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The Journal of Immunology, 2000, 164: 5088-5093.
Copyright © 2000 by The American Association of Immunologists

{kappa}-Opioid Regulation of Thymocyte IL-7 Receptor and C-C Chemokine Receptor 2 Expression1

Lily Zhang and Thomas J. Rogers2

Department of Microbiology and Immunology, Fels Institute for Cancer Research and Molecular Biology, and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, PA 19140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Endogenous and exogenous {kappa}-opioid agonists have been widely reported to modulate the immune response. We have published results that show that the superantigen-induced proliferative response of thymocytes is inhibited by the selective {kappa}-opioid agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexyl]benzeneaceamide methanesulfonate (U50,488H). Previous work has established that the {kappa}-opioid receptor is widely expressed within the thymus; however, little is known about the role of the {kappa}-opioid receptor in the function of thymocytes. In the present report, we have examined the impact of U50,488H administration on the expression of cytokines in superantigen-stimulated thymocytes by RNase protection analysis. We have measured detectable levels of the cytokines IL-2, IL-4, IL-5, IL-13, and IFN-{gamma}, and the chemokines lymphotactin and RANTES, in stimulated thymocyte cultures; however, addition of U50,488H did not alter the expression of these cytokines. Examination of cytokine receptor expression by these thymocytes revealed a significant inhibition in the expression of the transcript for the IL-7 receptor {alpha}-chain (IL-7R{alpha}), and these results were confirmed by flow cytometry. Surprisingly, the expression of several other cytokine receptor chains including the common {gamma}-chain, IL-2Rß, or the IL-2R{alpha}, IL-4R{alpha}, and IL-15R{alpha} chains, was not altered. In contrast to these results, a significant elevation in the expression of the chemokine receptor CCR2 was observed in U50,488H-treated cultures. These results suggest that the {kappa}-opioid receptor may function to promote cellular migration at the expense of the sensitivity to the growth-promoting/maturation activity of IL-7.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Studies with a variety of endogenous and exogenous µ- and {delta}-opioid compounds have established that opioids possess broad immunomodulatory activity, including the inhibition of Ab responses (1, 2, 3, 4, 5, 6, 7), delayed-type hypersensitivity responses (8, 9, 10), and NK cell activity (11, 12, 13, 14). The mechanism of the immunosuppression is uncertain and is likely to be due to an interaction of the opioid compound with multiple populations of immune cells. Much less is known about the role of the {kappa}-opioid receptor in the function of the immune response. The endogenous {kappa}-opioid agonist dynorphin has been shown to induce mononuclear cell chemotaxis (15) and to elevate both macrophage superoxide production (16) and macrophage tumoricidal activity (17, 18). In contrast, the production of the proinflammatory cytokines IL-1, IL-6, and TNF-{alpha} by both primary macrophages and the macrophage cell line P388D1 is inhibited by {kappa}-opioid agonist administration (19, 20). Finally, this laboratory and others have previously established that in vitro Ab responses are inhibited following the administration of {kappa}-opioid agonist such as trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexyl]benzeneaceamide methanesulfonate (U50,488H)3 (4, 21).

We have recently demonstrated the expression of the {kappa}-opioid receptor both in primary thymocytes and in a number of immature T cell lines, and our cloning and sequence analysis has revealed the expression of a number of intron-exon splice variants in these developing T cells (22, 23). Subsequent flow cytometric analysis employing a fluorescent {kappa}-opioid ligand suggests that the {kappa}-opioid receptor is widely expressed within the thymus (24). The expression of the {kappa}-opioid receptor in these immature T cell populations suggests the possibility that this receptor may participate in the functional activity of developing T cells in the thymus. We have recently reported results that have shown that superantigen-induced T cell IL-2 production and the expression of the IL-2 receptor {alpha}-chain (CD25) are inhibited by U50,488H in cocultures of murine thymocytes and activated macrophages (25, 26). The mechanism of this immunosuppressive activity is most likely related to alterations in accessory cell function, because the inhibitory activity of U50,488H was greatly reduced in cocultures of thymocytes and resting macrophages (25).

In an effort to more fully understand the role of the {kappa}-opioid receptor in the function of developing T cells, we have conducted experiments to determine the impact of {kappa}-opioid administration on the production of cytokines and cytokine receptors in both resting and superantigen-activated thymocytes. Our results show that the production of the IL-7 receptor, a receptor that is critical for T cell maturation, is significantly inhibited following U50,488H treatment. In contrast to studies with isolated macrophages, these studies show that the level of expression in thymocytes of proinflammatory cytokines and receptors remains largely unaltered by the {kappa}-opioid agonist. However, we find that the level of expression of the chemokine receptor CCR2 is significantly elevated following administration of U50,488H. The results suggest that the {kappa}-opioid receptor may participate in the regulation of the balance between T cell maturation and cellular migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Pathogen-free male BALB/c mice, 6–8 wk of age, were obtained from the National Cancer Institute (Frederick, MD). Mice were allowed to rest for 1 wk before initiation of the experiments.

