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Originally published online as doi:10.1189/jlb.1007685 on February 5, 2008

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(Journal of Leukocyte Biology. 2008;83:956-963.)
© 2008 by Society for Leukocyte Biology

DAMGO-induced expression of chemokines and chemokine receptors: the role of TGF-β1

Christine Happel*,{dagger}, Amber D. Steele{dagger},{ddagger},1, Matthew J. Finley*,{dagger}, Michele A. Kutzler*,{dagger},{ddagger},2 and Thomas J. Rogers*,{dagger},{ddagger},§,3

* Fels Institute for Cancer Research and Molecular Biology,
{dagger} Center for Substance Abuse Research, Departments of
{ddagger} Microbiology and Immunology and
§ Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania, USA

3 Correspondence: Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, PA 19140, USA. E-mail: rogerst{at}temple.edu


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ABSTRACT
 
Studies from a number of laboratories suggest that modulation of cytokine expression plays an integral role in the immunomodulatory activity of opioids. Previously, our laboratory reported that activation of the µ-opioid receptor induced the expression of CCL2, CCL5, and CXCL10, as well as CCR5 and CXCR4. Previous work has also suggested the possibility that TGF-β may participate in the opioid-induced regulation of immune competence, and in the present study, we set out to determine the role of this cytokine in the control of chemokine and chemokine receptor expression. We found that D-ala2,N-Me-Phe4-Gly-ol5enkephalin (DAMGO), a highly selective µ-opioid agonist, induced the expression of TGF-β1 expression at the protein and mRNA levels. In turn, the addition of TGF-β1 was found to induce CCL5 and CXCR4 expression but not CCL2, CXCL10, or CCR5. Further analysis showed that pretreatment with neutralizing anti-TGF-β1 blocked the ability of DAMGO to induce CCL5 or CXCR4. Similarly, pretreatment with cycloheximide prevented CCL5 or CXCR4 mRNA expression, consistent with the observation that DAMGO induction of chemokine and chemokine receptor expression requires newly synthesized TGF-β1 protein. These results describe a common molecular basis for the activation of chemokine and chemokine receptor expression and may permit the development of strategies to inhibit certain undesirable immunological properties of µ-opioid agonists such as morphine and heroin.

Key Words: opioid • CCL5 • CXCR4


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INTRODUCTION
 
The seven transmembrane G protein-coupled receptors compose a large family of receptors and include the opioid and chemokine receptor subgroups. The opioid receptors are predominantly expressed in the CNS; however, some cells of the immune system also express functional receptors. Monocytes, macrophages, and B and T lymphocytes have been shown to express mRNA for opioid receptors, and binding analysis has revealed surface expression of opioid receptors on these cells [1 2 3 4 ]. The ligands for chemokine and opioid receptors share the ability to induce chemotaxis [5 ] and have also been found to influence the ligand–receptor interactions between subgroups [6 ]. These findings, along with the growing data about the influence of opioids on the immune system, including inhibition of antibody responses [7 ], altered macrophage functions [8 , 9 ], and modulation of cytokine production [10 , 11 ], led us to study the effect of opioids on chemokine and chemokine receptor expression.

Opioid modulation of proinflammatory cytokines is a vital compontent of the immune response as well as host defense against infectious agents. Previous studies have shown that endogenous endorphins and enkephalins increase the production of IL-1, IL-2, and IFN-{gamma} [12 13 14 ]. Our lab has reported that the µ-opioid receptor (MOR)-selective agonist D-Ala2,N-Me-Phe4-Gly-ol5 enkephalin (DAMGO) can enhance CCL2, CXCL10, and CCL5 production by PBMCs at the RNA and protein level, and this effect is blocked by administration of the MOR-selective antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) [15 ].

The chemokine receptors CCR5 and CXCR4 are of particular interest, not only because of their role in inflammatory diseases processes but also because of their critical role as the major coreceptors for HIV-1. Several reports have shown that opioids modulate chemokine receptor expression. For example, CCR5 protein levels were elevated when primate PBMCs were treated with morphine for 24 h [16 ]. Steele et al. [17 ] found that morphine and DAMGO treatment of human PBMCs induces up-regulation of CCR5 and CXCR4 mRNA expression based on RNase protection analysis. This was the first study to link the specific activation of MOR to the induction of CCR5 and CXCR4 in human primary cells. More recent studies have also found that morphine enhances CCR5 expression in a monocytic cell line [18 ]; however, the biochemical mechanism of µ-opioid induction of chemokine and chemokine receptor expression is unknown.

