The δ opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover, in DOR-transfected Jurkat cells, δ opioids have been shown to suppress HIV-1 p24 Ag expression. These observations led us to characterize 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,−,{2S[1(S*),2α,5β]}-(9Cl) (SNC-80), can suppress p24 Ag expression by HIV-1-infected CD4+ T cells obtained from normal donors. By immunofluorescence flow cytometry, PHA stimulated the expression of DOR from 1.94 ± 0.70 (mean ± SEM) to 20.70 ± 1.88% of the PBMC population by 48 h (p < 0.0001). DOR expression was ∼40% of both the PHA-stimulated CD4+ and CD8+ T cell subsets, and virtually all DORs were found on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA, and then CD4+ 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 10−10 M maximally suppressed (∼50%) both lymphocytotropic (HIV-1 MN) and monocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished the inhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection with HIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD4+ T cells significantly suppressed the expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.

The δ opioid agonists modulate the immune function of lymphocytes located in solid lymphoid organs and the peripheral circulation. Some of these effects of synthetic δ opioids appear to emulate the actions of the endogenous opioid peptides produced by lymphocytes. Indeed, enkephalin peptides (e.g., methionine enkephalin) have been identified in splenic extracts obtained from naive rats, and Con A induced the expression of preproenkephalin A mRNA by CD4+ murine thymocytes in vitro (1, 2). Acting through DORs,3 endogenous enkephalins appear to modulate Con A-stimulated murine thymocyte proliferation (3). Pharmacological studies also have shown that synthetic DOR agonists modulate proliferation and IL-2 production by highly purified murine splenic CD4+ and CD8+ T cells stimulated through the TCR complex in vitro (4).

DOR ligands have been shown to affect lymphocyte intracellular signaling. Thus, methionine enkephalin exerted biphasic effects on cAMP levels in human PBL and β-endorphin (antagonized by DOR-selective naltrindole (NTI)), or (d-Ala(2), d-Leu(5)) enkephalin (DADLE) enhanced the Con A-induced mobilization of intracellular free calcium by murine splenic T cells (5, 6). Recent studies also have shown that DADLE inhibited the anti-CD3-induced phosphorylation of the mitogen-activated protein kinases, extracellular signal-related kinases (ERKs) 1 and 2, in murine splenocytes (7).

DOR transcripts have been found in mononuclear cells from several species. To detect DOR mRNA in simian PBMC, human PBL, and murine splenocytes, several laboratories have used RT-PCR techniques (8, 9, 10, 11). DOR transcripts were identified in freshly obtained simian mononuclear cells and murine splenocytes (8, 11), and expression in murine splenic T cells was enhanced by cell culture in the absence of mitogens, by Con A, and by cross-linking the TCR with anti-CD3-ε (10, 11, 12). Apparently, mitogenic stimulation with PHA was required to detect DOR transcripts in human PBL (9). Thus, low levels of DOR mRNA are present in lymphocytes found in the systemic circulation of several species, and substantial induction occurs with lymphocyte stimulation. The inducible expression of DOR transcripts is consistent with recent observations on the detection of immunofluorescent DOR protein on murine splenocytes and T cells obtained after in vivo stimulation with a single injection of the superantigen, staphylococcal enterotoxin B (7).

Previous studies have shown that morphine enhanced HIV-1 propagation in acutely infected PBMC, apparently through μ-like opioid receptors (13). Several lines of evidence suggest that DORs may modulate the expression of HIV-1 by normal human CD4+ T cells. These include the modulatory effects of DORs on T cell function, the induction of DOR transcripts in T cells from several species, the recent report of DOR immunofluorescence on murine T cells, and our previous study showing that DOR ligands suppressed HIV-1 p24 production by Jurkat cells stably transfected with a DOR cDNA (DOR-Ju.1 cells) (14, 15). In the latter study, two DOR agonists, deltorphin and benzamide,4-{[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl}-N,−, [2S-[1(S*),2α,5β]}-(9Cl) (SNC-80), concentration dependently inhibited the production of p24 Ag, an index of HIV-1 expression; maximal suppression was observed with 10−13–10−9 M SNC-80 (>60% reduction) or 10−15–10−11 M deltorphin (>50% reduction).

