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Originally published online as doi:10.1189/jlb.0203067 on September 12, 2003

Published online before print September 12, 2003
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(Journal of Leukocyte Biology. 2003;74:1074-1082.)
© 2003 by Society for Leukocyte Biology

Selective inactivation of CCR5 and decreased infectivity of R5 HIV-1 strains mediated by opioid-induced heterologous desensitization

Imre Szabo*,{dagger}, Michele A. Wetzel*,{dagger}, Ning Zhang{ddagger}, Amber D. Steele*,{dagger}, David E. Kaminsky*,{dagger}, Chongguang Chen{dagger},§, Lee-Yuan Liu-Chen{dagger},§, Filip Bednar*,{dagger}, Earl E. Henderson*,{dagger},¶, O. M. Zack Howard{ddagger}, Joost J. Oppenheim{ddagger} and Thomas J. Rogers*,{dagger},¶

Departments of
* Microbiology and Immunology and
§ Pharmacology,
{dagger} Center for Substance Abuse Research, and the
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania; and
{ddagger} Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland

1 Correspondence: Department of Microbiology and Immunology, Temple University School of Medicine, 3400 N. Broad Street, Philadelphia, PA 19140. E-mail address: rogerst{at}temple.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The opiates are well-established immunomodulatory factors, and recent evidence suggests that µ- and {delta}-opioid receptor ligands alter chemokine-driven chemotactic responses through the process of heterologous desensitization. In the present report, we sought to examine the capacity of µ- and {delta}-opioids to modulate the function of chemokine receptors CCR5 and CXCR4, the two major human immunodeficiency virus (HIV) coreceptors. We found that the chemotactic responses to the CCR1/5 ligand CCL5/regulated on activation, normal T expressed and secreted, but not the CXCR4 ligand stromal cell-derived factor-1{alpha}/CXCL12 were inhibited following opioid pretreatment. Studies were performed with primary monocytes and Chinese hamster ovary cells transfected with CCR5 and the µ-opioid receptor to determine whether cross-desensitization of CCR5 was a result of receptor internalization. Using radiolabeled-binding analysis, flow cytometry, and confocal microscopy, we found that the heterologous desensitization of CCR5 was not associated with a significant degree of receptor internalization. Despite this, we found that the cross-desensitization of CCR5 by opioids was associated with a decrease in susceptibility to R5 but not X4 strains of HIV-1. Our findings are consistent with the notion that impairment of the normal signaling activity of CCR5 inhibits HIV-1 coreceptor function. These results have significant implications for our understanding of the effect of opioids on the regulation of leukocyte trafficking in inflammatory disease states and the process of coreceptor-dependent HIV-1 infection. The interference with HIV-1 uptake by heterologous desensitization of CCR5 suggests that HIV-1 interaction with this receptor is not passive but involves a signal transduction process.

Key Words: chemokines • neuroimmunology • cell trafficking • AIDS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The opioid and chemokine receptors, like many of the chemoattractant receptors, are members of the Gi protein-coupled seven-transmembrane receptor family. It is clear that these receptors, together with the chemokine and endogenous opioid peptide ligands, are widely distributed in brain tissue and the periphery. The chemokines have been classified into four families, C, CC, CXC, and CX3C, based on the position of conserved cysteines [1 ], and these ligands interact with receptors designated CR1, CCR1–11, CXCR1–5, or CX3CR, respectively. Three classes of receptors have been identified for the opioids, designated µ, {kappa}, and {delta}, and each of the opioid receptor genes expressed in brain tissue has been cloned and sequenced [2 3 4 ]. Moreover, radiolabeled agonist- and antagonist-binding assay experiments suggest that these receptors are also expressed by leukocytes [5 , 6 ], and µ-, {kappa}-, and {delta}-opioid receptors have been cloned from cells of the immune system [7 8 9 ].

The µ-, {kappa}-, and {delta}-opioids are known to possess the ability to modulate antibody and cellular immune responses [10 11 12 ], natural killer (NK) cell activity [13 ], cytokine expression [14 15 16 ], and phagocytic activity [17 , 18 ]. Like the chemokines, the µ-, {kappa}-, and {delta}-opioids possess chemoattractant activity and induce the chemotaxis of monocytes and neutrophils [19 20 21 ]. It has been reported that pretreatment with opioids, including morphine, heroin, met-enkephalin (ME), the more selective µ-agonist [D-ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO), or the selective {delta}-agonist [D-Pen2, D-Pen5]enkephalin (DPDPE), leads to the inhibition of the chemotactic response of leukocytes to complement-derived chemotactic factors [22 ] and to the chemokines macrophage inflammatory protein (MIP-1{alpha})/CCR ligand CCL3, CCL5/regulated on activation, normal T expressed and secreted, monocyte chemotactic protein/CCL2, and CXCR ligand CXCL8/interleukin (IL)-8 [21 ]. This inhibitory effect does not involve competitive inhibition of chemokine receptor binding by the opioid, but these results suggest that the activation of the µ- and {delta}-opioid receptors leads to the heterologous desensitization of the chemokine receptors CCR2, CXCR1, and CXCR2. Moreover, the inhibition of CCL3 and CCL5 responses following opioid pretreatment is consistent with the desensitization of CCR1 or CCR5 or both.

