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The Journal of Immunology, 2002, 169: 3589-3599.
Copyright © 2002 by The American Association of Immunologists

Morphine Regulates Gene Expression of {alpha}- and {beta}-Chemokines and Their Receptors on Astroglial Cells Via the Opioid µ Receptor1

Supriya D. Mahajan*, Stanley A. Schwartz*, Thomas C. Shanahan{dagger}, Ram P. Chawda* and Madhavan P. N. Nair2,*

* Department of Medicine, Division of Allergy, Immunology, and Rheumatology, and {dagger} Department of Histocompatibility and Immunogenetics, State University of New York and Buffalo General Hospital, Kaleida Health System, Buffalo, NY 14203


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The brain is a target organ for recreational drugs and HIV-1. Epidemiological data demonstrate that opioid abuse is a risk factor for HIV-1 infection and progression to AIDS. Chemokines and their receptors have been implicated in the neuropathogenesis of HIV-1 infections. However, little is known about the effects of opioids on the expression of chemokines and their receptors (the latter also are HIV-1 coreceptors) by cells of the CNS. Herein we describe the effects of morphine on gene expression of the {alpha}- and {beta}-chemokines and their receptors by the astrocytoma cell line U87 and by primary normal human astrocyte (NHA) cultures. U87 cells treated with morphine showed significant down-regulation of IL-8 gene expression, whereas expression of the IL-8 receptor CXCR2 was reciprocally up-regulated as detected by RT-PCR. Treatment of NHAs with morphine suppressed IL-8 and macrophage-inflammatory protein-1{beta} gene expression, whereas expression of their receptor genes, CCR3 and CCR5, was simultaneously enhanced. These morphine-induced effects on U87 and NHA cells were reversed by the opioid µ receptor antagonist {beta}-funaltrexamine. Morphine also enhanced the constitutive expression of the opioid µ receptor on astroglial cells. Our results support the hypothesis that opioids play a significant role in the susceptibility of the CNS to HIV-1 infection and subsequent encephalopathy by inhibiting local production of HIV-1-protective chemokines (IL-8 and macrophage-inflammatory protein-1{beta}) and enhancing expression of HIV-1 entry coreceptor genes (CCR3, CCR5, and CXCR2) within the CNS. These effects of opioids appear to be mediated through the opioid µ receptor that we demonstrated on astroglial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Encephalopathy is a common feature of many retrovirus infections and is an AIDS-defining condition associated with HIV-1 infections. Studies on the distribution, physiology, and modulation of the expression of chemokines and their receptors in the human brain are fundamental to an understanding of the immunopathogenesis of HIV-1 infection of the CNS (1, 2, 3). Chemokines are chemotactic, proinflammatory cytokines that can inhibit HIV-1 infection in vitro (4). IL-8 was one of the first chemokines to be characterized and today >30 human chemokines have been described (5, 6). HIV-1-infected patients express elevated levels of IL-8 that may responsible for some clinical manifestations of AIDS (7). Studies by Benfield et al. (8) indicate that HIV-positive patients infected with Pneumocystis carinii express higher levels of IL-8 in bronchoalveolar lavage fluid as compared with HIV-negative individuals infected with P. carinii. HIV-derived viral proteins activate endothelial cells in the CNS to produce IL-8, which acts as a stimulator and chemoattractant for neutrophils and lymphocytes (7). However, there is limited information on the participation of chemokines in inflammatory processes within the CNS. Both microglia and astrocytes produce {alpha}- and {beta}-chemokines (9).

Chemokine receptors were recently identified as important HIV-1 coreceptors that, in conjunction with the CD4 receptor, mediate entry of the virus into target cells (6). The pattern of chemokine receptor expression in the brain likely determines the tropism of HIV-1 for particular CNS target cells and may induce associated inflammatory and degenerative mechanisms. Fusion and entry of HIV-1 into CD4+ T lymphocytes requires expression of CD4 and a coreceptor. Several members of the chemokine receptor family have been implicated as coreceptors for HIV-1 infection in the CNS (1, 10, 11, 12, 13). The major coreceptors for HIV-1 infection, such as CCR3, CCR5, CXCR2, and CXCR4, have been detected in the human brain on a variety of resident cell types including microglia, astrocytes, neurons, and vascular endothelial cells that line the wall of lymphatic and blood vessels (14). Thus, astrocytes, glial cells, and neuronal cells all play an important role in HIV pathogenesis (2, 4, 14, 15, 16, 17). Chemokines and their receptors are produced in intact normal parenchyma and in the brains of HIV-1-infected patients (18, 19).

Among the drugs of abuse, opioids have been postulated to promote HIV-1 infection and disease progression, including secondary opportunistic infections in HIV-1-infected i.v. drug users (20, 21, 22, 23, 24, 25). Morphine can cross the blood brain barrier in sufficient amounts to affect brain function (25, 26). Opioids can modulate the immune response by indirect and direct mechanisms (27, 28). Indirect modulation occurs when activation of opioid receptors within the CNS modifies the activity of neuroendocrine axes or neurotransmission pathways and then secondarily affects immune functions (27, 28). Direct modulation results from the effects of opioids on cells of the immune system, resulting in their dysregulation (22). We hypothesize that the use of opioids by i.v. drug users is a significant cofactor in susceptibility of the CNS to HIV-1 infection and subsequent encephalopathy by inhibiting local production of HIV-1-protective chemokines within the brain and/or CNS and enhancing the expression of HIV-1 coreceptors/chemokine receptors. In the present investigation, we investigate the in vitro effects of morphine on gene expression and production of the {alpha}- and {beta}-chemokines, IL-8 and macrophage inflammatory protein-1{beta} (MIP-1{beta}),3 respectively, and their specific receptors, CXCR2 (IL-8), CCR3 (MIP-1{beta}), and CCR5 (MIP-1{alpha} and {beta}), in the astrocytoma cell line U87 and primary normal human astrocyte (NHA) cultures.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