The objectives of the present study were to characterize the expression of DOR immunofluorescence by human T cells and to determine whether DOR agonists suppress p24 Ag production by normal peripheral blood CD4+ T cells that have been acutely infected with HIV-1. Immunofluorescence flow cytometry was used to measure DOR expression by resting and mitogen-stimulated CD4+ vs CD8+ T cells and to determine whether memory and/or naive cells express DOR. To evaluate whether activated DORs affect HIV-1 propagation, normal PBMC were PHA stimulated, and CD4+ T cells were purified and then pretreated with SNC-80 before infection with either of two strains of HIV-1. Cells were cultured for 3 days, and p24 Ag production was measured in culture supernatants. These studies demonstrated that PHA induced the expression of DORs on ∼40% of both CD4+ and CD8+ T cells. Signaling through DORs substantially reduced the propagation of both lymphocytotropic and monocytotropic strains of HIV-1 in normal human peripheral blood CD4+ T cells.

Human PBMC were isolated from units of whole blood randomly obtained from healthy male donors at Life Blood (Memphis, TN). Whole blood was diluted 1/2 with PBS, layered onto lymphocyte separation medium (ICN, Aurora, OH), and centrifuged (400 × g) for 25 min at 24°C. The middle layer was removed and again spun, the pellet was resuspended, and the RBC were lysed with alkaline lysis buffer (0.15 M ammonium chloride, 0.01 M potassium carbonate, 1 mM Na EDTA). After centrifugation, the pellet was resuspended at 2 × 106 cells/ml in culture media within small flasks (RPMI 1640, 5% FBS with penicillin, streptomycin, and glutamine), and either vehicle or PHA, 5 μg/ml (Sigma, St. Louis, MO), was added for 48 or 96 h of culture.

After cell culture, human PBMC were fixed with 4% paraformaldehyde for 10 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 at 4°C with blocking buffer (5% donkey serum in TBS). Cells were incubated with both rabbit anti-DOR antisera (1/400 dilution; raised against the N-terminal peptide of DOR (aa 3–17 of the murine receptor); Chemicon International, Temecula, CA) and mouse anti-human-CD4 or anti-human-CD8 (BD PharMingen, San Diego, CA) for 2 h at 22°C, and then washed and incubated with biotinylated donkey anti-rabbit IgG for 60 min at 22°C. Thereafter, cells were washed extensively, incubated for 10 min at 4°C with fluorescein avidin defined calf serum (Vector Labs, Burlingame, CA), again washed, and then resuspended in TBS. To evaluate the expression of DORs by naive vs memory cells, CyChrome anti-human CD45RA or CyChrome anti-human CD45RO (PharMingen, San Diago, CA) and their isotype (CyChrome mouse IgG1, κ) 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 for excitation at 488 nm and emission at 526 and 682 nm. For background control, normal rabbit serum (NRS) substituted for the primary antisera against DOR, CyChrome mouse IgG1, κ (isotype) for anti-CD4, anti-CD8, anti-CD45RA, or anti-CD45RO. Mean background immunofluorescence levels 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, determined for each quadrant in each experiment, was subtracted from the total signal in that quadrant. Immunoneutralization with the N-terminal DOR Ag reduced PHA-stimulated anti-DOR immunofluorescence from 20.5 ± 1.5% of total PBMC to 3.3 ± 0.4% of the population.

Four healthy, HIV-1-seronegative laboratory personnel served as donors of venous blood. From heparinized blood, PBMC were obtained by Ficoll-Hypaque centrifugation using lymphocyte separation medium. PBMC were then activated for 3 days with 4 μg/ml PHA in RPMI supplemented with 10% heat-inactivated FBS, 5 U/ml IL-2, 2 mM-l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. CD4+ lymphocytes were then isolated from the activated PBMC using Dynabeads (Dynal, Lake Success, NY), according to directions supplied by the manufacturer. Briefly, magnetic polystyrene beads coated with primary mAb to CD4 were incubated with 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 (Dynal MPC) and washed four times with PBS containing 2% FBS. After isolation, DETACHaBEAD was used to remove the isolated CD4+ cells from Dynabeads (1 μm/100-μl cell suspension was used to detach positively selected lymphocytes from the magnetic beads using a Dynal-MPC magnet). Isolated CD4+ lymphocytes were ≥98% pure by FACScan analysis and were ≥98% viable by trypan blue dye exclusion criteria.

The HIV-1 MN isolate used in this study was originally recovered from the peripheral blood of an asymptomatic HIV-1-infected patient and prepared as previously described (13). This viral isolate has characteristics most suggestive of a T-tropic strain, i.e., it replicates readily in the T cell line H9 and in primary activated CD4+ lymphocytes, but is not expressed in cultures of human microglial cells, which are primary brain macrophages that are productively infected by M-tropic, but not by T-tropic HIV-1 strains. In addition, a monocytotropic HIV-1 isolate, SF162, was tested. This was provided by the National Institutes of Health AIDS Research and Reference Reagent Program (National Institute of Allergy and Infectious Diseases, Rockville, MD).