It is well known that the µ-opioids can modulate the expression of various cytokines, including IFN-{gamma}, IL-2, and IL-12 [19 20 21 22 23 ]. Moreover, there is evidence that TGF-β may play an important role in these modulatory effects. For example, morphine has been shown to inhibit production of TNF-{alpha} secretion by PBMCs in response to LPS or PHA stimulation, and this morphine-induced, suppressive effect was partially reversed by neutralizing antibodies to TGF-β [24 ]. It has been shown that human PBMCs treated with morphine express increased levels of TGF-β in response to LPS or PHA [11 ]. Additionally, Singhal et al. [25 ] have shown that morphine-induced peritoneal macrophage apoptosis was mediated through the generation of TGF-β.

Based on these studies, we hypothesized that the biochemical mechanism of µ-opioid-induced chemokine and chemokine receptor expression may involve the participation of TGF-β. Our present data show that DAMGO induces CCL5 and CXCR4 expression by a TGF-β1-dependent mechanism. These results are consistent with evidence suggesting a proinflammatory role for MOR and also implicate TGF-β in a novel role as a positive regulator of the proinflammatory chemokine CCL5 and the homeostatic chemokine receptor CXCR4.


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MATERIALS AND METHODS
 
Antibodies
The antibodies used for the chemokine ELISAs were capture anti-CXCL10 (clone 4D5), capture anti-CCL2 (clone 10F7), capture anti-CCL5, biotin-conjugated detection anti-CXCL10 (clone 6D4), biotin-conjugated detection anti-CCL2 (clone 5D3-F7), and biotin-conjugated detection anti-CCL5 (PharMingen, San Diego, CA, USA).

PBMC isolation and culture
PBMCs were obtained from whole blood of normal donors under aseptic conditions. Whole blood was layered over Ficoll-Paque Plus (Pharmacia Biotech, Piscataway, NJ, USA), and PBMCs were isolated by density gradient centrifugation. PBMCs were washed twice and plated at a cell density of 3 x 106 cells/ml in 24-well tissue-culture plates. Cell cultures were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated (56°C, 30 min), endotoxin-free FBS (Hyclone, Logan, UT, USA), 10 µg/ml gentamicin, and 1 mM L-glutamine. Cell cultures were maintained at 37°C, 5% CO2.

Cell culture treatments
PBMCs were treated with DAMGO (Multiple Peptide Systems, San Diego, CA, USA), diluted in Dulbecco’s PBS solution, and administered at designated concentrations. In designated experiments, PBMCs were pretreated with the antagonist CTAP (Tocris Bioscience, Ellisville, MO, USA) 30 min prior to DAMGO administration to verify the role of MOR in the experimental system.. Alternatively, PBMCs were pretreated for 1 h with human monoclonal anti-TGF-β1 (10 µg/ml) or IgG1 isotype control (10 µg/ml, Sigma Chemical Co., St. Louis, MO, USA) prior to DAMGO administration.

Flow cytometry
Cultures of PBMCs were harvested with versene and washed with "HF buffer," consisting of HBSS (Life Technologies, Gaithersburg, MD, USA) containing 2% endotoxin-free FBS (Hyclone) and resuspended in 50 µl HF. Goat serum (Sigma Chemical Co.) was added to block nonspecific binding, and cultures were incubated at 4°C for 30 min. Cells were treated with the first antibody, incubated at 4°C for 45 min, and washed, and the second antibody was added and incubated at 4°C for 45 min, and excess was washed off. Samples were analyzed with a Coulter Epics XL flow cytometer (Coulter Corp., Hialeah, FL, USA). Antibodies were as follows: PE-CXCR4 (12G5), PE-CCR5 (2D7), FITC-CD3 (UCHT1), or FITC-CD14 (M5E2), all from PharMingen.