The astrocytoma cell line U87 (ATCC Human Tumor Cell Bank-14) was obtained from the American Type Culture Collection (Manassas, VA). Cultures are maintained in Eagle’s MEM with nonessential amino acids, 1 mM sodium pyruvate, and 10% FBS (complete medium). Primary NHA cultures were obtained from Clonetics-BioWhittaker (San Diego, CA). These astrocyte cultures are established from normal human brain tissue and are cryopreserved after secondary or tertiary passage. NHA cultures were used within 10 population doublings because growth rate, biological responsiveness, and function deteriorate with subsequent passages (29). NHAs are grown in special Astrocyte Basal Medium (Clonetics-BioWhittaker) supplemented with 10 µg/ml human epidermal growth factor, 10 mg/ml insulin, 25 µg/ml progesterone, 50 mg/ml transferrin, 50 mg/ml gentamicin, 50 µg/ml amphotericin, and 10% FBS. Both U87 and NHA cells (3.2 x 10-6/60-mm dish) were cultured in complete medium with morphine at concentrations ranging from 10-7 to 10-15 M for 24–96 h at 37°C in a 5% CO2 incubator. Supernatants were harvested and stored at -70°C and RNA was extracted from the cells for RT-PCR.

RNA extraction

Cytoplasmic RNA was extracted by an acid guanidinium-thiocyanate-phenol-chloroform method as described (30). Cultured cells were sedimented by centrifugation and resuspended in a 4 M solution of guanidinium thiocynate. Cells were lysed by repeated pipetting and then phenol-chloroform extracted in the presence of sodium acetate. After centrifugation, RNA was precipitated from the aqueous layer by adding an equal volume of isopropanol, and the mixture was kept at -20°C for 1 h and then centrifuged to sediment the RNA. The RNA pellet was washed with 75% ethanol to remove any traces of guanidinium. The final pellet was dried and resuspended in diethyl pyrocarbonate water, and the amount of RNA was determined using a spectrophotometer at 260 nm. Isolated RNA was kept at -70°C until used.

RT-PCR

Extracted RNA was used for RT-PCR as described by the manufacturer using a PerkinElmer kit (catalog no. N808-0143; Wellesley, MA). RNA was reverse transcribed to make a DNA copy for use in PCR. Briefly, 1 µg of RNA was added to a tube containing 5 mM MgCl2, 1 mM each of dNTP (A, T, G, C), 50 mM KCl, 10 mM Tris buffer (pH 8.3), 2.5 µM oligo(dT), 20 U of RNase-inhibitor, and 50 U of murine leukemia virus reverse transcriptase. The mixture was incubated at 45°C for 35 min, heated to 95°C for 5 min, and placed on ice until used for PCR. The newly synthesized cDNA was then amplified by PCR using specific sense and antisense primers for the genes of interest along with a housekeeping gene, G3PDH or {beta}-actin, as a control (Table IGo). Briefly, to each tube a 10-µl sample of the RT product in a final concentration of 2 mM MgCl2, 10 mM Tris (pH 8.3), 50 mM KCl, plus 0.02 µM of both the 5' and 3' primers, and 2.5 U of Taq polymerase was added. The mixture was placed in a thermocycler for 30 cycles of 95°C for 30 s, 60°C for 30 s and 74°C for 1 min. The PCR conditions were modified slightly for the CXCR2 gene to obtain optimal results. Samples were separated by 1.0–1.2% agarose gradient gel electrophoresis along with molecular size markers for reference. Resultant bands were visualized with UV light, photographed, size determined, and OD quantified using a scanning densitometer. All values were normalized to the constitutive expression of the housekeeping gene.


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  		Table I. Sequences of primers used in RT-PCR

 
FACS analysis

Multicolor immunofluorescent staining was used to identify and quantify the number of U87 and NHA cells expressing intracellular IL-8 and the surface marker CXCR2 in response to treatment with morphine. Approximately 1 x 106 U87 cells were treated with 10-7 M morphine for 24 h, and cells were harvested, trypsinized, washed, and suspended in staining buffer. FACS conditions were optimized by adjusting the settings for photomultiplier tube voltage and compensation using appropriate cell surface staining controls, and quadrant markers were set using specified isotype controls for each flurochrome-conjugated Ab used. The mAbs against IL-8 and CXCR2 were conjugated to either FITC or PE, and matched isotype controls were obtained from BD PharMingen (San Diego, CA). For CXCR2 surface marker analysis, Fc receptors were preblocked by incubating cells with an excess of irrelevant purified IgG and then were stained with 0.5 µg of FITC-conjugated mAb specific for the cell surface Ag CXCR2. Cells were washed twice with staining buffer and pelleted by centrifugation at 250 x g, and the supernatant fluid was removed. Cells stained for intracellular IL-8 were treated with a cell activator, PMA (50 ng/ml), plus calcium ionophore (1 µg/ml) in the presence of a protein transport inhibitor, 2 mM monensin, and incubated for 4 h. Note that the use of PMA plus calcium ionophore is a routine procedure to enhance the phenotypic expression of many inracellular cytokines before FACS analysis (31); it is not used for the primary induction of cytokines. Cells were then washed in PBS and fixed by resuspending them in 100 µl of Cytofix/Cytoperm (Perm) solution (BD PharMingen) for 10–20 min at 4°C. Cells were permeabilized by washing twice in 1x Perm solution and were then resuspended in staining buffer before flow cytometric analysis using a FACSCalibur instrument (BD Biosciences, San Jose, CA). Stained cells were subjected to light scatter analysis, and a fixed population of cells was gated after quandrant markers were set, based on the isotype control, and represented as fluoresence (FL)-2 (PE-labeled) on the y-axis and FL-1 (FITC-labeled) on the x-axis. Cells positive for IL-8 and CXCR2 were expressed as a percentage of the total cells gated.