Purified activated CD4+ lymphocytes were incubated with SNC-80, a DOR-specific agonist, and/or NTI, a DOR-specific antagonist, at varying concentrations for indicated time periods before or postinfection with either strain of HIV-1 at a multiplicity of infection of 0.02. NTI and SNC-80 were provided by Drs. P. Portoghese (University of Minnesota) and K. Rice (National Institutes of Health), respectively. After 2 h of incubation with HIV-1 at 37°C, CD4+ lymphocytes were washed three times with PBS and resuspended in culture medium (RPMI 1640, 10% FBS, penicillin/streptomycin, 2 μg/ml PHA, and 5 U/ml IL-2) containing SNC-80. Three days postinfection, culture supernatants were collected in duplicate for 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 standard dilution curve derived from known amounts of p24 Ag was used to quantify the Ag levels in culture supernatants. The sensitivity of this assay is 30 pg/ml.

Data are expressed as means ± SEM. For comparisons of multiple group means, analysis of variance was performed and, except where noted, posthoc testing utilized Scheffé’s test. As indicated, other comparisons were performed with t tests.

Cytofluorometric analyses were performed on PBMC that had been cultured with PHA or vehicle for 48 or 96 h. Fig. 1 A is a representative experiment in which the distribution of cells expressing either DOR and/or CD4 or DOR and/or CD8 is shown. By 48 h, PHA increased the fraction of DOR+/CD4+ and DOR+/CD8+ T cells. Both groups were composed of subsets expressing higher and lower levels of CD4+ or CD8+. To determine whether this was related to differences in cell size, the T cell subsets were gated to measure the fraction of small cells. In both the CD4+ and CD8+ populations, similar percentages of small cells were found in the subsets expressing higher or lower levels of these surface markers (fraction of small cells in the lower CD4+ subset, 79% ± 1.4 vs higher CD4+, 81.8% ± 1.9; and lower CD8+, 86.5% ± 0.6 vs higher CD8+, 87.2% ± 0.8). Therefore, regardless of the level of CD4+ or CD8+ expression, DOR was predominantly detected on small cells.

FIGURE 1.

Immunofluorescence detection of DOR on CD4+ and CD8+ human peripheral blood T cells. After 48 or 96 h in culture with PHA (5 μg/ml (P)) or vehicle (saline (S)), PBMC were labeled with rabbit anti-DOR and mouse anti-human CD4 or anti-human CD8; NRS and CyChrome mouse IgG1, κ (isotype) were used as controls. Anti-DOR was detected with a fluorescein avidin-biotin anti-rabbit Ab complex, whereas anti-CD4 or anti-CD8 was detected with CyChrome (directly conjugated). Cytofluorometric analyses were performed using an EPICS XL flow cytometer (Coulter) equipped with an argon laser, and filtered for excitation at 488 nm and emission at 526 and 682 nm. The upper panels (A) show representative distributions of cells positive for DOR and CD4 or CD8 immunofluorescence in the PBMC from one donor cultured with PHA or saline for 48 h. The value within each quadrant is the percentage of the total cells analyzed in the experiment that were detected within that quadrant. In unstimulated PBMC, the fraction of cells positive for DOR/CD4 (right upper quadrant) was greater in those labeled with primary antisera than NRS/isotype. PHA increased the fraction of cells expressing both DOR/CD4 and DOR/CD8. B, Shows the quantitative analysis of the fraction of PBMC positive for the indicated immunofluorescent labels in blood obtained from four donors (values are mean ± SEM). Fluorescence detected in the presence of NRS/isotype was subtracted from the immunofluorescence signal emitted by the primary Ab. Compared with unstimulated cultures (saline), PHA 5 (μg/ml) significantly increased both the fraction of DOR+ cells in PBMC and the fractions of double-positive cells (i.e., DOR+CD4+) in both T cell subsets at 48 h. The effects of PHA were sustained at 96 h in both T cell subsets. Analysis of differences was performed with ANOVA (F = 52.5, p < 0.0001 for DOR+; F = 51.1, p < 0.0001 for DOR+CD4+; F = 27.1, p < 0.0001 for DOR+CD8+). Group comparisons of the following treatments utilized Scheffé’s test: saline and PHA at the same time interval (∗, p < 0.001), PHA at 48 h and 96 h (0, p < 0.05).

FIGURE 1.