ELISA
The concentrations of chemokine and cytokine production were measured by ELISA. PBMCs were treated as stated previously, and supernatants were collected at designated times as indicated. The TGF-β OptEIA ELISA set (PharMingen) was used to determine TGF-β concentrations after DAMGO or LPS stimulation of PBMCs. Briefly, capture antibody was bound overnight at 4°C to 96-well Nunc maxisorp plates, which were washed, and blocking buffer was added for 1 h at room temperature. During this incubation, samples for determination of TGF-β1 levels were activated by the addition of 1 N HCL in a 1:25 dilution for 1 h at 4°C and then neutralized with 1 N NaOH at a 1:25 dilution. The plates were then washed, and the prepared samples and standards were added and incubated at room temperature for 2 h. The plates were then washed, and avidin-HRP-conjugated detection antibody was added and incubated at room temperature for 1 h. The plates were washed, and tetramethylbenzidine and hydrogen peroxide substrate were added for color development. The plate was read using a Molecular Devices spectrophotometer in 10 min intervals at 600 nm to determine when the reaction would be stopped with 2 N H2SO4, and the final reading was taken at 450 nm. For the chemokine ELISAs, samples were not activated prior to addition to the plates, 2'-azino-bis-(3-ethylbenzothiazoline)-6-sulfonate/H2O2 was used at the substrate solution, and the samples were read at 405 nm after stop solution was added.

Real-time PCR
PBMCs were cultured in RPMI containing 10% FBS and penicillin/streptomycin overnight. Cells were treated for 4 or 24 h with the indicated concentrations of DAMGO, with or without cyclohexamide (Sigma Chemical Co.) or anti-TGF-β1 antibody (5 µg/ml, Sigma Chemical Co.). RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and the quality of the RNA was examined using a diethylpyrocarbonate-treated 1% agarose gel. RNA was then subjected to DNase treatment using Turbo DNA-free (Ambion, Austin, TX, USA), quantitated with a spectrophotometer, and 1 µg was used for cDNA synthesis using Invitrogen SuperScript first-strand synthesis kit. Quantitation of cDNA using real-time PCR was performed with Roche (Indianapolis, IN, USA) LightCycler FastStart DNA master SYBR green kit. PCR primers were used as outlined in Table 1 . PCR conditions were 95°C for 10 min, 90°C for 30 s, followed by 50 cycles of 95°C for 0 s, 62°C for 5 s, and 72°C for 10 s, followed by the melt curve at 95°C for 0 s, 65°C for 15 s, and 95°C for 0 s at 0.1°C/s slope and cooling at 42°C for 30 s. Remaining primers were purchased from SuperArray Bioscience (Frederick, MD, USA), RT2 PCR primers sets, β-actin position 888–907, CCR5 position 113–135, and CXCR4 position 172–192. PCR conditions for the SuperArray primer sets were 95°C for 15 min, 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by the melt curve analysis and cooling as described previously. Data were analyzed using Roche LightCycler software, Version 4.05. Target gene cDNA concentrations were determined based on the cross-point and compared with the housekeeping gene β-actin to determine the concentration ratio, which was then correlated with the untreated sample to establish the normalized ratio. The results are presented as a collective analysis of five independent donors.


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  		Table 1. Sequence of Primers Used in Real-Time PCR Reaction and Positions

Statistical analysis
The statistical significance was determined through the use of analysis of variance.


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RESULTS
 
TGF-β1 expression is altered after DAMGO administration
To explore the influence of the MOR on the expression of the cytokine TGF-β1, we investigated the expression of this cytokine by PBMCs and monocytes treated with DAMGO at 6, 24, 48, and 72 h of culture. The results show (Fig. 1 A and 1B ) that the TGF-β1 is produced in response to DAMGO administration, and levels of this cytokine reach a maximum at 24 h. Experiments were also carried out to determine the production of this cytokine over a concentration range of 1 µM to 1 pM, and we found (Fig. 1 C) that DAMGO dose-dependently induced TGF-β1 production from PBMCs and purified monocytes with an optimal concentration of 10 nM. We also wanted to use a µ-selective opioid antagonist, CTAP, to determine if the effects of DAMGO specifically required the MOR. We found that pretreatment with CTAP was able to block the DAMGO-induced expression of TGF-β1 (Fig. 1A and 1B) . Finally, the levels of TGF-β1 induced by DAMGO were comparable with TGF-β1 levels produced after a low dose of PHA or LPS stimulation (data not shown).