In the present report, we have attempted to determine whether the major human immunodeficiency virus (HIV) coreceptors CXCR4 and CCR5 are targets of opioid-induced heterologous desensitization. Our results show that CCR5 but not CXCR4 is desensitized following µ- or {delta}-opioid receptor activation. We find that the opioid-induced heterologous desensitization of CCR5 results in a significant reduction in the susceptibility of macrophages to infection by HIV. Our findings have significant implications for the regulation of chemokine receptor function in the generation of certain inflammatory disease states.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and cells
The {kappa}-opioid agonist U50,488H (Upjohn, Kalamazoo, MI), the µ-opioid agonist DAMGO (Multiple Peptide Systems, San Diego, CA), {delta}-opioid agonists deltorphin and DPDPE (Multiple Peptide Systems), ME (Peninsula Laboratories, Belmont, CA), and the {delta}-opioid antagonist naltrindole (Multiple Peptide Systems) were dissolved in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) before use. Recombinant human chemokines were obtained from R&D Systems (Minneapolis, MN) or PeproTech (Rocky Hill, NJ). Human peripheral blood monocytes were isolated from leukopheresis packs (National Institutes of Health Clinical Center, Transfusion Medicine Department, Bethesda, MD) and were enriched for monocytes using iso-osmotic Percoll gradient centrifugation as described previously [23 ].

Stable expression of opioid and chemokine receptors in Jurkat and Chinese hamster ovary (CHO) cell lines
Jurkat T cells were transfected with the human {kappa}-opioid receptor cDNA in the vector pcDNA3.zeo or the human µ-opioid receptor cDNA in the vector pcDNA3.neo, and the clonal cell lines stably expressing the {kappa} (designated J-KOR8)- or µ (designated J-MOR5.1)-opioid receptors were established with zeocin or neomycin selection, respectively. The expression of opioid receptors was verified by reverse transcriptase-polymerase chain reaction (RT-PCR) and radiolabeled-binding analysis using [3H]-diprenorphine [24 ]. CHO cells were transfected initially with the rat µ-opioid receptor cDNA in the pcDNA3.neo vector, and a clonal cell line (CHOMOR) was established and verified as described above. The CHOMOR cells were subsequently transfected with the CCR5 cDNA in pcDNA3.hygro, and a stable clonal cell line (CHOMOR-CCR5) was established with hygromycin and neomycin selection.

Chemotaxis
The migration of primary cells and cell lines was analyzed in a 48-well microchemotaxis chamber, as described previously [25 ] with minor modification. Briefly, the upper compartments of the chamber were loaded with cells at a concentration of between 1 and 2 x 10 6 per mL, the lower compartments of the chamber were loaded with the chemoattractant, and the two compartments were separated by a fibronectin-coated (for Jurkat and CHO cells) or uncoated (for monocytes), 5-µm (for Jurkat cells and monocytes) or 8-µm (for CHO cells) pore-size polycarbonate polyvinylpyrrolidone-free membrane. Following incubation for 1.5 h (monocytes) or 3 h (Jurkat or CHO cells), the filter was removed, the top of the membrane was wiped, and then the membrane was fixed and stained with a Diff-Quick kit. The cells, which migrated to the bottom of the membrane, were counted microscopically, and the average number of cells in four high-power fields (400x) was determined. The results are expressed as the chemotaxis index, which represents the fold increase in the number of cells migrating in response to chemoattractant versus the medium control.

Binding analysis
CHO cells stably transfected with the µ-opioid receptor and the chemokine receptor CCR5 were cultured in DMEM F12 HAM supplemented with 10% fetal calf serum (FCS), 0.5 mg/mL geneticin, 0.2 mg/ml hygromycin, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere consisting of 5% CO2 and 95% air at 37°C. Cells were treated with 2 nM DAMGO in the medium for 10 min at 37°C, detached with ice-cold versene solution, and washed twice. Cells (7.5x104 cells/tube) were incubated with [125I]CCL4/MIP-1ß (0.1 nM) and graded concentrations of unlabeled CCL4 (from 2 to 64 nM) at room temperature for 45 min, centrifuged at 4000 g at 4°C for 5 min, and washed twice by resuspension and centrifugation. Radioactivity associated with cells was measured in a {gamma}-counter. Binding data were analyzed with the EBDA program [26 ].

Flow cytometry
Monocytes or transfected CHO cells were harvested, washed, and resuspended in a medium containing RPMI 1640, 25 mM HEPES, glutamine, and 1% bovine serum albumin. Cells were treated with DAMGO or CCL5 at the designated concentrations and incubated for 60 min at 37°C. Cells were immediately harvested, washed with cold Hanks’ balanced saline solution (HBSS; Life Technologies, Gaithersburg, MD) with 2% endotoxin-free FCS (Hyclone Laboratories, Logan, UT; HF), and resuspended in HF. Goat serum (Sigma Chemical Co., St. Louis, MO) was added to block nonspecific binding, and cultures were incubated at 4°C for 30 min. Cells were treated with phycoerythrin (PE)-conjugated anti-CCR5 (2D7/CCR5) antibody (BD PharMingen, San Diego, CA), incubated at 4°C for 45 min, washed, and analyzed in a Coulter Epics XL flow cytometer (Coulter Corp., Hialeah, FL).