ELISA

IL-8 and MIP-1{beta} protein secretion in culture supernatants was quantitated as described by the manufacturer using highly specific and sensitive ELISA kits obtained from BioSource (Camarillo, CA). The sensitivity of the IL-8 assay was >=0.1 pg/ml and that of the MIP-1{beta} assay was >=4 pg/ml.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Morphine down-regulates IL-8 gene expression by U87 astrocytoma cells

Several {alpha}-chemokine receptors serve as coreceptors for T-tropic HIV-1 strains (32). The {alpha}-chemokine IL-8 can suppress HIV-1 infection by blocking its specific receptor, CXCR2 (5, 33). We undertook the following experiments to determine whether morphine can inhibit the gene expression of the HIV-1 protective chemokine IL-8 by U87 astrocytoma cells. U87 cells were cultured with different concentrations of morphine for 24 h, and RNA was extracted, reverse transcribed, and cDNA amplified by PCR using primers specific for the housekeeping gene G3PDH and IL-8. The G3PDH gene and the IL-8 gene migrated in agarose gel electrophoresis as expected to 983 bp and 279 bp, respectively (Fig. 1Go, A and B). U87 cells cultured with morphine did not affect the constitutive expression of the G3PDH gene (Fig. 1GoA). Treatment of U87 cells with morphine (Fig. 1GoB) at 10-7 M (lane 2, OD = 0.086), 10-9 M (lane 3, OD = 0.131), and 10-11 M (lane 4, OD = 0.207) significantly down-regulated IL-8 gene expression in a dose-dependent manner compared with control cultures (lane 1, OD = 0.310). Morphine at 10-13 M (lane 5, OD = 0.253) and 10-15 M (lane 6, OD = 0.306) concentrations did not affect IL-8 gene expression and were comparable with control cultures. Data presented in Fig. 1GoC show the mean percent change ± SD in OD values from three separate experiments performed as described for Fig. 1Go, A and B. Morphine at 10-7, 10-9, and 10-11 M significantly down-regulated IL-8 gene expression compared with control cultures (lane 1); suppression was 29% (p < 0.01), 47% (p < 0.01), and 68% (p < 0.05), respectively.



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  		FIGURE 1. Morphine inhibits IL-8 gene expression in U87 cells. U87 cells (3 x 106/ml) were cultured with or without morphine for 24 h. RNA was extracted, reverse transcribed, and amplified by PCR using housekeeping gene G3PDH and IL-8-specific primers. PCR products were separated by electrophoresis on a 1.2% agarose gel. A, G3PDH expression remained unchanged at all morphine concentrations. B, Morphine significantly inhibits IL-8 gene expression in a concentration-dependent manner. C, Quantitation of the effect of morphine on IL-8 gene expression by densitometry after nomalization with corresponding values of G3PDH expression in A; these data are the mean ± SD of three separate experiments done in duplicate.

 
Morphine down-regulates IL-8 gene expression by NHAs

To determine whether the HIV-1-protective chemokine IL-8 is expressed by NHAs and whether morphine can inhibit its gene expression in a dose- and time-dependent manner, NHA cells were cultured with different concentrations of morphine (10-7–10-15 M) for 24, 48, and 72 h. RNA was extracted and subjected to RT-PCR using primers specific for the housekeeping gene {beta}-actin and IL-8. The products of the {beta}-actin and the IL-8 genes migrated in agarose gel electrophoresis to 548 bp and 279 bp, respectively (Fig. 2Go, A–D). Culture of NHA cells with morphine (10-7–10-15 M) for 24, 48, 72, and 96 h did not affect the constitutive expression of the {beta}-actin gene (Fig. 2Go). However, treatment of NHA cells with morphine at concentrations of 10-7 and 10-9 M suppressed IL-8 gene expression at all time points (Fig. 2Go). Thus, we consider 10-7–10-9 M morphine to be the optimal concentration range that was used in subsequent experiments. Lower concentrations of morphine (10-11–10-15 M) did not significantly affect the expression of IL-8. Data in Fig. 2GoE are the mean percent change ± SD in OD values from three separate experiments, showing that morphine at 10-7 and 10-9 M significantly suppressed (p < 0.001 for both concentrations) IL-8 gene expression as compared with control cultures.



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  		FIGURE 2. Kinetics of morphine inhibition of IL-8 gene expression by NHA cells. NHA (3 x 106/ml) were cultured with or without morphine for 24, 48, 72, and 96 h. RNA was extracted, reverse transcribed, and amplified by PCR with housekeeping gene {beta}-actin and IL-8-specific primers. PCR products were separated by electrophoresis on a 1.2% agarose gel. {beta}-Actin expression remained unchanged at all morphine concentrations. A–D, Morphine significantly inhibits IL-8 gene expression in a concentration-dependent manner at all time periods studied. E, Quantitation of PCR results by densitometry after nomalization with corresponding values of {beta}-actin expression at each time period; these data are the mean ± SD of three separate experiments done in duplicate.