Figure 1
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  		Figure 1. DAMGO administration induces TGF-β1 protein expression. PBMCs (A) or monocytes (B) were treated with medium alone ({blacksquare}), DAMGO (10 nM, •), CTAP followed by 30 min with DAMGO ({blacktriangleup}), or CTAP alone ({blacktriangledown}), and supernatants were collected at the designated times, and TGF-β1 protein was determined by ELISA. (C) PBMCs (•) and monocytes ({circ}) were treated with medium alone or the indicated concentrations of DAMGO, and supernatants were collected at 24 h, and the TGF-β1 level was determined. The results are representative of at least four donors and are determined in triplicate or quadruplicate ± SD; *, P < 0.05.

Role of TGF-β1 in the induction of chemokine expression by DAMGO
Previously, we showed that DAMGO administration induces CCL5 expression in PBMCs with maximal chemokine expression at 72 h [15 ]. To determine whether TGF-β1 is required for the effects of DAMGO on chemokine expression, experiments were carried out using an anti-human TGF-β1 mAb, which neutralizes TGF-β1 activity. PBMCs were pretreated for 1 h with anti-TGF-β1 or an IgG1 isotype control Ig, followed by DAMGO administration. Supernatants were removed after 72 h in culture, and CCL5 levels were determined by ELISA (Fig. 2 ). DAMGO treatment resulted in a significant elevation in CCL5, and the presence of the isotype control had no effect on the DAMGO-induced expression of these chemokines (Fig. 2) . However, pretreatment of PBMCs with anti-TGF-β1 resulted in a significant inhibition of the DAMGO-induced increase in CCL5 levels (Fig. 2) . In contrast, we found that increases in CCL2 and CXCL10 production after DAMGO administration were not altered after pretreatment with anti-TGF-β1 (data not shown), suggesting that the DAMGO-induced elevation in the expression of these chemokines was not mediated entirely by TGF-β1.


Figure 2
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  		Figure 2. Preincubation with neutralizing anti-TGF-β1 blocks the DAMGO-induced induction of CCL5 protein expression. PBMCs were pretreated with 10 µM anti-TGF-β1 antibody or isotype-matched Ig (IgG1) for 1 h prior to administration of 1 µM DAMGO. Supernatants were harvested after an additional 72 h in culture, and CCL5 protein levels were determined by ELISA. Values represent the mean (±SD) of the triplicates cultures, and results are representative of five different donors; *, P < 0.05.

We were also interested in determining whether DAMGO treatment regulated the transcription of CCL5 mRNA in a TGF-β1-dependent manner. PBMCs were pretreated with anti-TGF-β1 or cycloheximide followed by DAMGO administration. Total RNA was isolated, and cDNA was prepared for quantitation. Real-time PCR was used to measure CCL5 transcript levels and then normalized to the housekeeping gene β-actin. We found that DAMGO increased CCL5 mRNA levels, and the addition of anti-TGF-β1 resulted in significant inhibition of this effect (Fig. 3A ). Further, the results from the administration of cycloheximide confirmed the requirement for new protein synthesis in the process of DAMGO-induced TGF-β1 expression. We also examined TGF-β1 mRNA transcript levels using real-time PCR and found that DAMGO elevated TGF-β1 mRNA levels as well (Fig. 3B) , suggesting that TGF-β plays a significant role in its own induction. Taken together, these data suggest that TGF-β1 plays an important role in the DAMGO-induced elevation in CCL5 expression at the protein and the mRNA levels.


Figure 3
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  		Figure 3. Neutralization of TGF-β1 and cycloheximide (CHX) blocks the DAMGO-induced increase in CCL5 and TGF-β1 mRNA transcript levels. PBMCs were treated with 10 µM anti-TGF-β antibody for 30 min or 100 ug/ml cycloheximide for 30 min, prior to administration of 100 nM DAMGO. RNA was harvested after 24 h, cDNA was synthesized, and real-time PCR was performed. CCL5 (A) and TGF-β (B) cDNA concentrations were determined based on the cross-point from the thermocycler and compared with the housekeeping gene β-actin to determine the concentration ratio, which was then compared with the untreated sample to establish the normalized ratio. The results show the mean values (±SD) of determinations from five different donors; *, P < 0.05, compared with the DAMGO-only group.

To confirm the apparent requirement for TGF-β in the induction of CCL5 expression, we wished to determine whether TGF-β might directly up-regulate CCL5 levels. Recombinant human TGF-β1 was administered to PBMCs, and chemokine production was determined. The results show that TGF-β1 significantly induced CCL5 expression but failed to induce the production of CCL2 or CXCL10 expression (Fig. 4 ).