Reagents

The {kappa}-opioid agonist U50,488H (Upjohn, Kalamazoo, MI) was dissolved in DMEM (Life Technologies, Grand Island, NY) before use. Naloxone and staphylococcal enterotoxin B (SEB; Sigma, St. Louis, MO) were dissolved in DMEM and diluted to the designated concentrations for use.

Thymocyte culture

Single-cell suspensions of thymocytes were placed in culture (1 x 107 cells/ml) in DMEM containing 2-ME (50 µM) and nonessential amino acids (1%) in a volume of 5 ml. Designated cultures received SEB (10 µg/ml) and/or U50,488H at the initiation of culture. Analysis of opioid receptor antagonists required the addition of naloxone (1 µM) 2 h before the addition of U50,488H. Dye-exclusion analysis showed that the viability of SEB-, U50,488H-, and naloxone-treated cells was essentially identical with that of the nontreated cells.

mRNA isolation

Total cellular RNA was extracted using the RNazol method (Cinna/Biotecx Laboratories, Friendswood, TX). Briefly, all medium containing the treatments were washed away, and the cells were washed twice with PBS. Then, 0.2 ml of the RNazol reagent per 106 cells was then added to each culture dish, and the cells were disrupted by passing the lysate through a pipette. Chloroform was added, and the samples were shaken vigorously for 15 s and then placed on ice for 5 min. The aqueous phase was then extracted with isopropanol, and the RNA was precipitated overnight at 4°C.

RNase protection analysis

The expression of cytokines and cytokine receptors was measured by RNase protection analysis using the RiboQuant MultiProbe RNase Protection Assay System (PharMingen, San Diego, CA). Briefly, 10 µg of RNA from each sample was allowed to hybridize in solution with the radiolabeled antisense RNA probe generated with the RNA probe set mCK-1b (IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-13, IL-15, and IFN-{gamma}), mCR-1 (IL-2R{alpha}, IL-2Rß, {gamma}-chain ({gamma}C), IL-4R{alpha}, IL7R{alpha}, IL-9R{alpha}, IL-13R{alpha}, and IL-15R{alpha}), mCR-4 (IL-1RI, IL-1RII, TNFRp75, TNFRp55, IL-6R{alpha}, gp130, TGFßRI, and TGFßRII), mCK-5 (lymphotactin (Ltn), RANTES, eotaxin, macrophage inflammatory protein (MIP)-1{alpha}, MIP-1ß, MIP-2, IFN-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, and T cell activation gene (TCA)-3), or CR-5 (CCR1, CCR1b, CCR2, CCR3, CCR4, and CCR5) according to manufacturer’s instructions. The hybridized probe-transcript duplex was subjected to digestion with RNase, and the protected probes were purified and resolved on 5% denaturing polyacrylamide. The gels were then dried and exposed to a phosphorimaging screen, and protected fragments were visualized and quantitated using a Model GS-525 phosphorimager (Bio-Rad, Hercules, CA). Because expression of the chemokines in the thymus is at a low level in comparison to the L32 expression, it was necessary in this case to quantitate the band intensity of both the chemokines and L32 at and following both brief (0.5–2 h) and extended (4–6 h) exposures, so that accurate quantitation of both the chemokine and L32 bands was possible. Results are expressed as relative units, which are calculated according to the following formula: relative units = [(OD of cytokine (or receptor) band)/(OD of L32 band)] x 100.

FACS analysis

Analysis of the expression of the IL-7 receptor {alpha}-chain (CD127) was conducted with biotin-conjugated monoclonal anti-CD127 Ab B12-1 (PharMingen). Thymic T cells were plated in 96-well plates at a density of 107 cells/ml in HBSS containing 2% FCS. The cells were incubated in 50 µl of a combination of anti-CD16 and anti-CD32 (Fc-block; PharMingen), and, after incubation for 15 min at 4°C, anti-CD127 Ab was added. After an additional 30 min, the cells were washed and incubated with FITC-conjugated avidin. After 30 min at 4°C, the cells were washed twice, and resuspended in HBSS containing 1% paraformaldehyde (except for cells labeled for {kappa}-receptors). Flow cytometry was conducted on an EPICS Elite analyzer (Coulter, Hialeah, FL).

Statistical analysis

Data were subjected to statistical analysis using ANOVA. All results shown are representative of at least three experiments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Failure of U50,488H to directly alter thymocyte cytokine production

Previous studies showed that the production of IL-2, and the expression of the IL-2 receptor {alpha}-chain (CD25), in superantigen-stimulated cocultures of thymocytes and activated macrophages were inhibited by U50,488H (25). We wished to determine the direct effect of this {kappa}-opioid agonist on cytokine expression in stimulated and nonstimulated thymocyte cultures. Thymocytes were stimulated with the superantigen SEB for 24 h, either alone or with U50,488H, and the expression of cytokine mRNA was determined (Fig. 1Go). Analysis of transcript expression by RNase protection analysis showed that IL-2, IL-5, IL-13, and IFN-{gamma} are constitutively expressed in thymocyte cultures, and that IL-2, IL-4, IL-13, and IFN-{gamma} are induced with superantigen stimulation. In contrast, superantigen stimulation leads to a consistent reduction in the steady-state level of IL-5 mRNA. Moreover, the results showed that administration of U50,488H fails to exert a significant impact on the expression of any of these cytokines, including IL-2. A small but statistically insignificant increase in the expression of IL-13 was typically observed (Fig. 1GoC). We were unable to detect the production of IL-6, IL-9, IL-10, or IL-15 in cultures of thymocytes with or without superantigen stimulation, or in the presence or absence of U50,488H. Results from analysis of mRNA collected from cells 8, 48, and 72 h following superantigen stimulation (data not shown) were consistent with the results presented for U50,488H at 24 h.