Immunofluorescence detection of DOR on CD4+ and CD8+ human peripheral blood T cells. After 48 or 96 h in culture with PHA (5 μg/ml (P)) or vehicle (saline (S)), PBMC were labeled with rabbit anti-DOR and mouse anti-human CD4 or anti-human CD8; NRS and CyChrome mouse IgG1, κ (isotype) were used as controls. Anti-DOR was detected with a fluorescein avidin-biotin anti-rabbit Ab complex, whereas anti-CD4 or anti-CD8 was detected with CyChrome (directly conjugated). Cytofluorometric analyses were performed using an EPICS XL flow cytometer (Coulter) equipped with an argon laser, and filtered for excitation at 488 nm and emission at 526 and 682 nm. The upper panels (A) show representative distributions of cells positive for DOR and CD4 or CD8 immunofluorescence in the PBMC from one donor cultured with PHA or saline for 48 h. The value within each quadrant is the percentage of the total cells analyzed in the experiment that were detected within that quadrant. In unstimulated PBMC, the fraction of cells positive for DOR/CD4 (right upper quadrant) was greater in those labeled with primary antisera than NRS/isotype. PHA increased the fraction of cells expressing both DOR/CD4 and DOR/CD8. B, Shows the quantitative analysis of the fraction of PBMC positive for the indicated immunofluorescent labels in blood obtained from four donors (values are mean ± SEM). Fluorescence detected in the presence of NRS/isotype was subtracted from the immunofluorescence signal emitted by the primary Ab. Compared with unstimulated cultures (saline), PHA 5 (μg/ml) significantly increased both the fraction of DOR+ cells in PBMC and the fractions of double-positive cells (i.e., DOR+CD4+) in both T cell subsets at 48 h. The effects of PHA were sustained at 96 h in both T cell subsets. Analysis of differences was performed with ANOVA (F = 52.5, p < 0.0001 for DOR+; F = 51.1, p < 0.0001 for DOR+CD4+; F = 27.1, p < 0.0001 for DOR+CD8+). Group comparisons of the following treatments utilized Scheffé’s test: saline and PHA at the same time interval (∗, p < 0.001), PHA at 48 h and 96 h (0, p < 0.05).

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Fig. 1 B represents the quantitative data (mean ± SEM) from four experiments in which PBMC were stimulated with PHA for 48 or 96 h, and the background cytofluorometric signal (NRS or isotypes) was subtracted. Considering the total PBMC population, 2–3% was DOR positive after 48 or 96 h of cell culture in the absence of mitogen; PHA increased the DOR-positive fraction by 10-fold at 48 h (p < 0.001). By 96 h, DOR expression remained elevated in PHA-stimulated cells (p < 0.001 compared with saline control), but the fraction of DOR+ cells was less than at 48 h (p < 0.01). The fluorescence intensity per cell was determined at 48 h in the PHA vs vehicle-treated cells. A 3.3-fold increase in DOR fluorescence intensity per cell was observed (mean channel fluorescence: 0.119 ± 0.023 for vehicle and 0.373 ± 0.067 for PHA, n = 4 per group; p = 0.012).

The fraction of CD4+ or CD8+ T cells was slightly increased by PHA (data not shown). However, Fig. 1 B shows that PHA greatly increased the percentage of these subsets that expressed DOR at both time intervals (p < 0.001). By 48 h, ∼40% of both the CD4+ and the CD8+ subsets were DOR positive, an increase from ∼6% in the respective vehicle-treated cultures. The fraction of cells expressing DOR in the PHA-stimulated CD4+ and CD8+ subsets declined significantly by 96 h (p < 0.05). In PHA-stimulated cell cultures, calculations showed that DOR-positive cells were either CD4+ or CD8+ cells.

In three separate experiments, CD45RA and CD45RO were used to determine whether DOR is expressed by naive or memory T cells. These studies were performed 48 h after stimulation by PHA, a time when the fraction of CD45RA+ and CD45RO+ cells was unaffected by PHA (Fig. 2). In contrast, additional experiments showed that between 2 and 6 days of PHA diminished the CD45RA+ fraction (45 ± 2% to 24 ± 2%, respectively; p < 0.0001, n = 6) and elevated the CD45RO+ fraction during this time interval (51 ± 1% to 61 ± 1%, respectively; p < 0.0001, n = 6). The experiments in Fig. 2 showed that PHA significantly increased the fraction of cells positive for both DOR and CD45RA from ∼2% (vehicle treated) to 15% of the total PBMC. Since all DOR+ cells were T cells and ∼40% of total PBMC were T cells, calculations demonstrated that ∼37% of T cells were positive for both DOR and CD45RA. Similarly, PHA significantly increased the fraction of DOR+/CD45RO+ in total PBMC from 4% in the vehicle-treated group to 16%; ∼41% of T cells were positive for both Ags. Thus, PHA stimulated similar fractions of both CD45RA- and CD45RO-positive T cells to express DOR.