Figure 4
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  		Figure 4. TGF-β1 induces CCL5 protein expression, and CCL2 and CXCL10 levels are not significantly altered. PBMCs were treated with the designated concentrations of TGF-β1, and supernatants were harvested at 24 h, and the concentrations of CCL2 (A), CXCL10 (B), and CCL5 (C) protein were determined. Values represent the mean (±SD) of the triplicates cultures, and results are representative of three different donors; *, P < 0.05.

Role of TGF-β in the induction of chemokine receptors by DAMGO
Our laboratory has previously reported that DAMGO administration elevates the expression of chemokine receptors CXCR4 and CCR5 in monocytes and T cells [17 ]. In an effort to extend these studies, PBMCs were pretreated with anti-TGF-β1 for 1 h prior to DAMGO administration, and flow cytometry was used to determine the expression of CCR5 and CXCR4. The results show (Fig. 5A ) that anti-TGF-β reduced DAMGO-induced CXCR4 expression to baseline levels. However, the same was not true for DAMGO-induced CCR5 expression, in that anti-TGF-β1 had a partial (but not statistically significant) effect on the increase in cells expressing CCR5 (Fig. 5B) . Additional experiments were carried out to determine whether TGF-β was also involved in the regulation of CXCR4 at the RNA level. Human PBMCs were pretreated with anti-TGF-β1 or cycloheximide, followed by DAMGO administration, and real-time PCR was used to measure CXCR4 and CCR5 transcript levels. The results show (Fig. 6 ) that DAMGO induced mRNA levels for CXCR4 and CCR5, and the presence of cycloheximide resulted in significant inhibition of this effect. Pretreatment of PBMCs with anti-TGF-β completely abrogated the effect of DAMGO on CXCR4 expression, and it only partially inhibited CCR5 mRNA expression, suggesting that TGF-β1 plays only a partial role in the DAMGO-induced induction of CCR5 transcription (Fig. 6B) .


Figure 5
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  		Figure 5. Neutralization of TGF-β1 blocks the DAMGO-induced elevation in CXCR4 expression. PBMCs were treated with 10 µM anti-TGF-β1 antibody or isotype-matched antibody for 1 h prior to administration of 1 µM DAMGO. After an additional 48 h, cells were analyzed for CD14 and CXCR4 (A) or CCR5 (B) by two-color flow cytometric analysis. The results are representative of three donors and are shown as the mean of triplicate cultures ± SD;*, P < 0.05, compared with the control group.


Figure 6
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  		Figure 6. Neutralization of TGF-β and cycloheximide blocks the DAMGO induction of CXCR4 and CCR5 transcript levels. PBMCs were treated with 10 µM anti-TGF-β1 antibody for 30 min or 100 ug/ml cycloheximide for 30 min prior to administration of 100 nM DAMGO. RNA was harvested after 24 h, cDNA was synthesized, and real-time PCR was performed to determine the expression of CXCR4 (A) or CCR5 (B). Results are representative of four different donors; *, P < 0.05; **, P < 0.01, compared with the DAMGO-only group.

TGF-β1 up-regulation of CXCR4 expression
We also wanted to assess whether TGF-β1 directly induces chemokine receptor expression at concentrations of TGF-β shown above (Fig. 1) to be produced in response to DAMGO. TGF-β1 was administered to PBMCs at concentrations from 100 pg/ml to 400 pg/ml for 24 and 48 h, and cells were then harvested for two-color flow cytometry analysis. The results show (Fig. 7 ) that CXCR4 expression was elevated (by approximately twofold) after TGF-β1 administration on T cells and monocytes with a maximal response at 200 pg/ml TGF-β1. In contrast, CCR5 levels were not altered significantly at any dose tested. These studies are consistent with the results from the neutralizing anti-TGF-β antibody studies described above and confirm that DAMGO-induced TGF-β1 production plays a role in DAMGO-induced CXCR4 production.


Figure 7
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  		Figure 7. TGF-β1 induces CXCR4 but not CCR5 expression. PBMCs were treated with the designated concentrations of TGF-β1, and the expression of CXCR4 (A) or CCR5 (B) was determined for CD14- or CD3-positive leukocytes after 48 h by two-color flow cytometry. The results are representative of four donors and are shown as the mean of triplicate cultures ± SD;*, P < 0.05.