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  		FIGURE 1. Cytokine transcript levels in thymocytes stimulated or unstimulated with SEB and treated with U50,488H. Thymocytes were cultured for 24 h in the presence (•) or absence ({blacktriangleup}) of SEB together with the designated concentrations of U50,488H. Following culture, the total mRNA was extracted from the cells, and the level of expression of IL-5 (A), IL-4 (B), IL-13 (C), IL-2 (D), and IFN-{gamma} (E) was determined by RNase protection analysis. Results are expressed relative to the level of L32 expression and are reported as relative units. The results are reported as the mean (±SD) from four independent experiments.

 
We also conducted experiments to determine the impact of opioid on the expression of the chemokines. Our results (Fig. 2Go) showed that only RANTES was constitutively expressed at detectable levels in the thymocytes, and the level of this chemokine is reduced in superantigen-stimulated cultures. We found very low, or undetectable, levels of MIP-1{alpha}, MIP-1ß, MIP-2, IP-10, MCP-1, TCA-3, or eotaxin in the stimulated thymocyte cultures. In contrast, the chemokine Ltn was induced with stimulation; here again, the administration of U50,488H failed to modulate the level of chemokine expression to a significant degree.



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  		FIGURE 2. Chemokine transcript levels in thymocytes stimulated or unstimulated with SEB and treated with U50,488H. Thymocytes were cultured for 24 h in the presence (•) or absence ({blacktriangleup}) of SEB together with the designated concentrations of U50,488H. Following culture, the total mRNA was extracted from the cells, and the level of expression of Ltn (A) and RANTES (B) was determined by RNase protection analysis. Results are determined following electrophoresis and quantitated by phosphorimaging, and the data are expressed relative to the level of L32 expression and are reported as relative units. The results are reported as the mean (±SD) from four independent experiments.

 
Inhibition of IL-7R{alpha} mRNA expression in thymocytes treated with U50,488H

We next attempted to determine the impact of U50,488H administration on the expression of cytokine receptors with our without superantigen stimulation. Our results show that IL-7R{alpha}, IL-4R{alpha}, and {gamma}C were constitutively expressed by thymocytes in culture. As expected, the expression of IL-2R{alpha} and IL-2Rß were significantly elevated following superantigen administration. In contrast, a very modest reduction in the expression of IL-7R{alpha} was typically observed 24 h following superantigen stimulation. Interestingly, while the expression of the IL-2, IL-4, and IL-15 receptors remained unaltered following U50,488H administration (Fig. 3Go, A and C–G), the expression of IL-7R{alpha} mRNA was significantly reduced by U50,488H administration (Fig. 3Go, A and C). The inhibition of IL-7R{alpha} by U50,488H was dose dependent, with a detectable effect in the nanomolar range. Additional analysis showed that the U50,488H-mediated effect was most pronounced at 24 h poststimulation, and, in addition, the expression of IL-7R{alpha} was also inhibited in nonstimulated cultures (data not shown).



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  		FIGURE 3. Selective modulation of cytokine receptor transcript levels in thymocytes stimulated or unstimulated with SEB and treated with U50,488H. Thymocytes were cultured for 24 h in the presence (•) or absence ({blacktriangleup}) of SEB together with the designated concentrations of U50,488H. Following culture, the total mRNA was extracted from the cells, and the level of expression of cytokine receptors was determined by RNase protection analysis (A). Quantitation of the levels of IL-7R{alpha} (B), IL-15R{alpha} (C), IL-4R{alpha} (D), {gamma}C (E), IL-2R{alpha} (F), and IL-2Rß (G) was conducted by phosphorimaging, and the data are expressed relative to the level of L32 expression and are reported as relative units. The results are reported as the mean (±SD) from six independent experiments. *, p < 0.05.

 
While U50,488H is a highly selective {kappa}-opioid agonist, we also wished to determine whether the altered levels of IL-7R{alpha} expression were mediated by a "classical" opioid receptor. We conducted experiments in which cells were pretreated with the opioid receptor antagonist naloxone for 2 h before SEB and/or U50,488H administration. The results (Fig. 4Go) show that while U50,488H treatment reduces the expression of IL-7R{alpha} by 74%, naloxone pretreatment completely blocks this inhibitory activity.