Immunofluorescent laser scanning confocal microscopy
Membrane-associated and cytoplasmic CCR5 expression was determined using the method of Signoret et al. [27 ]. Transfected CHO cells were grown on glass coverslips, washed, and treated with DAMGO or CCL5 as described above. Immediately after treatment, the cells were washed, fixed, and permeabilized. Staining for CCR5 was performed with PE-conjugated anti-CCR5 antibody for 1 h at 37°C. The slips were washed twice with phosphate-buffered saline and mounted on glass slides using the ProLong antifade kit (Molecular Probes, Eugene, OR), and the samples were analyzed using an Olympus Fluoview confocal laser scanning microscope.

Ligand-induced calcium mobilization
Calcium flux was measured as described by Badolato et al. [28 ]. In brief, IL-2-induced monocytes (100 ng/ml for 48 h) were incubated at 107/mL for 30 min at room temperature in DMEM containing 1 µM Fura-2 AM. Cells were then washed with DMEM once, HBSS twice, and diluted into 2 x 106/mL. Cells were then loaded into a 2-ml cuvette at 37°C, and the relative ratio of fluorescent emission at 510 nm when excited by 340 nm and 380 nm was recorded by a Perkin-Elmer luminescence spectrometer. For heterologous desensitization experiments, cells were first incubated in 100 nM DAMGO at 37°C for 30 min before adding Fura-2 AM.

HIV-1
The X4 IIIB and R5 JRFL strains of HIV-1 were obtained from the National Institute of Allergy and Infectious Diseases AIDS Research and Reference Reagent Program (Rockville, MD). The IIIB strain of HIV-1 was propagated in the human T cell line Molt4-IIIB. The multiplicity of infection (MOI) of the IIIB strain was determined by quantitation of syncytia formed by HIV-infected lymphocytes when cocultured with exponentially growing CD4-bearing SupT1 cells. The R5 JRFL strain of HIV-1 was propagated in cultures of peripheral blood mononuclear cells (PBMCs) from adult donors. Virus was concentrated from culture supernatants and purified by pelleting at 110,000 g for 90 min. The pellets were gently washed and resuspended in medium.

HIV infectivity by HIV p24 analysis
Infectivity analysis of HIV-1 strain JRFL was performed with monocyte-derived macrophages (MDM), which were prepared by allowing PBMCs to adhere to plastic for 2 h, a gentle wash, followed by culture for 5 days in RPMI-1640 medium supplemented with 10% heat-inactivated, low-endotoxin FCS (Hyclone), 10 µg/mL gentamicin, and 1 mM L-glutamine containing macrophage-colony stimulating factor (100 ng/mL; PeproTech). The MDM cultures were washed and exposed to the designated concentrations of DAMGO or medium alone for 1 h, followed by addition of the R5 HIV-1 strain JRFL (MOI, 0.1). The infected cells were washed after 1 h to remove free virus and were allowed to incubate for up to 72 h. The infectivity analysis of HIV-1 strain IIIB was performed in a similar manner using fresh, normal human PBMCs. The supernatants were then collected, and enzyme-linked immunosorbent assay (ELISA) determined the level of HIV p24 (AIDS Vaccine Program, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, MD).

HIV infectivity by HIV-long-terminal repeat (LTR) PCR
PBMCs were plated to a cell density of 3 x 106 cells/mL in 24-well tissue-culture plates, and the cells were treated with DAMGO at the designated concentrations for 1 h at 37°C. The cells were then infected with the IIIB (X4) or JRFL (R5) HIV-1 strains for 1 h at 37°C, followed by washing to remove excess virus. Cells were harvested after 12 h, and the DNA was isolated using the Qiagen QIAamp DNA mini kit (Qiagen, Chatsworth, CA). PCR was used to amplify a 342-bp region of the LTR of HIV using oligonucleotide DNA primers (LTR sense, 5'-AGCCTCAATAAAGCTTGCCT-3'; LTR antisense, 5'-CCCCCTGGCCTTAACCGAAT-3'), followed by electrophoresis in 1.5% agarose gel and transfer to a positively charged nylon membrane (Ambion, Austin, TX) for Southern blot analysis. An oligonucleotide probe (5'-GGAGAGAGATGGGTGCGAG-3') was 5' end-labeled with [{gamma}-32P]adenosine 5'-triphosphate (DNA 5' end-labeling system, Promega, Madison, WI). The membrane was incubated for 2 h at 37°C in a rotary hybridization incubator with prehybridization solution [6x saline sodium citrate (SSC), 5x Denhardt, 0.05% NaPPi, 100 µg/mL herring sperm DNA, and 0.5% sodium dodecyl sulfate] followed by overnight hybridization in hybridization solution (6x SSC, 1x Denhardt, 0.05% NaPPi, 100 µg/mL herring sperm DNA, and 5–10 µl probe) and was washed five times with washing buffer (6x SSC plus 0.05% NaPPi) the next day. The membrane was exposed to Biomax-MR film (Eastman Kodak, Rochester, NY), and the film was analyzed by autoradiography. Quantitation of the bands was performed using tenfold dilutions of the latently infected ACH-2 cell line. These cells express a single copy of the HIV-1 genome and provide a source of characterized HIV-LTR DNA for the analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Opioid-induced inhibition of chemokine chemotaxis
We first used the Jurkat T cell line to explore the nature of the cross-desensitization between opioid and chemokine receptors, in part because it is reported to express the {delta}-opioid receptor [7 ]. We analyzed the chemotactic response of Jurkat cells to ME, an endogenous opioid with weak selectivity for the {delta}-opioid receptor. Our results (Fig. 1A ) show that Jurkat cells exhibited significant chemotactic activity to ME, with a peak response at 1 nM. The Jurkat cell line did not appear to express the µ- or {kappa}-opioid receptors, based on the failure of these cells to exhibit a response to the µ-opioid agonist DAMGO or the {kappa}-opioid agonist U50,488H (Fig. 1B and 1C) . However, Jurkat cells transfected to stably express the µ- and {kappa}-opioid receptors did exhibit responses to the respective ligands (Fig. 1B and 1C) , suggesting that the Jurkat cells possess the necessary signaling components to carry out µ- and {kappa}-opioid receptor-mediated chemotaxis. Additional studies showed that the Jurkat cell line exhibited a response to the selective {delta}-opioid agonist deltorphin, and this response was blocked by pretreatment with the {delta}-opioid antagonist naltrindole (Fig. 1D and 1E) . The results confirm the ability of the opioids to induce a chemotactic response in cells of the immune system, a finding that has been reported previously by a number of investigators [19 20 21 ]. Based on RT-PCR analysis, the Jurkat cell line also expresses the chemokine receptors CCR5 and CXCR4 (data not shown).