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DISCUSSION
 
To address the mechanism by which the binding of DAMGO to the MOR results in the significant elevation in CCL5, CCL2, CXCL10, CXCR4, and CCR5 expression after 48–72 h in culture, experiments were carried out to determine the role of cytokines in opioid-induced chemokine and chemokine receptor regulation. Several studies have shown that endogenous and exogenous opioids can alter cytokine production in cells of the immune system [26 , 27 ]. These data along with several others are consistent with a growing body of results, which suggest that a portion of the modulatory effects mediated by opioid receptor ligands involves the selective regulation of cytokine expression, and we hypothesize that the DAMGO-induced elevation in chemokine and chemokine receptor expression may be mediated through the indirect participation of cytokines.

We found that DAMGO significantly induced expression of the cytokine TGF-β at the protein and mRNA levels, consistent with the observations by Chao et al. [11 ], who reported that morphine elevates the expression of this cytokine. The present studies show that the ability of DAMGO to alter TGF-β1 production is mediated through MOR, based on the ability of CTAP, a µ-selective antagonist, to block the induction of TGF-β1 production. Additionally, PBMCs treated with TGF-β1 resulted in a significant elevation of CCL5 production but had no effect on CCL2 or CXCL10 levels. Furthermore, TGF-β1 was also able to elevate CXCR4 but not CCR5 expression in T cells and monocytes.

Our earlier reports [15 , 28 ] showed that DAMGO induction of CCL5 and CXCR4 expression reached a maximum after 48–72 h of culture, and these observations are consistent with the results in the present report. The induction of TGF-β reaches a maximum at 24 h, and the subsequent induction of CCL5 and CXCR4 reaches maximum levels following an additional 24–48 h of culture. In an effort to better define the role of TGF-β1 in the DAMGO-induced elevation of chemokine and chemokine receptor expression, we carried out studies using a neutralizing anti-TGF-β1 antibody. PBMCs treated with DAMGO in the presence of this antibody failed to exhibit a normal increase in the expression of CCL5 or CXCR4, suggesting that the MOR-mediated induction of CCL5 and CXCR4 is linked to the presence of TGF-β1. These data indicate that TGF-β1 plays a critical role in MOR-mediated regulation of the expression of some of the chemokines and chemokine receptors. However, the results suggest that other factors play a role in DAMGO induction of CCL2, CXCL10, and CCR5 expression.

TGF-β1 is pleiotropic cytokine affecting processes, including regulation of growth and development of cells of the immune system [29 , 30 ]. TGF-β is most often associated with suppression of immune cell activation and typically inhibits the expression of proinflammatory cytokines [31 32 33 34 ]. However, the cellular response to TGF-β is diverse, depending on the cellular context, and factors in the physiological environment often contribute to differential regulation of TGF-β expression. Wahl et al. [35 ] were the first to show that TGF-β acts as a chemoattractant for human peripheral blood monocytes and plays an important role in the regulation of monocyte recruitment to the site of injury or inflammation. Additionally, TGF-β can induce transcription of proinflammatory regulators such as IL-1 and IL-6 in human monocytes [35 , 36 ], and previous studies have also reported a role for TGF-β in the regulation of chemokine receptor expression. Wang et al. [37 ] showed that TGF-β up-regulates the chemokine receptors CCR5 and CXCR4 in CD4+ T lymphocytes, and this effect was implicated in the pathogenesis of HIV-1 infection. TGF-β has also been shown to increase CXCR4 expression in human monocyte-derived macrophages as well as increased responsiveness to CXCL12 (ligand for CXCR4) and susceptibility to HIV-1 [38 ]. However, in each of these studies, the concentration reported to induce chemokine expression was an order of magnitude greater than the doses found to be effective in the present report. In our experiments, we chose concentrations that we found were induced by DAMGO administration, as we considered these doses to be more relevant to the effects of MOR activation. We cannot rule out additional regulatory activities for TGF-β at much higher doses. For example, Sato et al. [39 ] reported evidence that high concentrations of TGF-β were able to control chemotaxis of human monocyte-derived dentritic cells through the regulation of chemokine receptors, including CCR5 and CXCR4.