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  		FIGURE 4. The inhibition of thymocyte IL-7R{alpha} expression by administration of U50,488H is reversed by naloxone. Designated groups of thymocytes were treated with the opioid antagonist naloxone for 2 h, followed by administration of U50,488H for 2 h, and finally SEB was added and cultured for 24 h. Control cells were cultured in the absence of naloxone or U50,488H or SEB (no SEB). Results are expressed relative to the level of L32 expression. Cells cultured with naloxone only, or naloxone plus SEB, did not differ from the control cultures. The results are reported as the mean (±SD) from three experiments. *, p < 0.05.

 
We also examined the expression of the "proinflammatory" cytokine receptors in these thymocyte cultures. Thymocyte cultures were established as described above, and results showed that TNFRp75, gp130, TGFRII, and IL-6R{alpha} were constitutively expressed in the thymus. Both of the TNF receptor subunits were induced with superantigen stimulation, and both forms of the IL-1 receptor and the type I TGFß receptor were not detectable under any of the conditions used in these cultures. In contrast to expression of the other receptors, the expression of TNFRp75 was consistently elevated by U50,488H to a very small degree (Fig. 5Go). While this increase was not statistically significant, it was reversed by pretreatment with naloxone (data not shown).



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  		FIGURE 5. Cytokine receptor transcript levels in thymocytes stimulated or unstimulated with SEB and treated with U50,488H. Thymocytes were cultured for 24 h in the presence (•) or absence ({blacktriangleup}) of SEB together with the designated concentrations of U50,488H. Following culture, the total mRNA was extracted from the cells, and the level of expression of TNFRp55 (A), TNF-Rp75 (B), gp130 (C), TGFRII (D), and IL-6R{alpha} (E) was determined by RNase protection analysis. Results are expressed relative to the level of L32 expression. The results are reported as the mean (±SD) from five independent experiments.

 
Inhibition of IL-7 receptor protein expression by U50,488H treatment

We conducted experiments to determine the level of IL-7 receptor protein expressed by thymocytes following U50,488H administration. Cultures of thymocytes were established as described above, and the level of IL-7 receptor was measured by flow cytometry. The results (Fig. 6Go and Table IGo) show that the administration of U50,488H to these cultures significantly inhibited the surface expression of the IL-7 receptor complex. We also determined expression of CD25 (IL-2R{alpha}) by the U50,488H-treated cells using flow cytometry, and our results show that the expression of this cytokine receptor remains unaltered through 48 h of culture.



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  		FIGURE 6. Inhibition of surface IL-7 receptor protein expression following U50,488H administration. Thymocytes were cultured for 24 h with SEB in the presence or absence of U50,488H, and the surface expression of IL-7 receptor was determined by flow cytometry. The cells cultured with SEB are 16.8% positive, while the cells cultured with both SEB and U50,488H are 5.9% positive. The results are from a single representative experiment.

 

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  		Table I. Inhibition of IL-7R protein expression following administration of U50,488H1

 
Elevation in the expression of CCR2 following U50,488H administration

Because of recent reports that have established an ability of µ-opioids to alter the chemotactic activity of microglial cells, we sought to determine whether the activation of the {kappa}-opioid receptor with U50,488H might influence the expression of CCRs. Our studies showed (Fig. 7Go) that CCR2, CCR4, and CCR5 are all constitutively expressed by thymocytes in culture. Moreover, we observed that superantigen stimulation induced very little change in the expression of these receptors over 24–48 h of culture. While the expression of CCR4 and CCR5 remained unchanged, the addition of U50,488H induced a significant increase in the expression of CCR2. Here again, the induction of CCR2 expression was dose dependent, with a significant increase detected at a concentration of 10 nM U50,488H. Additional experiments showed that the elevation in the level of CCR2 expression in these cultures was blocked by pretreatment with naloxone (data not shown). We failed to detect the expression of either CCR1 or CCR3 with or without stimulation, and opioid addition did not induce detectable levels of these receptors.



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  		FIGURE 7. Modulation of chemokine receptor transcript levels in thymocytes stimulated or unstimulated with SEB and treated with U50,488H. Thymocytes were cultured for 24 h in the presence (•) or absence ({blacktriangleup}) of SEB together with the designated concentrations of U50,488H. Following culture, the total mRNA was extracted from the cells, and the level of expression of chemokine receptors was determined by RNase protection analysis (A). Quantitation of the levels of CCR4 (B), CCR5 (C), and CCR2 (D) was conducted by phosphorimaging, are expressed relative to the level of L32 expression, and are reported as relative units. The results are reported as the mean (±SD) from three independent experiments. *, p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies from this laboratory have demonstrated the expression of transcripts for the {kappa}-opioid receptor in both primary thymocytes and two immature T cell lines (22, 23). Subsequent work using a fluorescence-conjugated {kappa}-opioid ligand have established the presence of {kappa}-opioid receptors on each of the major subpopulations of developing T cells in the thymus (24). While little is known about the function of the {kappa}-opioid receptor in the thymus, it is clear that {kappa}-opioid agonists exert a variety of effects on both macrophages and mature T cells (15, 16, 17, 18, 27). Interestingly, the {kappa}-opioid agonists exhibit both pro- and antiinflammatory activities for cells of the monocyte/macrophage lineage. The endogenous {kappa}-opioid agonist dynorphin increases monocyte/macrophage superoxide production and tumoricidal activity and induces monocyte chemotaxis (15, 16, 17, 18). At the same time, studies conducted by Belkowski et al. (20) showed that the {kappa}-opioid receptor agonist U50,488H decreased the LPS-induced levels of IL-1 and TNF{alpha} produced by the macrophage-like cell line P388D1. More recently, Alicea et al. (19) demonstrated that nonelicited peritoneal macrophages stimulated with LPS and treated with U50,488H at concentrations as low as 1 nM exhibited decreased production of IL-1, TNF{alpha}, and IL-6.