FIGURE 2.

Immunofluorescence detection of DOR on CD45RA+ and CD45RO+ human peripheral blood T cells. After 48 h in culture with PHA (5 μg/ml) or vehicle (saline), PBMC were labeled with rabbit anti-DOR and CyChrome anti-human CD45RA or CyChrome anti-human CD45RO. Cytofluorometric analyses were performed as previously described. Fluorescence levels detected in the presence of NRS/isotype were subtracted from the immunofluorescence (FL1) signal emitted by the primary Ab. The resulting values (mean ± SEM) indicate the fraction of PBMC positive for the indicated immunofluorescent labels in blood obtained from three donors. Compared with unstimulated cultures (saline), PHA (5 μg/ml) significantly increased both the fraction of DOR+ cells in PBMC and the fractions of cells double positive for DOR and CD45RA (DOR+RA+) or CD45RO (DOR+RO+). Statistical analyses were made with t tests on each immunofluorescently defined treatment pair. ∗, p < 0.0001.

FIGURE 2.

Immunofluorescence detection of DOR on CD45RA+ and CD45RO+ human peripheral blood T cells. After 48 h in culture with PHA (5 μg/ml) or vehicle (saline), PBMC were labeled with rabbit anti-DOR and CyChrome anti-human CD45RA or CyChrome anti-human CD45RO. Cytofluorometric analyses were performed as previously described. Fluorescence levels detected in the presence of NRS/isotype were subtracted from the immunofluorescence (FL1) signal emitted by the primary Ab. The resulting values (mean ± SEM) indicate the fraction of PBMC positive for the indicated immunofluorescent labels in blood obtained from three donors. Compared with unstimulated cultures (saline), PHA (5 μg/ml) significantly increased both the fraction of DOR+ cells in PBMC and the fractions of cells double positive for DOR and CD45RA (DOR+RA+) or CD45RO (DOR+RO+). Statistical analyses were made with t tests on each immunofluorescently defined treatment pair. ∗, p < 0.0001.

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Fig. 3 shows that SNC-80, a DOR-specific agonist, dose dependently inhibited ∼50% of the p24 Ag accumulated in the supernatant from CD4+ T cells that were cultured for 3 days after infection with HIV-1. SNC-80 was effective when cells were pretreated for 1 h before infection, but not 24 h pre- or postinfection. A more comprehensive dose-response study was performed with CD4+ cells from four healthy donors (Fig. 4). In the upper panel of Fig. 4, p24 Ag production by cells infected with HIV-1 MN was maximally suppressed by SNC-80 10−10 M; an inverted U-shaped dose-response relationship was observed. Using blood obtained from the same donors, the lower panel of Fig. 4 shows that SNC-80 had a similar effect on p24 Ag production by cells infected with the SF162 strain of HIV-1.

FIGURE 3.

Effect of pre- and posttreatment with SNC-80 on p24 Ag expression by human peripheral blood CD4+ T cells. Purified activated CD4+ lymphocytes were incubated with SNC-80, a DOR-specific agonist, at varying concentrations for indicated time periods before or postinfection with the MN strain of HIV-1 at a multiplicity of infection of 0.02. After 2-h incubation with HIV-1 at 37°C, CD4+ lymphocytes were cultured with PHA and the indicated concentrations of SNC-80. Three days postinfection, culture supernatants were collected in duplicate for HIV-1 p24 Ag assay. Four subjects were studied, and the data were expressed as the mean percentage of inhibition of p24 Ag expression (p24 Ag levels were 6682.5 ± 1536.5 pg/ml in vehicle control) for the group. SNC-80 was effective when a relatively brief (1-h) preincubation interval was tested. Analysis of differences was performed with ANOVA (F = 37.7, p < 0.0001), followed by comparisons between concentrations of SNC-80 (∗∗, p < 0.0001 for 1 h preinfection with SNC-80 10−10 M vs 10−6 M; ∗, p < 0.05 for SNC-80 10−8 M vs 10−6 M) by Scheffé’s test.

FIGURE 3.

