stimulated a 5- to 7-fold increase inDOR immunoreactivity in the CD41 and CD81 T cell subsets,respectively. Accounting for;40% of these T cell subsets, thislevel of DOR expression declined by 96 h in both T cell subsets.After PHA stimulation, these DOR1 T cells represented virtuallyall of the DOR1 cells. Since the fraction of CD45RA1 andCD45RO1 cells was unaffected by PHA at 48 h (in contrast to 6days), it is likely that individual T cells had not changed theirstatus with respect to the expression of these Ags at this timeinterval. This suggests that similar percentages of T cells positivefor CD45RA or CD45RO were induced to express DOR by PHA.Thus, these studies provide evidence for the activation-associatedexpression of DOR on both naive and memory T cells in responseto PHA (16–18).

DORs are seven-transmembrane G protein-coupled receptorsthat were originally cloned from neural cells and have been studiedextensively in the nervous system (21). In the immune system,DORs have been shown to modulate Con A-stimulated calciummobilization and anti-CD3-e-induced phosphorylation of the ex-tracellular-regulated kinases, ERK1, 2, in splenocytes (6, 7). Froma functional perspective, DOR agonists, produced endogenously,are known to suppress Con A-stimulated thymocyte proliferation(3). In addition, synthetic DOR agonists suppressed the anti-CD3-e-driven proliferation of highly purified CD41 and CD81 splenicT cells, and the production of IL-2 (4). Our previous experimentsdemonstrated that DOR ligands suppressed HIV-1 p24 Ag produc-tion by Jurkat cells stably transfected with a DOR cDNA (DOR-Ju.1 cells) (14, 15). Two DOR agonists, deltorphin and SNC-80,concentration dependently inhibited the production of p24 Ag, andmaximal suppression was observed with 10213–1029 M SNC-80(.60% reduction) or 10215–10211 M deltorphin (.50% reduc-tion). At higher concentrations, neither agonist was effective. Al-though not well understood, this U-shaped dose-response functionmay reflect the interaction of intracellular signaling cascades,which varies with agonist concentration.

In the present investigations, we observed a similar concentra-tion dependence and degree of suppression of p24 Ag productionby activated human peripheral blood T cells inoculated with HIV-1MN or SF162. At a concentration of 10210 M, SNC-80 was max-imally effective, whereas at 1027 M, suppression was no longerevident. In both strains of HIV-1, maximal suppression was;50%. This degree of suppression coheres with our finding that;40% of CD41 T cells express DOR within the time interval (48h) used to activate the PBMC before inoculation with HIV-1. Ifthese observations are causally related, it would suggest that DORagonists are able to suppress p24 Ag expression to a much greaterdegree in the CD41 T cell subpopulation that are DOR1.

These investigations indicate that a relatively brief preincuba-tion with SNC-80 was required; in contrast, a 24-h pre- or post-treatment with SNC-80 was ineffective. Preincubation is requiredfor the effects of DOR agonists on other immune functions. Forexample, suppression of the anti-CD3-e-stimulated proliferation ofhighly purified murine splenic T cell subsets and IL-2 productionwas optimal when lymphocytes were treated with DOR agonistsfor 1 h before culture with anti-CD3-e (4). In addition, for thesuppressive effect of DOR agonists on anti-CD3-e-induced ERKphosphorylation, pretreatment was necessary (7). These observa-tions suggest that preincubation with a DOR agonist modulates theactivity of intracellular pathways involved in TCR-dependent sig-naling. This may reflect the effects of DORs on the tyrosine phos-phorylation of intracellular signals known to regulate cell growthand proliferation (22). Somatostatin, a ubiquitous extracellular sig-nal peptide that activates Gi protein-coupled receptors, also hasbeen shown to inhibit the growth and proliferation of a variety of

FIGURE 5. NTI prevents the suppressive effect of SNC-80 on p24 Agexpression by human peripheral blood CD41 T cells infected with HIV-1MN or SF162. Purified activated CD41 lymphocytes were incubated for 30min with the designated concentrations of NTI and then SNC-80 10210 Mfor 1 h before infection with the MN (A) or SF162 (B) strains of HIV-1 ata multiplicity of infection of 0.02. After 2-h incubation with HIV-1 at37°C, CD41 lymphocytes were cultured for 3 days with PHA and NTI1SNC-80. Three subjects were studied, and the data are expressed as meanpercentage of inhibition p24 Ag expression (in the vehicle control groups,p24 Ag levels were 16,5106 946 pg/ml and 19,3806 1,259 pg/ml for theMN and SF162 strains, respectively) for the group. SNC-80 significantlysuppressed p24 Ag expression by both strains of HIV-1, and NTI preventedthis. Statistical analyses were made with ANOVA and comparisons be-tween vehicle control and SNC-80 with Scheffe’s test (p,p , 0.01).ANOVA resulted in the following values for HIV-1 MN and SF162, re-spectively:F 5 10.2,p , 0.005;F 5 33.3,p , 0.001.

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cell lineages (23). Indeed, somatostatin-enhanced phosphotyrosinephosphatase activity, which inactivates growth factor receptor ki-nases, has been implicated in its action (23–25). Thus, phospho-tyrosine phosphatases may be involved in mediating the intracel-lular action of DORs, which, like somatostatin receptors, arecommonly coupled to Gi proteins.

In previous studies, preincubation was also required for the ef-fects of morphine on HIV-1 p24 Ag expression by human PBMCcocultures (13). In these studies, normal donor PBMC were incu-bated with morphine sulfate and then activated with PHA beforethe addition of HIV-1-infected PBMC. The morphine-enhancedexpression of HIV-1 p24 Ag was both stereoselective and revers-ible by m opioid receptor-specific antagonists (13). Although thisreport contrasts with the present observations and with our reporton the inhibitory effects of DOR agonists on HIV-1-infected DOR-Ju.1 cells, there are important differences in experimental design.The ligand-receptor interactions of morphine and SNC-80 dependon two different opioid receptor subtypes that can couple to dif-ferent intracellular effectors, depending on cell type. In addition,the design of the present studies suggests that the effects of DORligands were directly on T cells, whereas it is not possible to pre-cisely identify the cellular target in the previous experiments withPBMC cocultures. Thus, the contrasting effects ofm vs d opioidson HIV-1 p24 Ag expression are likely to reflect differences in thereceptor-specific intracellular effectors that are modulated by thesecompounds.

Both the requirement for preincubation with SNC-80 and thesuppressive effects of DOR agonists on lymphocytotropic andmonocytotropic strains of HIV-1 suggest that early cellular eventsinvolved in viral replication may be affected by activating DORs.Viral entry is a good candidate since HIV-1 gains access to CD41

T cells by interacting with both CD4 molecules and chemokinecoreceptors (26). In support of this, a recent study reported thatDOR agonists inhibited chemokine-induced chemotaxis by induc-ing the phosphorylation and desensitization of the chemokine re-ceptor (27).

In summary, these studies have shown that a large fraction ofCD41 and CD81 T cells can be induced to express DORs bymechanisms dependent on activation through the TCR. DORs,which modulate intracellular signaling pathways affecting T cellfunction, can suppress the expression of p24 Ag by HIV-1-infectedCD41 T cells. These findings suggest that adjunctived opioidimmunotherapy may augment the antiviral therapy of HIV-1infection.

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