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  		Figure 1. Opioid-induced chemotaxis of Jurkat T cells. (A) Chemotactic response of Jurkat T cells to ME. The results are expressed as the chemotaxis index (CI; fold increase in cells migrating in response to chemoattractant vs. the medium control). (B) Chemotactic response of Jurkat and the µ-opioid receptor-expressing J-MOR5.1 cell line to the µ-opioid agonist DAMGO. (C) Chemotactic response of Jurkat and the {kappa}-opioid receptor-expressing J-KOR8 cell line to the {kappa}-opioid agonist U50,488H. (D) Inhibition of the response of Jurkat cells to the {delta}-opioid agonist deltorphin (Delt) following treatment with the {delta}-opioid antagonist naltrindole (NALT). Jurkat cells were pretreated for 45 min with medium alone (Non-NALT) or 100 nM NALT, and then the chemotactic response to the designated concentrations of DELT was determined. (E) The inhibition of the response of Jurkat cells to DELT is dose-dependent. Jurkat cells were pretreated with the designated concentrations of NALT or medium for 45 min, and the chemotactic response to DELT (0.1 nM) was determined. Results are presented from a single representative experiment from a total of four independent experiments and show the mean ± SE with six replicates per group. Cont, Control. *, P< 0.05.

 
We next assessed whether the effect of pretreatment with ME could desensitize the chemotactic response of Jurkat cells to the chemokines CXCL12 and CCL5. Our results show that ME-treated cells continued to manifest fully normal responses to CXCL12 (Fig. 2A ) but failed to exhibit a chemotactic response to CCL5 (Fig. 2B) . In contrast, pretreatment with the {kappa}-opioid agonist U50,488H failed to induce heterologous desensitization in the Jurkat cell line (Fig. 2B) . This result is expected, as our results suggest that the parental Jurkat cell line fails to express the {kappa}-opioid receptor (Fig. 1C) . These results suggest that CCL5 receptors are desensitized by the {delta}-opioid treatment, but the CXCL12 receptor (CXCR4) is not a target for the opioid-induced desensitization. These results were confirmed using the highly selective {delta}-opioid agonist deltorphin. Jurkat cells pretreated with deltorphin also failed to elicit a detectable chemotactic response to CCL5 (Fig. 2E) but generated a normal response to CXCL12 (Fig. 2C) . We next analyzed the cross-desensitization of primary human monocyte chemokine receptors by {delta}-opioids, and our results showed that cells pretreated with deltorphin manifested a normal response to CXCL12 (Fig. 2D) but failed to exhibit a response to CCL5 (Fig. 2F) . These results suggest that the selectivity in the {delta}-opioid-induced cross-desensitization is not limited to T cells. Finally, additional experiments showed that monocytes pretreated with the highly selective µ-opioid agonist DAMGO also failed to respond to CCL5 (Fig. 3 ). Cells pretreated with DAMGO (10 nM) failed to exhibit a chemotactic response to CCL5. However, monocytes treated with the µ-opioid-selective antagonist CTAP before the DAMGO administration remained fully responsive to CCL5. These results verify that the DAMGO-induced cross-desensitization is mediated by a classical µ-opioid receptor.



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  		Figure 2. Pretreatment with ME or the {delta}-opioid agonist deltorphin cross-desensitizes the response of cells to CCL5 but not CXCL12. (A and B) Jurkat cells were pretreated for 60 min with 10 nM ME (ME-Tr) or medium alone (Non-Tr) and were washed thoroughly, and the chemotactic response to CXCL12 (A) and CCL5 (B) was determined. The response to CCL5 in Jurkat cells pretreated with 10 nM U50,488H is also shown (B). (C–F) Jurkat cells (C and E) and monocytes (D and F) were pretreated with medium alone or 0.1 nM deltorphin (DELT-Tr) for 1 h, and the chemotactic response to the designated concentrations of CXCL12 and CCL5 was determined. Results are shown as the mean (±SE) and are representative of five independent experiments. *, P < 0.05.