Based on these results with monocytes, and the knowledge that {kappa}-opioid receptors are widely expressed in the thymus, we explored the possibility that U50,488H might alter thymocyte cytokine expression. We have chosen the dissociated thymocyte culture approach to address this issue because of the innate advantages over alternative approaches. For example, experiments conducted in vivo to determine the impact of opioids on thymocyte function have been complicated by the significant contribution of the hypothalamic-pituitary-adrenal axis to the results (28, 29). In contrast, one of the limitations of our cell culture approach is that the thymic architecture is not preserved, and the normal composition of thymic-supporting cells may not be maintained in these cultures. However, our approach permits an analysis of the direct effects of the opioids on thymocyte function without confounding complications provided by the production of hormones from other organ systems.

In the present work, we were able to consistently detect expression of IL-2, IL-4, IL-5, IL-13 and IFN{gamma}, and the chemokines Ltn and RANTES, in superantigen-stimulated thymocyte cultures by RNase protection analysis. In contrast to the earlier work described above with primary macrophages and a macrophage cell line, our work showed no evidence that cytokine expression by thymocytes was altered by U50,488H administration. It should be pointed out that our treatment paradigm may not provide the optimal alteration of cytokine or receptor expression, and a longer or shorter administration scheme may provide a different pattern of immunomodulation. This is a limitation in these types of studies, even though data collected on the level gene expression at earlier (8 h) and later (48 and 72 h) time points (data not shown) did not reveal additional alterations in the immune response. An additional caveat in all of these studies is that results from RNase protection analysis do not reveal regulation at a posttranscriptional level. Notwithstanding these limitations in the evaluation of our present results, our previous studies (25) showed that the SEB-induced production of IL-2 and IL-2R{alpha} by cocultures of thymocytes and activated macrophages was inhibited following administration of U50,488H at concentrations as low as 1 nM. Because the inhibition of IL-2 and IL-2R{alpha} was greatly reduced in cocultures of thymocytes and nonactivated macrophages, it was likely that this inhibitory activity of U50,488H was mediated through the macrophage.

Examination of the expression of cytokine receptors revealed a significant alteration in the level of IL-7R{alpha} following U50,488H administration. The IL-7 receptor is essential for the normal maturation of T cells in the thymus based on the reduced numbers of mature T cell subsets in mice with deletions of the IL-7 or IL-7 receptor genes (30, 31) and overexpression of IL-7 results in a restoration of T cell populations in nude mice (32). It is also apparent that IL-7 acts to support the survival of immature T cells in part by increasing Bcl-2 and reducing the level of Bax (33). The IL-7 receptor is composed of the IL-7R{alpha} chain and the {gamma}C, both of which are members of the hemopoietin family (34, 35). Regulation of IL-7 receptor expression has not been thoroughly investigated; however, analysis of the IL-7R{alpha} promoter revealed the presence of a functional IFN regulatory element (IRE), which was recognized by members of the family of IFN regulatory factors (IRF) (36, 37). It is not certain that the IL-7 receptor is regulated by IFN; however, both IRF-1 and IRF-2 bind to the IL-7R{alpha} IRE and have been shown to regulate promoter function (36). It is tempting to speculate that U50,488H might regulate IL-7R{alpha} expression through the IRE promoter element. An opioid-mediated alteration of IRF function may be the result of a reduction in the production of an inducer of IRF production. Several of the members of the IRF family are induced in response to IFN-{gamma} (38); however, our results suggest that the expression of this cytokine is not altered by U50,488H. Moreover, it is known that the expression of IL-4R, TNFRp55, TNFRp75, and RANTES are regulated by IFN-{gamma} (38, 39, 40, 41, 42), and our results show that the levels of expression of these genes are not altered by exposure to U50,488H.

Examination of the expression of chemokine receptors revealed a significant elevation of steady-state levels of CCR2. MCP-1 (or alternatively, JE) is the primary ligand for this receptor, although MCP-3 and MCP-5 serve as additional ligands for this receptor in the mouse (43). While previous studies have clearly demonstrated the presence of CCR2 in the thymus (44), little is known about the regulation of the expression of CCR2 during T cell development. In contrast, IL-2 has been reported to induce the expression of both CCR1 and CCR2 in mature T lymphocytes and NK cells (45, 46). In addition, while IL-10 has been shown to elevate CCCR1, CCR2, and CCR5 in human monocytes (47), there is no evidence from our studies to suggest that IL-2 or IL-10 is elevated following U50,488H treatment. We were unable to reproducibly detect CCR1 by RNase protection analysis; however, the expression of CCR5 was not elevated by U50,488H administration, in contrast to effects reported for IL-2 and IL-10. The mechanism of the {kappa}-opioid-induced elevation of CCR2 levels remains uncertain; however, an indirect effect mediated through the production of an intermediate cytokine does not appear to be likely. We suggest that the combined effect of the {kappa}-opioid ligand to inhibit IL-7R and elevate CCR2 levels in the thymus implies that {kappa}-opioid receptor activation functions in part to promote an increase in cellular migration at the expense of IL-7-mediated T cell growth and/or maturation. Studies that address the cellular source of endogenous {kappa}-opioid(s) (primarily dynorphin) in the thymus have not been forthcoming. A complete understanding of the role of {kappa}-opioids to the function of the developing T cells will require a greater appreciation for the cellular sources, and regulation, of endogenous {kappa}-opioid in the thymus gland.