Our results do not rule out a role for other cytokines in mediating DAMGO-induced effects on the expression of other chemokines or chemokine receptors. For example, there are several reports that show that TNF-{alpha} up-regulates the production of CCL2, CXCL10 [40 ], and CCR5 expression [41 , 42 ]. In addition, IFN-{gamma} has been shown to induce CCR5 expression in primary monocytes [43 ], and previous studies have shown that opioids elevate levels of TNF-{alpha} and IFN-{gamma} [12 , 44 ], although these cytokines have been found by other investigators to have the opposing activity (reviewed in refs. [26 , 27 ]). The capacity of opioids to alter cytokine, chemokine, and chemokine receptor expression further confirms the complex role that opioids play in regulating the immune response.

It is particularly interesting that TGF-β, a cytokine with well-documented, anti-inflammatory activity, has the capacity to induce CCL5 expression. T cells and macrophages appear to be particularly susceptible to the inhibitory activity of this cytokine, and this includes a reduction in T cell-proliferative responses, at least in part because of inhibition of IL-2 transcription associated with reduced activity of the octamer-binding enhancer [45 ]. In addition, thymocytes from TGF-β1-deficient mice hyperproliferate upon TGF-β stimulation, leading to the development of an autoimmune phenotype [46 ]. This cytokine also is a potent inhibitor of T cell differentiation and polarization and the subsequent acquisition of Th1 or Th2 functions. This is likely a result of a combination of mechanisms, but most notable is the documented inhibition of Th2 cell function through the inhibition of GATA-3 expression [47 , 48 ], a transcription factor that is mandatory for normal Th2 polarization. TGF-β also inhibits Th1 polarization and functional activity through an inhibition of t-bet and STAT4, transcription factors that are required for the expression of IL-12R [49 ]. TGF-β is also a general inhibitor of macrophage function, including a reduction of phagocytosis [50 ], macrophage activation [51 ], and inhibition of MyD88-dependent Toll receptor signaling by promoting the degradation of this key signaling protein [52 ]. In fact, TGF-β1-deficient mice develop a multifocal, inflammatory disease associated with a generalized up-regulation of proinflammatory cytokine expression [53 , 54 ].

It should be pointed out that TGF-β has proinflammatory activity for nondifferentiated monocytes. This includes overt chemoattractant activity for monocytes [35 , 55 ] and the induction of adhesion molecules, including LFA-1 and the fibronectin receptor on monocytes, an effect that would promote attachment to endothelial cells [56 ]. TGF-β up-regulates the expression of proinflammatory cytokines produced by monocytes [35 , 36 , 57 ], and in view of these studies, the expression of a proinflammatory cytokine such as CCL5 is not altogether surprising in our present report. However, we report that monocytes and T cells respond to TGF-β and exhibit elevated CXCR4 expression. Perhaps this is not entirely unexpected, as CXCR4 does not appear at this time to possess substantial inflammatory activity for T cells or cells of the monocyte lineage. On the other hand, CCL5 appears to possess significant proinflammatory activity, based on the ability of this chemokine to participate in recruitment of a number of leukocyte populations, including monocytes, macrophages, and activated T cells. This suggests that TGF-β may exhibit diverse effects on inflammation, depending on the particular combination of cell population and the nature of the inducing agent. The precise mechanism of regulation of CCL5 is not understood at this time, and further molecular analysis may reveal greater clues to this apparent paradox.

We have recently suggested that activation of MOR leads to a predominantly proinflammatory response, based on a number of parameters, including elevated levels of a number of proinflammatory cytokines and cytokine receptors [26 ]. However, it should be noted that MOR agonists have pro- and anti-inflammatory activities, and the precise nature of the outcome of exposure to opioids is not well understood in each case. It is not entirely surprising that activation of MOR leads to the expression of TGF-β, a cytokine that is produced during wound healing and at sites of inflammation. It is at these sites in the periphery where levels of endogenous opioids are elevated, and the influx of inflammatory cells is most likely to be under opioid control [58 ].


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ACKNOWLEDGEMENTS
 
This research was supported by National Institute on Drug Abuse grants DA16544, DA13429, DA14230, T32AI07101, T32DA07237, and F31DA05894.


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FOOTNOTES
 
1 Current address: United States Patent and Transfer Office, Alexandria, VA 22314, USA. Back

2 Current address: Department of Medicine, Division of Infectious Diseases and HIV Medicine, Drexel University College of Medicine, Philadelphia, PA 19102, USA. Back

Received October 10, 2007; revised December 5, 2007; accepted December 5, 2007.


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