 


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  		Figure 3. Pretreatment with the µ-opioid agonist DAMGO cross-desensitizes the response of monocytes to CCL5. Fresh monocytes were pretreated with medium alone, DAMGO (10 nM), the µ-opioid antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 100 nM), or the combination of DAMGO (10 nM) and CTAP (100 nM) for 1 h, and the chemotactic response to CCL5 (100 ng/ml) was determined. Results are representative of three independent experiments. *, P < 0.05, for comparison with the nonpretreated group in the absence of chemokine (top bar).

 
Opioid-induced desensitization of CCR5-meditated calcium mobilization
In an effort to examine the nature of the opioid-induced desensitization of chemokine responses, we compared the CCL4-elicited calcium response in monocytes that were pretreated with DAMGO or medium alone. The results show (Fig. 4 ) that monocytes pretreated with DAMGO manifest a calcium mobilization response to a low dose (1 ng) of CCL4, which is less than 10% of the nonpretreated control cells. The results also show that the impaired calcium mobilization response evident with 1 or 2 ng CCL4 is not apparent at high concentrations (25 or 1250 ng) of this chemokine (Fig. 4) . These results suggest that the opioid-induced cross-talk with CCR5 in monocytes results in a failure to exhibit a normal calcium mobilization response; however, high CCR5 ligand concentrations can overcome the inhibition of the calcium response. This result is not unexpected, as previous studies have shown that ME pretreatment fails to alter the CCL3-induced calcium flux response in monocytes [21 ].



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  		Figure 4. Analysis of DAMGO-induced heterologous desensitization of CCL4-stimulated Ca++ mobilization. Monocytes were pretreated with medium alone (A) or 100 nM DAMGO (B), and after 1 h, the MIP-1ß-stimulated calcium flux response was determined over a period of 150 s (+, CCL4 at 0 ng/ml; {circ}, 1 ng/mL; short bar, 2 ng/mL; {triangleup}, 5 ng/mL; x, 25 ng/mL; {square}, 1250 ng/mL).

 
Cross-desensitization between CCR5 and the µ-opioid receptor
The desensitization of µ- and {delta}-opioid receptor function by CCL5 could be mediated through CCR1 or CCR5, as this chemokine has a high affinity for both receptors [1 ]. In an effort to specifically assess the cross-desensitization of CCR5 by the µ-opioid receptor, we prepared CHO cells, which stably express CCR5 and the µ-opioid receptor. This cell line (CHOMOR-CCR5) exhibited a chemotactic response to CCL5 and DAMGO (Fig. 5 ), with an optimal chemotactic dose of 5 nM and 1 nM, respectively (data not shown). Importantly, CHOMOR-CCR5 cells pretreated with DAMGO failed to respond to DAMGO (Fig. 5A) or CCL5 (Fig. 5B) , suggesting that DAMGO induces homologous desensitization of the µ-opioid receptor and heterologous desensitization of CCR5. Conversely, CHOMOR-CCR5 preincubated with CCL5 failed to manifest a normal response to CCL5 (Fig. 5B) or DAMGO (Fig. 5A) , showing that the activation of CCR5 induces heterologous desensitization of the µ-opioid receptor.



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  		Figure 5. Bidirectional cross-desensitization of µ-opioid and CCR5 receptors in CHOMOR-CCR5 cells, which were pretreated (PreTr) with DAMGO (10 nM) or CCL5 (12.5 nM), and the chemotactic response (Resp) of these cells to DAMGO (1 nM; A) and CCL5 (6.25 nM; B) was determined. Results are representative of six experiments.

 
Expression of CCR5 following desensitization
As desensitization of G protein-coupled receptors (GPCRs) is frequently associated with internalization of the receptor [29 , 30 ], we examined the expression of CCR5 following homologous and heterologous desensitization. Monocytes were treated with CCL5 at a concentration (12.5 nM), which we found was optimal for homologous desensitization of CCR5, and the expression of CCR5 was determined after 60 min. The results for resting monocytes (Fig. 6 ) show that CCL5 administration leads to a reduction (by ~50%) of cell membrane CCR5 expression (Fig. 6B) . Similarly, monocytes were treated with DAMGO, at the concentration (10 nM) that we found to be optimal for heterologous desensitization of CCR5, and our results (Fig. 6C) show that the cell-surface expression of CCR5 remained unchanged in cells treated with this opiate.



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  		Figure 6. Opioid-induced heterologous desensitization is not associated with internalization of CCR5. (A–C) Effect of pretreatment with CCL5 or DAMGO on the expression of CCR5 on peripheral blood monocytes. Monocytes were pretreated with medium only (A), CCL5 (12.5 nM; B), or DAMGO (10 nM; C), and after 1 h, the expression of CCR5 and CD14 was determined by flow cytometry by two-color analysis of PE-CD14 and fluorescein isothiocyanate (FITC)-CCR5. Results are representative of seven experiments. (D and E) CHOMOR-CCR5 cells were pretreated for 1 h with medium alone or in combination with DAMGO (10 nM; D) or CCL5 (12.5 nM; E), and the expression of CCR5 was determined by flow cytometry. The fluorescence profile for cells stained with an isotype-matched murine immunoglobulin (Isotype) is also presented. Data are representative of three independent experiments. (F–H) CHOMOR-CCR5 cells were treated with medium alone (F), CCL5 (G), or DAMGO (H), and after 1 h, the cells were permeabilized and stained for the expression of CCR5 by confocal laser scanning microscopic analysis.