The role of the {kappa}-opioid receptor in the regulation of CCR2 expression may have significant consequences for the function of the immune system in a number of disease states. For example, it is becoming clear that MCP-1 is associated with the development of several inflammatory diseases, including multiple sclerosis, inflammatory bowel disease, myocardial reperfusion injury, and AIDS dementia (48). Interestingly, the human CCR2b chemokine receptor is a minor coreceptor for HIV, but serves as the major monocyte coreceptor for HIV strain 89.6 (49). Moreover, a genetic variant of CCR2 that changes a valine to isoleucine at amino acid 64 has been identified in certain HIV-infected individuals, and these patients have been found to progress to AIDS 2–4 years later than subjects homozygous for the wild-type CCR2 allele (50). The molecular basis for the protective effect of this mutation on the progression to AIDS is uncertain; however, the CCR2 mutation results support the notion that CCR2 plays a significant role in the interaction of the immune system with HIV. It is unclear whether the {kappa}-opioid receptor may regulate the expression of CCR2 outside of the thymus. Recent studies conducted with cocultures of fetal brain cells with HIV-infected monocytes show that subnanomolar concentrations of U50,488H, or the endogenous {kappa}-opioid dynorphin, promote HIV replication (51). These results are in contrast with those from studies which show that both U50,488H and dynorphin inhibit the replication of HIV in primary human microglial cells (52). While these contrasting results appear to be due in part to the influence of proinflammatory cytokines produced in these rather distinct cell cultures, the impact of opioid-induced alterations in CCR2 expression on HIV replication remains to be defined.


   Acknowledgments
 
We thank Gregory Harvey for his excellent editorial assistance.


   Footnotes
 
1 This work was supported by National Institute on Drug Abuse Grants DA-11130 and DA-06650. Back

2 Address correspondence and reprint requests to Dr. Thomas J. Rogers, Department of Microbiology and Immunology, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140. Back

3 Abbreviations used in this paper: U50,488H, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)cyclohexyl]benzeneaceamide methanesulfonate; SEB, staphylococcal enterotoxin B; Ltn, lymphotactin; MIP, macrophage inflammatory protein; IP, IFN-inducible protein; MCP, monocyte chemotactic protein; TCA, T cell activation gene; IRE, IFN regulatory element; IRF, IFN regulatory factor; {gamma}C, {gamma}-chain. Back