 
Morphine suppresses the synthesis and secretion of IL-8 by U87 cells and NHAs

Data presented in Table IIGo show the effect of treatment with morphine for 72 h on IL-8 protein synthesis and secretion by U87 and NHA cells into culture supernatants as measured by ELISA. Kinetic studies also performed at 48 and 72 h of incubation showed no significant difference in IL-8 production. The constitutive production of IL-8 in untreated, control U87 cultures was readily detectable at a concentration of 8.5 pg/ml. U87 cells treated with 10-7, 10-9, and 10-11 M morphine produced significantly less IL-8 (2.1, 3.7, and 5.6 pg/ml, respectively) compared with untreated control cultures. This corresponds to 76, 57, and 35% suppression of IL-8 production, respectively. Morphine at lower concentrations of 10-13 and 10-15 M did not affect IL-8 production and was comparable to untreated control cultures. Untreated NHA control cultures also constitutively secreted IL-8 detectable at a concentration of 11.5 pg/ml. NHA cells treated with morphine at 10-7 and 10-9 M produced significantly less IL-8 (3.4 and 4.0 pg/ml, respectively) compared with untreated controls, corresponding to 29.5 and 34.5% suppression, respectively. Morphine at lower concentrations of 10-11, 10-13, and 10-15 M did not show any effect on IL-8 production by NHAs and was comparable with control cultures.


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  		Table II. Effect of morphine on IL-8 secretion by NHAs

 
Morphine down-regulates MIP-1{beta} gene expression by NHAs

{beta}-Chemokines like MIP-1{alpha} and MIP-1{beta} are expressed by astrocytes and microglia (34). The following experiments were performed to determine whether morphine can modulate the gene expression of the HIV-1-protective chemokine MIP-1{beta} by primary NHA cultures. NHA cells were incubated with 10-7 and 10-9 M concentrations of morphine for 24 h, and RNA was extracted, reverse transcribed, and cDNA amplified by PCR using primers specific for the housekeeping gene {beta}-actin and MIP-1{beta}. The {beta}-actin and the MIP-1{beta} gene amplification products migrated in agarose gel electrophoresis as expected to 548 bp and 343 bp, respectively (Fig. 3Go, A and B). NHA cells cultured with morphine did not affect the constitutive expression of the {beta}-actin gene (Fig. 3GoA). Treatment of NHA cells with morphine at 10-7 M (lane 2, OD = 0.167) and 10-9 M (lane 3, OD = 0.205) significantly down-regulated MIP-1{beta} gene expression compared with the control cultures (lane 1, OD = 0.497). Data presented in Fig. 3GoE show the mean percent change ± SD in OD values from three separate experiments performed similarly as in Fig. 3Go, A and B. Morphine at 10-7 and 10-9 M significantly down-regulated MIP-1{beta} gene expression by NHAs compared with untreated control cultures (lane 1); the suppression was 67% (p < 0.001) and 58% (p < 0.001), respectively. These data demonstrate that morphine can down-regulate the expression of the HIV-protective chemokine MIP-1{beta} gene in NHA cells.



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  		FIGURE 3. Morphine inhibits MIP-1{beta} and enhances CCR3 and CCR5 gene expression by NHA cells. NHAs (3 x 106/ml) were cultured with or without morphine for 24 h. RNA was extracted, reverse transcribed, and amplified with housekeeping gene {beta}-actin and MIP-1{beta}, CCR3, and CCR5 specific primers. PCR products were separated by electrophoresis on a 1.2% agarose gel where the {beta}-actin, MIP-1{beta}, CCR3, and CCR5 genes migrated as expected to 548, 343, 313, and 1117 bp, respectively. A, {beta}-Actin expression remained unchanged at all morphine concentrations. B, Morphine significantly inhibits MIP-1{beta} gene expression in a concentration-dependent manner. C, Morphine significantly enhances CCR3 gene expression in a concentration-dependent manner. D, Morphine significantly enhances CCR5 gene expression in a concentration-dependent manner. Quantitation of PCR results by densitometry after nomalization with corresponding values of {beta}-actin expression in A; these data are the mean ± SD of three separate experiments done in duplicate.

 
Morphine suppresses the synthesis and secretion of MIP-1{beta} by NHA cells

Data presented in Table IIIGo show the effect of treatment with morphine (10-7–10-13 M) for 72 h on MIP-1 {beta} protein synthesis and secretion by NHA cells in the culture supernatants as measured by ELISA. The constitutive production of MIP-1{beta} in untreated, control NHA cultures was readily detectable at a concentration of 17.3 pg/ml. NHA cells treated with 10-7 and 10-9 M morphine produced significantly lower MIP-1{beta} (6.8 and 11.2 pg/ml, respectively) compared with untreated control cultures. This corresponds to 61 and 36% suppression of MIP-1{beta} production, respectively. Morphine at lower concentrations of 10-13 and 10-15 M did not affect MIP-1{beta} production and was comparable with untreated control cultures.


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  		Table III. Effect of morphine on MIP-1{beta} secretion by NHAs

 
Morphine up-regulates CCR5 and CCR3 gene expression by NHAs

Both C-C and C-X-C chemokine receptors function as coreceptors for HIV-1 infection (3, 4). The following experiments were performed to determine whether morphine modulates the expression of the predominant CCRs, CCR5 and CCR3, on NHA cells. NHA cells were cultured with 10-7 and 10-9 M concentrations of morphine for 24 h, and RNA was extracted, reverse transcribed, and cDNA amplified by PCR using primers specific for the housekeeping gene {beta}-actin and the CCR5 and CCR3 genes. The amplification products for the housekeeping gene {beta}-actin and the CCR5 and CCR3 genes migrated as expected to 548, 1117, and 313 bp, respectively, on agarose gel electrophoresis (Fig. 3Go). Treatment of NHAs with morphine did not affect the constitutively expressed {beta}-actin gene (Fig. 3GoA). NHA cultures treated with morphine (Fig. 3GoC) at 10-7 M (lane 2, OD = 0.936) and 10-9 M (lane 3, OD = 0.811) up-regulated CCR3 gene expression compared with the untreated control culture (lane 1, OD = 0.557). NHA cells treated with morphine (Fig. 3GoD) at 10-7 M (lane 2, OD = 0.850) and 10-9 M (lane 3, OD = 0.631) significantly up-regulated CCR5 gene expression compared with the control culture (lane 1, OD = 0.452). Data presented in Fig. 3GoE show the mean percent change in OD values ± SD from three experiments performed as described in Fig. 3Go, A, C, and D). Morphine at 10-7–10-9 M significantly up-regulated CCR3 gene expression compared with control cultures with the percent up-regulation being 68% (p < 0.001) and 45% (p < 0.01), respectively. Similarly, morphine at 10-7 and 10-9 M significantly up-regulated CCR5 gene expression compared with control cultures with the percent up-regulation being 87% (p < 0.001) and 39% (p < 0.05), respectively.