 
We extended our analysis of the effect of DAMGO on CCR5 expression to the CHOMOR-CCR5 cell line. The CHOMOR-CCR5 cells were treated with CCL5 (12.5 nM) or DAMGO (10 nM), and after 60 min, the cell-surface expression of CCR5 was determined by flow cytometry. Our results show that neither CCL5 nor DAMGO significantly altered the expression of CCR5 by the CHOMOR-CCR5 cell line. Following CCL5 treatment (Fig. 6E) , we observed a slight (but insignificant) reduction in the expression of CCR5 at the highest density and a slight (but insignificant) increase in the percentage of cells expressing CCR5 at a low density. Even smaller changes in receptor expression were apparent for cells treated with DAMGO (Fig. 6D) . Additional experiments were performed with lower and higher concentrations of CCL5 (6 and 25 nM), with lower and higher concentrations of DAMGO (1 nM and 100 nM), and with shorter and longer durations of treatment (30, 90, and 120 min), and we were unable to detect a significant change in the expression of CCR5 by these cells (data not shown). Thus, heterologous desensitization induced by µ-opioids does not result in CCR5 internalization.

Finally, we used immunofluorescent confocal microscopy to examine the expression of CCR5 following homologous or heterologous desensitization of the CHOMOR-CCR5 cell line. The results from this analysis show that the expression of membrane-associated CCR5 following homologous desensitization with CCL5 (Fig. 6F vs. 6G) was modestly reduced relative to the control cells. The expression of membrane-associated CCR5 following heterologous desensitization with DAMGO (Fig. 6F vs. 6H) administration was also essentially identical to that of the control cells. We observed low levels of cytoplasmic CCR5 fluorescence staining, consistent with intracellular receptor pools, in the cells for each of the treatment groups. However, careful examination of these cells did not reveal any change in the level of intracellular staining following CCL5 or DAMGO administration. These results are consistent with data obtained from radiolabeled-binding analysis, which shows that the expression of CCR5 chemokine-binding sites (based on radiolabeled CCL4-binding) was unchanged following pretreatment with DAMGO. The results show that 8.61 ± 1.1 x 105 CCR5-binding sites were detected for control cells compared with 8.31 ± 0.6 x 105 CCR5-binding sites following DAMGO administration at a concentration of 100 nM.

Susceptibility to HIV infection following CCR5 desensitization
The selective desensitization of CCR5 but not CXCR4 following preincubation with DAMGO suggests that these cells may exhibit altered susceptibility to HIV infection. For these studies, we tested PBMCs or MDM to determine the impact of DAMGO on the susceptibility to X4 or R5 strains of HIV-1, respectively. Our results (Fig. 7A and 7C ) show that DAMGO pretreatment failed to alter the susceptibility to X4 HIV-1. In contrast, pretreatment with DAMGO, over a concentration range of 0.1–100 nM, resulted (Fig. 7B and 7D) in a significant reduction in the level of R5 HIV infectivity and replication. These results correlate well with the results described above, which showed that DAMGO induced heterologous desensitization of the function of CCR5 but not CXCR4.



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  		Figure 7. Effect of DAMGO pretreatment on the susceptibility of PBMCs or macrophages to X4 or R5 HIV-1. PBMCs or MDM were pretreated with DAMGO concentrations from 0.1 to 100 nM (A and B), and after 1 h, the PBMCs were exposed to the X4-HIV-1 strain IIIB (A and C), and the macrophages were exposed to the R5-HIV-1 strain JRFL (B and D). Cells were washed after 1 h and returned to culture for 24 h (X4 HIV-1; A) or 72 h (R5 HIV-1; B) with DAMGO. Results are also shown for DAMGO-treated (DAMGO-Tr; 10 nM) and nontreated (Non-Tr) cells infected with X4 HIV for 24–72 h (C) and R5 HIV for 48–96 h (D). ELISA determined the HIV p24 level in the culture supernatant. Data are representative of three experiments.

 
These infectivity studies were verified by analysis of the reverse transcription of the HIV-LTR during the early phase of the HIV infection. For these experiments, PBMCs were treated with or without DAMGO (at 10 or 0.1 nM), infected with X4 or R5 HIV-1, and the synthesis of the early HIV-LTR gene product was determined by PCR and Southern blot analysis. The results (Fig. 8 ) show that pretreatment with DAMGO at 10 nM reduced the reverse-transcription of R5 HIV-LTR DNA to a level that was less than 1% of the non-DAMGO-treated cells (Fig. 8 , lane 2 vs. 3). Pretreatment with DAMGO at 0.1 nM reduced the synthesis of the R5 HIV-LTR by ~60% (Fig. 8 , lane 2 vs. 4), and this is consistent with the reduced degree of heterologous desensitization observed with DAMGO at this lower concentration. In contrast, pretreatment with DAMGO (at 10 or 0.1 nM) failed to inhibit the production of the early HIV-LTR gene product in PBMCs (Fig. 8 , lanes 6 vs. 7 or 8). These results are consistent with the studies described above in which HIV infectivity was assessed by the accumulation of the p24 antigen in the culture supernatants (Fig. 7) . Our results suggest that the DAMGO-induced heterologous desensitization of CCR5 is associated with a reduction in susceptibility of macrophages to HIV-1 infection.