Received for publication December 1, 1999. Accepted for publication March 1, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Johnson, H. M., E. M. Smith, B. A. Torres, J. E. Blalock. 1982. Regulation of the in vitro antibody response by neuroendocrine hormones. Proc. Natl. Acad. Sci. USA 79:4171.[Abstract/Free Full Text]
  2. Heijnen, C. J., C. Bevers, A. Kavelaars, R. E. Ballieux. 1986. Effect of {alpha}-endorphin on the antigen-induced primary antibody response of human blood B cells in vitro. J. Immunol. 136:213.[Abstract]
  3. Eisenstein, T. K., Jr J. J. Meissler, E. B. Geller, M. W. Adler. 1990. Immunosuppression to tetanus toxoid induced by implanted morphine pellets. M. S. O’Dorisio, and A. Panerai, eds. Neuropeptides and Immunopeptides: Messengers in a Neuroimmune Axis 377. New York Academy of Sciences, New York.
  4. Taub, D. D., T. K. Eisenstein, E. B. Geller, M. W. Adler, T. J. Rogers. 1991. Immunomodulatory activity of µ- and {kappa}-selective opioid agonists. Proc. Natl. Acad. Sci. USA 88:360.[Abstract/Free Full Text]
  5. Bussiere, J. L., M. W. Adler, T. J. Rogers, T. K. Eisenstein. 1992. Differential effects of morphine and naltrexone on the antibody response in various mouse strains. Immunopharmacol. Immunotoxicol. 14:657.[Medline]
  6. Bussiere, J. L., M. W. Adler, T. J. Rogers, and T. K. Eisenstein. 1992. Effects of in vivo morphine treatment on antibody responses in C57BL/6 bgJ/bgJ (beige) mice. Life Sci. 52:PL43.
  7. Bussiere, J. L., M. W. Adler, T. J. Rogers, T. K. Eisenstein. 1993. Cytokine reversal of morphine-induced suppression of the antibody response. J. Pharmacol. Exp. Ther. 264:591.[Abstract/Free Full Text]
  8. Pellis, N. R., C. Harper, N. Dafny. 1986. Suppression of the induction of delayed hypersensitivity in rats by repetitive morphine treatments. Exp. Neurol. 93:92.[Medline]
  9. Bryant, H. U., R. E. Roudebush. 1990. Suppressive effects of morphine pellet implants on in vivo parameters of immune function. J. Pharmacol. Exp. Ther. 255:410.[Abstract/Free Full Text]
  10. Molitor, T. W., A. Morilla, J. M. Risdahl, M. P. Murtaugh, C. C. Chao, P. K. Peterson. 1992. Chronic morphine administration impairs cell-mediated immune responses in swine. J. Pharmacol. Exp. Ther. 260:581.[Abstract/Free Full Text]
  11. Weber, R. J., A. Pert. 1989. The periaqueductal gray matter mediates opiate-induced immunosuppression. Science 245:188.[Abstract/Free Full Text]
  12. Shavit, Y., R. Yirmiya, B. Beilin. 1990. Stress neuropeptides, immunity and neoplasia. S. Freier, ed. The Neuroendocrine-Immune Network 163. CRC Press, Boca Raton.
  13. Bayer, B. M., S. Daussin, M. Hernandez, L. Irvin. 1990. Morphine inhibition of lymphocyte activity is mediated by an opioid dependent mechanism. Neuropharmacology 29:369.[Medline]
  14. Carr, D. J. J., C. P. France. 1993. Immune alterations in morphine-treated rhesus monkeys. J. Pharmacol. Exp. Ther. 267:9.[Abstract/Free Full Text]
  15. Ruff, M. R., S. M. Wahl, S. Mergenhagen, C. B. Pert. 1985. Opiate receptor-mediated chemotaxis of human monocytes. Neuropeptides 5:363.[Medline]
  16. Sharp, B. M., W. F. Keane, H. J. Suh, G. Gekker, D. Tsukayama, P. K. Peterson. 1985. Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leukocytes and macrophages. Endocrinology 117:793.[Abstract]
  17. Foster, J. S., R. N. Moore. 1987. Dynorphin and related opioid peptides enhance tumoricidal activity mediated by murine peritoneal macrophages. J. Leukocyte Biol. 42:171.[Abstract]
  18. Hagi, K., K. Uno, K. Inaba, S. Muramatsu. 1994. Augmenting effect of opioid peptides on murine macrophage activation. J. Neuroimmunol. 50:71.[Medline]
  19. Alicea, C., S. M. Belkowski, T. K. Eisenstein, M. W. Adler, T. J. Rogers. 1996. Inhibition of primary murine macrophage cytokine production in vitro following treatment with the {kappa}-opioid agonist U50,488H. J. Neuroimmunol. 64:83.[Medline]
  20. Belkowski, S. M., C. Alicea, T. K. Eisenstein, M. W. Adler, T. J. Rogers. 1995. Inhibition of interleukin-1 and tumor necrosis factor-{alpha} synthesis following treatment of macrophages with the {kappa} opioid agonist U50,488H. J. Pharmacol. Exp. Ther. 273:1491.[Abstract/Free Full Text]
  21. Radulovic, J., C. Miljevic, D. Djergovic, V. Vujic, J. Antic, S. von Horsten, B. D. Jankovic. 1995. Opioid receptor-mediated suppression of humoral immune response in vivo and in vitro: involvement of {kappa} opioid receptors. J. Neuroimmunol. 57:55.[Medline]
  22. Belkowski, S. M., J. Zhu, L.-Y. Liu-Chen, T. K. Eisenstein, M. W. Adler, T. J. Rogers. 1995. Sequence of {kappa}-opioid receptor cDNA in the R1.1 thymoma cell line. J. Neuroimmunol. 62:113.[Medline]
  23. Alicea, C., S. M. Belkowski, J. Sliker, J. Zhu, L.-Y. Liu-Chen, T. K. Eisenstein, M. W. Adler, T. J. Rogers. 1998. Characterization of {kappa}-opioid receptor transcripts expressed by T cells and macrophages. J. Neuroimmunol. 91:55.[Medline]