Morphine up-regulates CXCR2 gene expression by U87 astrocytoma cells

Previous studies have shown that the CXCR2 chemokine receptor functions as a coreceptor for HIV-1 infection and is present on various cells of the CNS (35, 36). The following experiments were undertaken to determine whether morphine modulates the expression of CXCR2 on U87 astrocytoma cells. U87 cells were cultured with different concentrations of morphine for 24 h, and RNA was extracted, reverse transcribed and, cDNA amplified by PCR using primers specific for the housekeeping gene G3PDH and the CXCR2 gene. The amplification products for the housekeeping gene G3PDH and the CXCR2 gene migrated as expected to 983 and 325 bp, respectively, on agarose gel electrophoresis (Fig. 4Go, A and B). Treatment of U87 cells with morphine did not affect the constitutively expressed G3PDH gene. U87 cultures treated with morphine (Fig. 4GoB) at 10-7 M (lane 2, OD = 0.71, 125% increase), and 10-9 M (lane 3, OD = 0.44, 47% increase) significantly up-regulated CXCR2 gene expression compared with the control culture (lane 1, OD = 0.32). Morphine at 10-11 M (lane 4, OD = 0.28), 10-13 M (lane 5, OD = 0.30), and 10-15 M (lane 6, OD = 0.31) concentrations did not affect CXCR2 gene expression and were comparable with control cultures (lane 1, OD = 0.32). Data presented in Fig. 4GoC show the mean percent change in OD values ± SD from three experiments performed as described in Fig. 4Go, A and B. Morphine at 10-7–10-9 M significantly up-regulated CXCR2 gene expression compared with control cultures; the percent increases were 125% (p < 0.01) and 47% (p < 0.05), respectively, for 10-7 and 10-9 M morphine concentrations.



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  		FIGURE 4. Morphine enhances CXCR2 gene expression by U87 cells. U87 cells were cultured with or without morphine for 24 h. Experimental conditions were as described in Fig. 1Go. The G3PDH and CXCR2 genes migrated as expected to 983 bp and 325 bp, respectively. A, G3PDH gene expression. B, Dose-dependent effect of morphine on CXCR2 gene expression. C, Quantitation of PCR results by densitometry after normalization with corresponding values of G3PDH expression; these data are the mean ± SD of three separate experiments done in duplicate.

 
Morphine modulates the phenotypic expression of IL-8 and CXCR2 on U87 astrocytoma cells

Data presented in Figs. 5Go and 6Go show the effect of morphine on the phenotypic expression of intracellular IL-8 and surface CXCR2, respectively, as demonstrated by flow cytometry. U87 cells treated with 10-7 M morphine demonstrated a significant decrease in the percentage of intracellular IL-8-positive cells (Fig. 5GoC, 6.37% of the total cells) compared with untreated U87 cells (Fig. 5GoB, 9.12% of the total cells). Fig. 5GoD shows the mean percent ± SD of IL-8-positive cells from three separate experiments. Morphine at 10-7 M significantly (p < 0.05) decreased the percentage of IL-8-positive cells.



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  		FIGURE 5. Effect of morphine on the phenotypic expression of intracellular IL-8 by U87 cells. U87 cells were cultured with and without 10-7 M morphine for 24 h and subjected to FACS analysis. Briefly, cells stained for intracellular IL-8 were treated with a cell activator, PMA (50 ng/ml), plus calcium ionophore (1 µg/ml) in the presence of a protein transport inhibitor, 2 mM monensin, and were incubated for 4 h. Cells were then washed in PBS and fixed by resuspending them in 100 µl of Perm solution (BD PharMingen) for 10–20 min at 4°C. Cells were permeabilized by washing twice in 1x Perm solution and then were resuspended in staining buffer before flow cytometric analysis. A–C, Upper panel is a scatter plot representing the FL-1 vs FL-2 axis (log scale), whereas the lower panel is a histogram representation of the same data showing cell count vs log fluorescence (FL-2). A, Isotype control used to set the quadrant markers and as a negative control. B, Untreated control cells. C, Cells treated with morphine. D, Mean values ± SD of IL-8-positive cells from three experiments.

 


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  		FIGURE 6. Effect of morphine on the phenotypic expression of CXCR2 on the surface of U87 cells. U87 cells were cultured with and without 10-7 M morphine for 24 h and subjected to FACS analysis. A–C, Upper panel is a scatter plot representing the FL-1 vs FL-2 axis (log scale), whereas the lower panel is a histogram representation of the same data showing cell count vs log fluorescence (FL-2). A, Isotype control used as a negative control to set quadrant markers. B, Untreated control cells showing that 1.34% of the total cells constitutively express CXCR2 on their surface. C, Morphine treatment of U87 cells produces a 74% increase in CXCR2-positive cells to 5.14% of the total. D, Mean values ± SD of CXCR2-positive cells from three experiments.

 
Fig. 6Go shows the effect of morphine on CXCR2 expression on the surface of U87 cells. Morphine-treated (10-7 M) U87 cells demonstrated a significantly increased number of CXCR2-positive cells (Fig. 6GoC, 5.14% of the total cells), as compared with untreated control cultures (Fig. 6GoB, 1.34% of the total cells). Fig. 6GoD shows the mean percent ± SD of CXCR2-positive cells. Morphine at 10-7 M significantly (p < 0.01) increased the percentage of CXCR2-positive cells.