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  		Figure 8. Effect of DAMGO pretreatment on the reverse-transcription of the HIV-LTR gene product. PBMCs were pretreated with DAMGO (10 or 0.1 nM) and after 60 min, were infected with HIV strain JRFL (R5; lanes 1–4) or strain IIIB (X4; lanes 5–8). After 12 h, DNA was isolated from the cells, a region of the HIV-LTR was amplified by PCR, and the LTR product was specifically detected by Southern blot analysis. DNA was also isolated from the latently infected ACH-2 cell line as a source of characterized HIV DNA (lanes 9–12). Results show analysis of uninfected PBMCs (lanes 1 and 5), HIV-infected PBMCs without pretreatment (lanes 2 and 6), or HIV-infected PBMCs pretreated with DAMGO at 10 nM (lanes 3 and 7) or 0.1 nM (lanes 4 and 8). Results also show LTR DNA from ACH-2 cells at 5000 copies (lane 9), 500 copies (lane 10), 50 copies (lane 11), or 5 copies (lane 12). Results are representative of four experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies reported from one of our laboratories have shown that µ- and {delta}-opioid receptor ligands induce heterologous desensitization of chemokine receptors [21 ]. These studies showed that the desensitization was associated with the phosphorylation of the target chemokine receptor [21 ]. In addition, the previous studies demonstrated that the heterologous desensitization induced by opioid receptors is highly selective, based on the observation that the function of CXCR1, CXCR2, and CCR2 but not the formyl peptide receptor (FPR) was inactivated by opioid pretreatment [21 ]. It has recently been suggested that the Gi protein-linked chemoattractant receptors exhibit a hierarchy in producing cross-desensitization, and this is inversely correlated with their susceptibility to desensitization [29 ]. It appears, for example, that the FPR induces a desensitizing signal, which inactivates many unrelated chemoattractant receptors, including C5a, IL-8, platelet activating factor (PAF), and leukotriene B4 (LTB4) receptors [31 32 33 ]. In contrast, the FPR is only minimally desensitized following activation of the C5a and IL-8 receptors and is not significantly altered by the PAF or LTB4 receptor signaling. There is clearly a hierarchy in the cross-desensitization between opioid and chemokine receptors, as opioid receptor-induced desensitization does not target the CXCR4 receptor. It is quite possible that ligands for CXCR4 are potent desensitizers, but CXCR4 is a poor target for cross-desensitization. For example, we have previously shown that the CXCR4 ligand CXCL12 desensitizes µ- and {delta}-opioid receptors [34 ]. Furthermore, CCR7 is desensitized by CXCL12 activation of CXCR4, but CXCR4 is not desensitized by CCR7 ligands [35 ]. The failure of µ- and {delta}-opioids to induce desensitization of CXCR4 may suggest that these opioid receptors are not strong desensitizers. This would be consistent with the failure of the µ-opioid receptor to inhibit the calcium mobilization with higher doses of CCL4. This result is consistent with previous work with monocytes, which showed that ME fails to inhibit a CCL3-induced calcium flux response [21 ].

The previous studies on cross-desensitization did not address the susceptibility of CCR5 or CXCR4, the major HIV coreceptors, to µ- and {delta}-opioid receptor-induced desensitization. In the present report, we show that µ- and {delta}-opioid receptor ligands selectively induce cross-desensitization of CCR5, without any detectable alteration of CXCR4 function. Our results demonstrate that the desensitization of CCR5 is coincident with a significant, albeit incomplete, reduction in the level of R5 HIV-1 infectivity in macrophages. The effect on R5 HIV infectivity cannot be explained by the simple down-modulation of HIV coreceptor expression, as there is a very modest reduction in the number of cells expressing CCR5 or in the density of CCR5 expression as measured by fluorescence intensity following opioid administration to primary monocytes. These results provide further support for the notion, proposed previously [30 , 36 ], that optimal coreceptor function requires intact receptor signaling capability. It is well established that HIV-1 envelope proteins activate G proteins following the interaction with CCR5 or CXCR4 [37 , 38 ]. Moreover, recent evidence is accumulating, which suggests that signaling through the chemokine receptors CXCR4 and CCR5 has a profound impact on viral entry [38 , 39 ]. For example, Gi protein inactivation by pertussis toxin treatment significantly inhibited X4 HIV infection without altering the expression of CXCR4 or CD4 [39 ]. The biochemical basis for the contribution of GPCR signaling to HIV infectivity remains uncertain. However, studies suggest a role for signaling in the formation of proper membrane complexes of CXCR4 receptors together with CD4, based in part on results showing an inhibition of HIV entry following cytochalasin D-induced disruption of actin-dependent membrane colocalization of CXCR4 and CD4 [40 ]. In fact, recent data show that the dimerization of CXCR4, which is prominent in T cells, is required for coreceptor function, and the absence of the dimer form of CXCR4 in monocytes may explain the resistance of these cells to X4 strains of HIV-1 [37 ].