Effect of pre- and posttreatment with SNC-80 on p24 Ag expression by human peripheral blood CD4+ T cells. Purified activated CD4+ lymphocytes were incubated with SNC-80, a DOR-specific agonist, at varying concentrations for indicated time periods before or postinfection with the MN strain of HIV-1 at a multiplicity of infection of 0.02. After 2-h incubation with HIV-1 at 37°C, CD4+ lymphocytes were cultured with PHA and the indicated concentrations of SNC-80. Three days postinfection, culture supernatants were collected in duplicate for HIV-1 p24 Ag assay. Four subjects were studied, and the data were expressed as the mean percentage of inhibition of p24 Ag expression (p24 Ag levels were 6682.5 ± 1536.5 pg/ml in vehicle control) for the group. SNC-80 was effective when a relatively brief (1-h) preincubation interval was tested. Analysis of differences was performed with ANOVA (F = 37.7, p < 0.0001), followed by comparisons between concentrations of SNC-80 (∗∗, p < 0.0001 for 1 h preinfection with SNC-80 10−10 M vs 10−6 M; ∗, p < 0.05 for SNC-80 10−8 M vs 10−6 M) by Scheffé’s test.

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FIGURE 4.

Effect of SNC-80 on p24 Ag expression by human peripheral blood CD4+ T cells infected with HIV-1 MN or SF162. Purified activated CD4+ lymphocytes were incubated with the designated concentrations of SNC-80 for 1 h before infection with the MN or SF162 strains of HIV-1 at a multiplicity of infection of 0.02. After 2-h incubation with HIV-1 at 37°C, CD4+ lymphocytes were cultured for 3 days with PHA and SNC-80. Four subjects were studied, and the data are expressed as the mean percentage of inhibition of p24 Ag expression (in the vehicle control groups, p24 Ag levels were 6,683 ± 1,537 pg/ml and 16,174 ± 460 pg/ml for the MN and SF162 strains, respectively) for the group. SNC-80 significantly suppressed p24 Ag expression by both strains of HIV-1. Statistical analyses were made by ANOVA (for HIV-1 MN and SF162, respectively: F = 37.1, p < 0.0001; F = 14.1, p < 0.0001) and between group comparisons of SNC-80 concentrations were performed with Fisher’s least significant difference (∗∗, p < 0.0001 for SNC-80 10−10 M vs 10−6 or 10−14 M; ∗, p < 0.01 for SNC-80 10−8 M vs 10−6 M or 10−12 M vs 10−14 M).

FIGURE 4.

Effect of SNC-80 on p24 Ag expression by human peripheral blood CD4+ T cells infected with HIV-1 MN or SF162. Purified activated CD4+ lymphocytes were incubated with the designated concentrations of SNC-80 for 1 h before infection with the MN or SF162 strains of HIV-1 at a multiplicity of infection of 0.02. After 2-h incubation with HIV-1 at 37°C, CD4+ lymphocytes were cultured for 3 days with PHA and SNC-80. Four subjects were studied, and the data are expressed as the mean percentage of inhibition of p24 Ag expression (in the vehicle control groups, p24 Ag levels were 6,683 ± 1,537 pg/ml and 16,174 ± 460 pg/ml for the MN and SF162 strains, respectively) for the group. SNC-80 significantly suppressed p24 Ag expression by both strains of HIV-1. Statistical analyses were made by ANOVA (for HIV-1 MN and SF162, respectively: F = 37.1, p < 0.0001; F = 14.1, p < 0.0001) and between group comparisons of SNC-80 concentrations were performed with Fisher’s least significant difference (∗∗, p < 0.0001 for SNC-80 10−10 M vs 10−6 or 10−14 M; ∗, p < 0.01 for SNC-80 10−8 M vs 10−6 M or 10−12 M vs 10−14 M).

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Fig. 5 shows that the specific DOR antagonist, NTI, markedly reduced the suppressive effects of SNC-80 on p24 Ag accumulation. As expected, in the absence of NTI, SNC-80 10−10 M reduced p24 Ag expression by ∼50% when supernatant from cells infected with either the MN (Fig. 5, upper panel) or SF162 strain (Fig. 5, lower panel) was assayed. At 10−9 and 10−10 M, NTI effectively blocked the suppressive effects of SNC-80, whereas NTI alone had no effect.

FIGURE 5.

NTI prevents the suppressive effect of SNC-80 on p24 Ag expression by human peripheral blood CD4+ T cells infected with HIV-1 MN or SF162. Purified activated CD4+ lymphocytes were incubated for 30 min with the designated concentrations of NTI and then SNC-80 10−10 M for 1 h before infection with the MN (A) or SF162 (B) strains of HIV-1 at a multiplicity of infection of 0.02. After 2-h incubation with HIV-1 at 37°C, CD4+ lymphocytes were cultured for 3 days with PHA and NTI + SNC-80. Three subjects were studied, and the data are expressed as mean percentage of inhibition p24 Ag expression (in the vehicle control groups, p24 Ag levels were 16,510 ± 946 pg/ml and 19,380 ± 1,259 pg/ml for the MN and SF162 strains, respectively) for the group. SNC-80 significantly suppressed p24 Ag expression by both strains of HIV-1, and NTI prevented this. Statistical analyses were made with ANOVA and comparisons between vehicle control and SNC-80 with Scheffé’s test (∗, p < 0.01). ANOVA resulted in the following values for HIV-1 MN and SF162, respectively: F = 10.2, p < 0.005; F = 33.3, p < 0.001.