In summary, these results demonstrate that morphine treatment of U87 cells results in opposite effects on the expression of IL-8 and CXCR2, with an increase in the number of CXCR2-positive cells and a reciprocal decrease in IL-8-positive cells. These results are also consistent with our gene expression data as analyzed by RT-PCR (Figs. 1Go and 4Go).

The selective opioid µ receptor antagonist {beta}-funaltrexamine (FNA) inhibits the immunomodulatory activities of morphine on U87 and NHA cells

To demonstrate that the effects of morphine on astroglial cells are mediated through the µ opioid receptor, we undertook the following experiments using the selective µ receptor antagonist {beta}-FNA to see whether it could block the immunoregulatory activities of morphine. U87 cells were cultured with 10-7 and 10-9 M morphine ± 10-6 M {beta}-FNA for 24 h, RNA was extracted and reverse transcribed, and cDNA was amplified by PCR using primers specific for the housekeeping gene G3PDH and the CXCR2 gene. PCR products of the CXCR2 and the G3PDH genes migrated on agarose gel electrophoresis as expected to 983 and 325 bp, respectively (Fig. 7Go, A and B). Treatment of U87 cells with morphine ± {beta}-FNA did not affect the constitutive expression of the G3PDH housekeeping gene (Fig. 7GoA). As shown in Fig. 7GoC and Table IVGo, treatment of U87 cells with 10-7 and 10-9 M morphine alone for 24 h significantly up-regulated CXCR2 gene expression in a dose-dependent manner compared with untreated control cultures. However, addition of 10-6 M {beta}-FNA significantly (p < 0.05 and p < 0.01) reversed these effects. Treatment with 10-6 M {beta}-FNA alone had no effect on the constitutive expression of the CXCR2 gene. Although it may appear that the combination of {beta}-FNA and morphine may have suppressed CXCR2 expression (Fig. 7GoB, lanes 5 and 6) below the level of the untreated control cells (Fig. 7GoB, lane 1), these differences were not statistically significant (Fig. 7GoB, lane 5, p < 0.069, and lane 6, p = 0.202).



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  		FIGURE 7. The opioid µ receptor antagonist {beta}-FNA reverses the effects of morphine on CXCR2 gene expression by U87 cells. U87 cells were cultured with or without morphine and {beta}-FNA for 24 h. RNA was extracted and amplified by RT-PCR with G3PDH- and CXCR2-specific primers. A, U87 cells treated with morphine and/or {beta}-FNA did not affect G3PDH gene expression. B, Morphine treatment of U87 cells up-regulates the expression of CXCR2 gene and this effect is inhibited by the addition of {beta}-FNA to the morphine-treated cells. C, Quantitation of PCR results by densitometry after normalization with corresponding values of G3PDH expression; these data are the mean ± SD of three separate experiments done in duplicate.

 

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  		Table IV. Effect of {beta}-FNA on chemokines and chemokine receptor gene expression in NHA and U87 cells

 
Similar experiments using NHAs confirmed the findings above with U87 cells. NHAs were cultured with morphine and analyzed by PCR as described above for U87 cells. However, primers specific for the housekeeping gene {beta}-actin, the HIV-protective chemokine genes IL-8 and MIP-1{beta}, and the chemokine receptor/HIV coreceptor genes CCR3 and CCR5 were used. Their PCR products migrated on agarose gels as expected to 548, 279, 343, 313, and 1117 bp, respectively (Figs. 8Go, A–C, and 9, A–C). Morphine ± {beta}-FNA did not affect the constitutive expression of the {beta}-actin housekeeping gene (Figs. 8GoA and 9A). However, 10-7 M morphine significantly suppressed the expression of the HIV-protective chemokines IL-8 and MIP-1{beta}, and these effects were completely reversed by {beta}-FNA (Fig. 8Go, B and C). By contrast, as shown in Fig. 9Go and Table IVGo, treatment of NHAs with 10-7 and 10-9 M morphine significantly enhanced the expression of the chemokine receptors/HIV coreceptors CCR3 (Fig. 9GoB) and CCR5 (Fig. 9GoC). As with the chemokines above, 10-6 M {beta}-FNA also completely reversed these effects. Treatment of cells with {beta}-FNA alone enhanced the gene expression of CCR3 and CCR5, and although it suppressed IL-8 gene expression, it did not affect MIP-1{beta} gene expression (Figs. 8Go and 9Go), suggesting that {beta}-FNA, in addition to its role as a morphine antagonist, may potentially act as an partial agonist on its own.



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  		FIGURE 8. {beta}-FNA reverses morphine-induced suppression of IL-8 and MIP-1{beta} gene expression by NHA cells. NHA (3 x 106 cells/ml) were cultured alone or with morphine or with {beta}-FNA or with morphine plus {beta}-FNA for 48 h. RNA was extracted and subjected to RT-PCR using primers for the housekeeping gene {beta}-actin (A), IL-8 (B), and MIP-1{beta} (C). Amplified products were electrophoresed on an agarose gel containing ethidium bromide. A–C, migration profiles of {beta}-actin (548 bp), IL-8 (279 bp), and MIP-1{beta} (343 bp) genes, respectively. Lane MW, molecular size markers; lane 1, untreated control cells; lane 2, 10-7 M morphine; lane 3, 10-9 M morphine; lane 4, 10-6 M {beta}-FNA; lane 5, 10-7 M morphine + 10-6 M {beta}-FNA; lane 6, 10-9 M morphine + 10-6 M {beta}-FNA. D, Percent change in the densitometry readings of the photographic negatives after normalization with corresponding {beta}-actin values. The data are the mean ± SD of three experiments. Statistical significance of the differences between control and morphine-treated cultures, between control and {beta}-FNA-treated cultures, and between morphine plus {beta}-FNA-treated cultures was evaluated by Student’s t test. The results clearly show that morphine can inhibit the expression of IL-8 and MIP-1{beta} and that these effects can be reversed by the specific opioid µ receptor antagonist {beta}-FNA.