It should be pointed out that the results reported here are consistent in some respects with the published findings of Sharp et al. [40 ], which show that pretreatment with the {delta}-opioid agonist SNC-80 inhibits the replication of HIV in CD4-positive T cells. These authors show that the SNC-80 inhibition of HIV replication is most apparent when administered 1 h before infection, and the inhibitory effect is blocked by pretreatment with the {delta}-opioid antagonist naltrindole. However, these authors find that the {delta}-opioid effect leads to an inhibition of replication of X4 and R5 strains of HIV-1 (strains MN and SF162, respectively). This is in contrast to the findings in the present report, which show that µ- and {delta}-opioids fail to induce heterologous desensitization of CXCR4. The mechanism of inhibition in the studies by Sharp et al. [40 ] was not fully defined, and it is not clear that the SNC-80-induced inhibition is at the level of heterologous desensitization of the HIV coreceptors. Moreover, the experimental parameters of the studies performed by Sharp and his colleagues [40 ] differ from those reported herein in some respects, including their use of phytohemagglutinin during the HIV infection period. It is relevant that recent studies by Lokensgard et al. [41 ] have shown that {kappa}-opioid receptor stimulation induces a significant inhibition in the surface expression of CXCR4 on activated CD4-positive T cells. In any case, a much more thorough analysis of the role of {delta}-opioid-induced heterologous desensitization in the susceptibility to HIV infection in activated and nonactivated CD4-positive T cells should be performed.

The results in the present report are consistent with recently reported studies showing that the B-oligomer of pertussis toxin induces cross-desensitization of CCR5, and this coincides with dramatic reduction in susceptibility of T cells to HIV-1 infection [36 ]. It appears that the activation of the receptor for the pertussis toxin B-oligomer (which has not been identified) results in cross-desensitization of CCR5 signaling, without altering the level of binding of CCR5 ligands [36 ]. Moreover, the B-oligomer induces heterologous desensitization of CCR5 without altering CXCR4 function and reduces susceptibility to R5 but not X4 strains of HIV [36 ]. Indeed, it appears that CCR5 is susceptible to heterologous desensitization induced by several unrelated GPCR ligands. Shen et al. [42 ] have found that activation of the high-affinity FPR induces desensitization of CCR5, and this also leads to a significant decrease in HIV-1 infectivity as measured by the production of HIV p24 and HIV envelope-mediated cell fusion. In addition, ligands for the low-affinity FPRL-1 have been shown to cross-desensitize CCR5 and CXCR4 and inhibit susceptiblity to R5 and X4 strains of HIV [43 , 44 ]. FPRs may induce more potent heterologous desensitization and down-regulate the function of CXCR4, and ligands for other GPCR (such as the µ- and {delta}-opioid receptors) are unable to manifest such a strong desensitization response. A more thorough analysis of the hierarchy of desensitization among the chemoattractant receptors is clearly necessary.

The inactivation of CCR5 function by µ- and {delta}-opioid agonists has implications for our understanding of the inflammatory response. T cells, NK cells, and cells of the monocyte/macrophage lineage express the CCR5 receptor, and elevated levels of the ligands for this receptor (CCL3, CCL4, and CCL5) have been detected at sites of inflammation [45 , 46 ]. Recent evidence suggests that between CD4+ and CD8+ T cells, cells with the characteristics of the TH1- and Tc1-lineage, respectively, predominantly express CCR5 [47 48 49 ]. It is becoming clear that CCR5 plays a critical role in the function of macrophages and T cells, which participate in the immune response, although CCL3 and CCL5 are ligands for other chemokine receptors (chiefly CCR1). For example, T cells from mice deficient in CCR5 (CCR5-/-) or CCL3 (CCL3-/-) expression fail to produce normal levels of interferon-{gamma} in response to T cell receptor/CD28 stimulation [50 ]. In addition, CCR5-/- mice exhibit increased susceptibility to the intracellular pathogen Cryptococcus neoformans [51 ], and the clearance of the pathogen Listeria monocytogenes by macrophages from these mice is significantly impaired [52 ]. It is interesting that leukocyte recruitment into sites of brain inflammation during the course of C. neoformans infection is also defective in the CCR5-/- mice [51 ]. This is consistent with accumulating evidence that the CCR5 ligands CCL3 and CCL5 are critical participants in the infiltration of blood-derived macrophages into neuroinflammatory response sites [53 ]. It is well established that the endogenous opioids ß-endorphin, ME, and leu-enkephalin are produced during inflammatory responses in the periphery [54 , 55 ], and these opioids are also elevated in the brain during periods of stress. Based on the results presented here, we suggest that the production of endogenous opioids, leading to the inactivation of CCR5, may serve as a negative regulator of the inflammatory response.


   ACKNOWLEDGEMENTS
 
This work was supported in part by NIH grants DA-06650, DA-11130, DA-14230, DA-04745, and P30 DA-13429 (T. J. R.), DA-11263 (L-Y. L-C.), T32 DA-07237 (M. A. W. and A. D. S.), F31 DA-05894 (M. A. W.), and T32 AI-07101 (A. D. S.).

Received February 13, 2003; revised June 10, 2003; accepted July 28, 2003.


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