FIGURE 5.

NTI prevents the suppressive effect of SNC-80 on p24 Ag expression by human peripheral blood CD4+ T cells infected with HIV-1 MN or SF162. Purified activated CD4+ lymphocytes were incubated for 30 min with the designated concentrations of NTI and then SNC-80 10−10 M for 1 h before infection with the MN (A) or SF162 (B) strains of HIV-1 at a multiplicity of infection of 0.02. After 2-h incubation with HIV-1 at 37°C, CD4+ lymphocytes were cultured for 3 days with PHA and NTI + SNC-80. Three subjects were studied, and the data are expressed as mean percentage of inhibition p24 Ag expression (in the vehicle control groups, p24 Ag levels were 16,510 ± 946 pg/ml and 19,380 ± 1,259 pg/ml for the MN and SF162 strains, respectively) for the group. SNC-80 significantly suppressed p24 Ag expression by both strains of HIV-1, and NTI prevented this. Statistical analyses were made with ANOVA and comparisons between vehicle control and SNC-80 with Scheffé’s test (∗, p < 0.01). ANOVA resulted in the following values for HIV-1 MN and SF162, respectively: F = 10.2, p < 0.005; F = 33.3, p < 0.001.

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The present investigations are, to our knowledge, the first to demonstrate the presence of DOR immunoreactivity on human peripheral blood T cells and to analyze DOR expression on CD4+ or CD8+ subsets and T cells positive for CD45RA or CD45RO (16, 17, 18). A small fraction of the CD4+ and CD8+ T cells expressed DOR in the absence of mitogenic stimulation and, by 48 h, PHA stimulated expression by ∼40% of these subsets. After PHA stimulation, the DOR-positive cells appeared in the T cell fraction, and similar fractions of both CD45RA+ and CD45RO+ cells had become DOR+. Subnanomolar concentrations of the DOR agonist, SNC-80, were shown to suppress the production of p24 Ag by lymphocytotropic and monocytotropic strains of HIV-1. The specificity of this suppression was evident in its concentration dependence and reversal by the DOR antagonist, NTI.

In several species, mononuclear cells contain DOR transcripts, although very few studies have provided substantial evidence for the detection of DOR itself (19, 20). DOR transcript levels are uniformly low in freshly obtained mononuclear cells, requiring reverse transcription with PCR and a relatively large number of amplification cycles for detection in simian and human PBMC and in murine splenocytes (8, 9, 10, 11). In murine splenocytes, recent studies have shown that stimulation in vivo or in vitro significantly increased the level of DOR transcripts. This was evident after in vivo treatment with a single injection of staphylococcal enterotoxin B, which produced a 2-fold increase by 8 h (7). In vitro, Con A and anti-CD3-ε had similar effects, stimulating transcript expression by T cells, including CD4+ and CD8+ subsets (10, 11, 12). T cell DOR transcripts increased from <1 copy/cell to 22 and 42 copies/cell after 24 and 48 h of anti-CD3-ε (12). Moreover, experiments with actinomycin D implied that transcriptional activation mediates the anti-CD3-ε-driven increase (12).

By 48 h in culture, PHA stimulated a 5- to 7-fold increase in DOR immunoreactivity in the CD4+ and CD8+ T cell subsets, respectively. Accounting for ∼40% of these T cell subsets, this level of DOR expression declined by 96 h in both T cell subsets. After PHA stimulation, these DOR+ T cells represented virtually all of the DOR+ cells. Since the fraction of CD45RA+ and CD45RO+ cells was unaffected by PHA at 48 h (in contrast to 6 days), it is likely that individual T cells had not changed their status with respect to the expression of these Ags at this time interval. This suggests that similar percentages of T cells positive for CD45RA or CD45RO were induced to express DOR by PHA. Thus, these studies provide evidence for the activation-associated expression of DOR on both naive and memory T cells in response to PHA (16, 17, 18).