 


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  		FIGURE 9. Morphine enhances CCR3 and CCR5 gene expression by NHA cells. NHAs (3 x 106 cells/ml) were cultured alone or with morphine or with {beta}-FNA or with morphine + {beta}-FNA for 48 h. RNA was extracted and subjected to RT-PCR using primers for the housekeeping gene {beta}-actin (A) and the CCR3 (B) and CCR5 (C) genes. Amplified products were electrophoresed on an agarose gel containing ethidium bromide. A–C, Migration profile of the {beta}-actin (548 bp), CCR3 (313 bp), and CCR5 (1117 bp) genes, respectively. Lane MW, Molecular size markers; lane 1, untreated control; lane 2, 10-7 M morphine; lane 3, 10-9 M morphine; lane 4, 10-6 M {beta}-FNA; lane 5, 10-7 M morphine + 10-6 M {beta}-FNA; lane 6, 10-9 M morphine + 10-6 M {beta}-FNA. D, Percentage change in densitometry readings of the PCR results after normalization with corresponding {beta}-actin values. The data are the mean ± SD of three experiments. Statistical significance of differences between control and morphine-treated cultures, between control and {beta}-FNA-treated cultures, and between morphine and morphine + {beta}-FNA-treated cultures were evaluated by Student’s t test. The results clearly show that morphine can enhance the expression of the CCR3 and CCR5 chemokine receptors/HIV coreceptors and that the effects of morphine are specifically reversed by the specific opioid µ receptor antagonist {beta}-FNA.

 
All of the previous experiments were repeated using a {kappa} opioid receptor antagonist, nor-binaltorphimine, instead of {beta}-FNA. Nor-binaltorphimine did not alter morphine-induced effects on U87 and NHA cells (data not shown). This further supports our premise that the immunoregulatory activities of morphine on astroglial cells are mediated through the opioid µ receptor.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Intravenous opiate users are at increased risk for infection with HIV-1 and the subsequent development of AIDS-defining disorders including encephalopathy (10, 21, 22). AIDS dementia complex is associated with productive virus infection, involving microglia, neurons, and astrocytes, and the concomitant stimulation of cytokine transcription and production by adjacent, uninfected cells (10, 21). Although microglial cells have been reported to be the main target for HIV-1 infection in the CNS (37), restricted viral replication also has been described in astrocytes (37). Proinflammatory cytokines such as IL-1{beta} and IL-6 have been shown to suppress HIV-1 production in actively infected human brain cultures (38, 39), potentially through modulation of the expression of the {beta}-chemokines. Using cocultures of the HIV-1-infected promonocytic cell line U1 and brain cells, Peterson et al. (38) showed that morphine potentiates endotoxin-stimulated HIV-1 expression by modulating cytokine expression.

CXCR2 is a chemokine receptor of considerable significance because of its role as a coreceptor for HIV-1 infection (33, 39, 40). CXCR2 has been reported to be present on a variety of cells, including astrocytes and other glial cells. Cota et al. (1) showed that astrocytes infected with HIV-1 manifest increased expression of the chemokines IL-8 and RANTES and the chemokine receptors CXCR1, CXCR2, and CCR2b; the latter are also HIV-1 coreceptors. Regarding chemokine receptor expression by cells of the CNS, previous studies showed that CXCR2 is predominantly expressed on microglial cells (14, 41) as compared with CXCR1 (42).

IL-8 secretion by astrocytes was reported to be responsible for transendothelial migration of HIV-1-infected macrophages (43). Depletion of IL-8 in astrocyte cultures rendered the cells more susceptible to apoptosis (44). However, the effect of exogenous opioids, as in drugs of abuse, on the expression of IL-8 or its specific receptor, CXCR2, by astrocytes was not known until now. Here, we demonstrate that U87 astrocytoma cells constitutively express the genes for IL-8 and its specific receptor, CXCR2, and that treatment of U87 cells with morphine suppressed IL-8 gene expression, whereas it up-regulated the expression of the CXCR2 gene. Using flow cytometry for immunophenotyping, we showed that treatment of U87 cells with morphine significantly increased the number of CXCR2-positive cells, whereas it significantly decreased the number of IL-8-positive cells. Thus, we have shown that morphine treatment of U87 astrocytoma cells suppresses both the expression of the IL-8 gene and the production of IL-8. Furthermore, morphine up-regulates the expression of the CXCR2 gene as well as its gene product on the surface of U87 cells. To demonstrate the biological relevance of these studies, additional experiments were conducted with NHA cultures to confirm the observations made with the U87 astrocytoma cell line. We found that, like the astrocytoma cells, morphine could induce the expression of the genes of the chemokine receptors/HIV coreceptors CCR5 and CCR3 and concomitantly inhibit both the expression of the genes of the HIV-protective chemokines IL-8 and MIP-1{beta} and the synthesis of both proteins by NHAs.

In addition to {alpha}-chemokines, several {beta}-chemokines also serve as coreceptors for HIV-1. The {beta}-chemokine MIP-1{beta} can suppress HIV-1 infection by blocking its coreceptors, CCR5 and CCR3 (5, 6). Both {alpha}- and {beta}-chemokines are expressed in the brain during the subacute, acute, and chronic stages of HIV-1 infection, thus emphasizing the dynamic regulation of the expression of {alpha}- and {beta}-chemokines in inflammatory disease processes in the CNS (45).

Although it is recognized that astrocytes are a major source of chemokines (e.g., MIP-1{alpha} and MIP-1{beta}) in the CNS, details of the mechanisms regulating their expression are not known (46, 47). These chemokines play an important role in trafficking of mononuclear phagocytes within the brain. Also, there is evidence that binding sites exhibiting the characteristics of the chemokine receptor CCR5 exist on human astrocytes and that such sites might function in the recruitment of both astrocytes and leukocytes to specific regions of the brain during physiological and pathophysiological processes (48). We examined the effect of morphine on the expression of MIP-1{beta} and its receptors, CCR5 and CCR3, on NHAs and found that morphine at 10-7 and 10-9 M concentrations down-regulates MIP-1{beta} gene expression (Fig. 3Go), whereas it reciprocally up-regulates the gene expression of its specific receptors, CCR5 and CCR3 (Fig. 3Go). Morphine at 10-7 M and 10-9M reduced MIP-1{beta} protein synthesis by NHA cells, and although the constitutive levels of MIP-1{beta} were low, the differences in morphine-treated and control cultures were statistically significant.

Many of the effects of opioids are mediated via µ, {kappa}, and {delta} receptors present on the surface of neurons. Among these, the µ receptor predominantly mediates the activities of morphine (49, 50, 51). The specificity of µ receptor-mediated CXCR2 gene expression was determined using a selective µ receptor antagonist {beta}-FNA. We observed that {beta}-FNA completely reversed morphine-induced enhancement of CXCR2 gene expression, confirming the role of the opioid µ receptor in mediating these effects. We also examined the activity of {beta}-FNA on morphine-induced expression of the genes for IL-8, MIP-1{beta}, CCR5, and CCR3. We observed that {beta}-FNA completely reversed the morphine-induced effects, including down-regulation of the expression of the HIV-protective chemokines IL-8 and MIP-1{beta} and the reciprocal up-regulation of the gene expression of the relevant chemokine receptors CCR5 and CCR3, which are also coreceptors for HIV-1. {beta}-FNA alone produced no effects on MIP-1{beta} gene expression. {beta}-FNA exhibits a mixed agonistic/antagonistic opioid activity, depending on receptor subtypes (52, 53). Remarkably, when {beta}-FNA was used in combination with morphine on NHA cells, not only did it reverse the immunoregulatory activities of morphine alone, but with regard to the gene expression of the chemokines IL-8 and MIP-1{beta}, it significantly enhanced expression of these genes even when compared with untreated control cells. When compared with morphine-treated cells, the enhancement was several orders of magnitude. Although we are using {beta}-FNA as an antagonist of morphine’s activities, it also may have partial agonist activities of its own. It is possible that {beta}-FNA not only binds to the µ receptor, but it also may activate an orphan receptor (e.g., opioid receptor-like type 1 (ORL-1)) or an unidentified receptor as described (52, 53). This unexpected observation will be the subject of further investigation. We also examined µ receptor expression by U87 and NHA cells using Western blot analysis of cell lysates and a commercially available mAb reportedly specific for the opioid receptor (BD PharMingen). Our results suggest that U87 and NHA cells demonstrate low but detectable levels of µ receptor present in the lysate and that morphine increases the expression of µ receptors on these cells. However, the commercial mAb was not rigorously characterized for specificity. Thus, these data are not presented. These observations confirm our hypothesis that the opioid µ receptor is pivotal in mediating the immunomodulatory effects of opioids on astroglial cells of the CNS. Other studies have shown significant modulatory effects of different µ receptor antagonists on various biological activities (54, 55). Although {beta}-FNA is a selective µ receptor antagonist, it can discriminate between two subtypes of µ2 receptors, a {beta}-FNA-sensitive and a {beta}-FNA-resistant receptor (56), both of which can be competitively inhibited by morphine. Although both µ opioid receptors and ORL-1 receptors are present on the neuroblastoma cell line BE2-C, {beta}-FNA inactivates only the ORL-1 receptor on those cells (57). It is possible that, like neuroblastoma cells, U87 astrocytoma cells and NHA cells may also have ORL-1 receptors in addition to the µ receptor.

In summary, our results demonstrate that the astrocytoma cell line U87 and NHA cells express chemokine receptors/HIV-1 coreceptors. Treatment of these cells with morphine in vitro enhances the expression of chemokine receptors/HIV coreceptors, whereas it inhibits the expression of both {alpha}- and {beta}-chemokines with HIV-1-protective activity. These studies support our hypothesis that opioids increase susceptibility to and progression of HIV-1 infections by up-regulating the expression of HIV-1 coreceptors and simultaneously down-regulating the expression of HIV-1-protective chemokines in the CNS. Our results suggest that pharmacologic antagonists for the opioid µ receptor may be useful in the prophylaxis and treatment of HIV-1 infections in patients who use opiates as recreational drugs as well as for nonusers.


   Footnotes
 
1 This work was supported in part by National Institute on Drug Abuse Grants RO1-DA10632, RO1-DA14218, RO1-DA12366, and RO3-DA11119 and by the Margaret Duffy and Robert Cameron Troup Memorial Fund for Cancer Research of the Kaleida Health System (Buffalo, NY). Back

2 Address correspondence and reprint requests to Dr. Madhavan P. N. Nair, Department of Medicine, Division of Allergy, Immunology, and Rheumatology, 310 Multi Research Building, Buffalo General Hospital, 100 High Street, Buffalo, NY 14203. E-mail address: mnair{at}acsu.buffalo.edu Back

3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; NHA, normal human astrocyte; Perm, Cytofix/Cytoperm; FL, fluorescence; {beta}-FNA, {beta}-funaltrexamine; ORL-1, opioid receptor-like type 1. Back

Received for publication August 13, 2001. Accepted for publication July 24, 2002.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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