DORs are seven-transmembrane G protein-coupled receptors that were originally cloned from neural cells and have been studied extensively in the nervous system (21). In the immune system, DORs have been shown to modulate Con A-stimulated calcium mobilization and anti-CD3-ε-induced phosphorylation of the extracellular-regulated kinases, ERK1, 2, in splenocytes (6, 7). From a 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-ε-driven proliferation of highly purified CD4+ and CD8+ splenic T cells, and the production of IL-2 (4). Our previous experiments demonstrated that DOR ligands suppressed HIV-1 p24 Ag production 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, and maximal suppression was observed with 10−13–10−9 M SNC-80 (>60% reduction) or 10−15–10−11 M deltorphin (>50% reduction). At higher concentrations, neither agonist was effective. Although not well understood, this U-shaped dose-response function may reflect the interaction of intracellular signaling cascades, which varies with agonist concentration.

In the present investigations, we observed a similar concentration dependence and degree of suppression of p24 Ag production by activated human peripheral blood T cells inoculated with HIV-1 MN or SF162. At a concentration of 10−10 M, SNC-80 was maximally effective, whereas at 10−7 M, suppression was no longer evident. In both strains of HIV-1, maximal suppression was ∼50%. This degree of suppression coheres with our finding that ∼40% of CD4+ T cells express DOR within the time interval (48 h) used to activate the PBMC before inoculation with HIV-1. If these observations are causally related, it would suggest that DOR agonists are able to suppress p24 Ag expression to a much greater degree in the CD4+ T cell subpopulation that are DOR+.

These investigations indicate that a relatively brief preincubation with SNC-80 was required; in contrast, a 24-h pre- or posttreatment with SNC-80 was ineffective. Preincubation is required for the effects of DOR agonists on other immune functions. For example, suppression of the anti-CD3-ε-stimulated proliferation of highly purified murine splenic T cell subsets and IL-2 production was optimal when lymphocytes were treated with DOR agonists for 1 h before culture with anti-CD3-ε (4). In addition, for the suppressive effect of DOR agonists on anti-CD3-ε-induced ERK phosphorylation, pretreatment was necessary (7). These observations suggest that preincubation with a DOR agonist modulates the activity of intracellular pathways involved in TCR-dependent signaling. This may reflect the effects of DORs on the tyrosine phosphorylation of intracellular signals known to regulate cell growth and proliferation (22). Somatostatin, a ubiquitous extracellular signal peptide that activates Gi protein-coupled receptors, also has been shown to inhibit the growth and proliferation of a variety of cell lineages (23). Indeed, somatostatin-enhanced phosphotyrosine phosphatase activity, which inactivates growth factor receptor kinases, has been implicated in its action (23, 24, 25). Thus, phosphotyrosine phosphatases may be involved in mediating the intracellular action of DORs, which, like somatostatin receptors, are commonly coupled to Gi proteins.

In previous studies, preincubation was also required for the effects of morphine on HIV-1 p24 Ag expression by human PBMC cocultures (13). In these studies, normal donor PBMC were incubated with morphine sulfate and then activated with PHA before the addition of HIV-1-infected PBMC. The morphine-enhanced expression of HIV-1 p24 Ag was both stereoselective and reversible by μ opioid receptor-specific antagonists (13). Although this report contrasts with the present observations and with our report on 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 depend on two different opioid receptor subtypes that can couple to different intracellular effectors, depending on cell type. In addition, the design of the present studies suggests that the effects of DOR ligands were directly on T cells, whereas it is not possible to precisely identify the cellular target in the previous experiments with PBMC cocultures. Thus, the contrasting effects of μ vs δ opioids on HIV-1 p24 Ag expression are likely to reflect differences in the receptor-specific intracellular effectors that are modulated by these compounds.

Both the requirement for preincubation with SNC-80 and the suppressive effects of DOR agonists on lymphocytotropic and monocytotropic strains of HIV-1 suggest that early cellular events involved in viral replication may be affected by activating DORs. Viral entry is a good candidate since HIV-1 gains access to CD4+ T cells by interacting with both CD4 molecules and chemokine coreceptors (26). In support of this, a recent study reported that DOR agonists inhibited chemokine-induced chemotaxis by inducing the phosphorylation and desensitization of the chemokine receptor (27).

In summary, these studies have shown that a large fraction of CD4+ and CD8+ T cells can be induced to express DORs by mechanisms dependent on activation through the TCR. DORs, which modulate intracellular signaling pathways affecting T cell function, can suppress the expression of p24 Ag by HIV-1-infected CD4+ T cells. These findings suggest that adjunctive δ opioid immunotherapy may augment the antiviral therapy of HIV-1 infection.

1

This work was supported by U.S. Public Health Service Grants DA-04196 (B.M.S.) and DA-09224 (P.K.P.).

3

Abbreviations used in this paper: DOR, δ 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,−,{2S[1(S*),2α,5β]}-(9Cl).

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