  24. Ignatowski, T. A., J. M. Bidlack. 1998. Detection of {kappa} opioid receptors on mouse thymocyte phenotypic subpopulations as assessed by flow cytometry. J. Pharmacol. Exp. Therapeut. 284:298.[Abstract/Free Full Text]
  25. Guan, L., T. K. Eisentein, M. W. Adler, T. J. Rogers. 1997. Inhibition of T cell superantigen responses following treatment with the {kappa}-opioid agonist U50,488H. J. Neuroimmunol. 75:163.[Medline]
  26. Guan, L., T. Eisenstein, M. Adler, T. Rogers. 1998. Modulation of DPK cell function by the {kappa} opioid agonist agonist U50,488H. B. Sharp, and T. K. Eisenstein, and J. Madden, eds. Drugs of Abuse, Immunomodulation, and AIDS 125. Plenum Press, New York.
  27. Guan, L., R. Townsend, T. K. Eisenstein, M. W. Adler, T. J. Rogers. 1994. Both T-cells and macrophages are targets of {kappa} opioid-induced immunosuppression. Brain Behav. Immun. 8:229.[Medline]
  28. Fuchs, B. A., S. B. Pruett. 1993. Morphine induces apoptosis in murine thymocytes in vivo but not in vitro: involvement of both opiate and glucocorticoid receptors. J. Pharmacol. Exp. Ther. 266:417.[Abstract/Free Full Text]
  29. Bryant, H. U., E. W. Bernton, J. R. Kenner, J. W. Holaday. 1991. Role of adrenal cortical activation in the immunosuppressive effects of chronic morphine treatment. Endocrinology 128:3253.[Abstract]
  30. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware, et al 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180:1955.[Abstract/Free Full Text]
  31. Von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181:1519.[Abstract/Free Full Text]
  32. Rich, B. E., P. Leder. 1995. Transgenic expression of interleukin 7 restores T cell populations in nude mice. J. Exp. Med. 181:1223.[Abstract/Free Full Text]
  33. Kim, K., C.-K. Lee, T. J. Sayers, K. Muegge, S. K. Durum. 1998. The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways. J. Immunol. 160:5735.[Abstract/Free Full Text]
  34. Noguchi, M., Y. Nakamura, S. M. Russell, S. F. Ziegler, M. Tsang, X. Cao, W. J. Leonard. 1993. Interleukin-2 receptor {gamma} chain: a functional component of the interleukin-7 receptor. Science 262:1877.[Abstract/Free Full Text]
  35. Kondo, M., T. Takeshita, M. Higuchi, M. Nakamura, T. Sudo, S.-L. Nishikawa, K. Sugamura. 1994. Functional participation of the IL-2 receptor {gamma} chain in IL-7 receptor complexes. Science 263:1453.[Abstract/Free Full Text]
  36. Pleiman, C. M., S. D. Gimpel, L. S. Park, H. Harada, T. Taniguchi, S. F. Ziegler. 1991. Organization of the murine and human interleukin-7 receptor genes: two mRNAs generated by differentiatial splicing and presence of a type I-interferon-inducible promoter. Mol. Cell Biol. 11:3052.[Abstract/Free Full Text]
  37. Nguyen, H., J. Hiscott, P. M. Pitha. 1997. The growing family of interferon regulatory factors. Cytokine Growth Factor Rev. 8:293.[Medline]
  38. Byron, K. A., G. A. Varigos, A. M. Wootton. 1991. Recombinant interferon-gamma inhibits the expression of IL-4 receptors on human lymphocytes. Clin. Exp. Immunol. 85:307.[Medline]
  39. Feldman, G. M., D. S. Finbloom. 1990. Induction and regulation of IL-4 receptor expression on murine macrophage cell lines and bone marrow-derived macrophages by IFN-{gamma}. J. Immunol. 145:854.[Abstract]
  40. Pandita, R., E. Pocsik, B. B. Aggarwal. 1992. Interferon-{gamma} induces cell surface expression for both types of tumor necrosis factor receptors. FEBS Lett. 312:87.[Medline]
  41. Jr Bebo, B. F., D. S. Linthicum. 1995. Expression of mRNA for 55-kDa and 75-kDa tumor necrosis factor (TNF) receptors in mouse cerebrovascular endothelium: effects of interleukin-1ß, interferon-{gamma} and TNF-{alpha} on cultured cells. J. Neuroimmunol. 62:161.[Medline]
  42. Marfaing-Koka, A., O. Devergne, G. Gorgone, A. Portier, T. J. Schall, P. Galanaud, D. Emilie. 1995. Regulation of the production of the RANTES chemokine by endothelial cells: synergistic induction by IFN-{gamma} plus TNF-{alpha} and inhibition by IL-4 and IL-13. J. Immunol. 154:1870.[Abstract]
  43. Zlotnik, A., J. Morales, J. A. Hedrick. 1999. Recent advances in chemokines and chemokine receptors. Crit. Rev. Immunol. 19:1.[Medline]
  44. Jiang, Y., M. N. Salafranca, S. Adhikari, Y. Xia, L. Feng, M. K. Sonntag, C. M. deFiebre, N. A. Pennell, W. J. Streit, J. K. Harrison. 1998. Chemokine receptor expression in cultured glia and rat experimental allergic encephalomyelitis. J. Neuroimmunol. 86:1.[Medline]
  45. Loetscher, P., M. Seitz, M. Baggliolini, B. Moser. 1996. Interleukin-2 regulates CC chemokine receptor expression and chemotactic responsiveness in T lymphocytes. J. Exp. Med. 184:569.[Abstract/Free Full Text]
  46. Polentarutti, N., P. Allavena, G. Bianchi, G. Giardina, A. Basile, S. Sozzani, A. Mantovani, M. Introna. 1997. IL-2 regulated expression of the monocyte chemotactic protein-1 receptor (CCR2) in human NK cells: characterization of a predominant 3.4 kb transcript containing CCR2B and CCR2A sequences. J. Immunol. 158:2689.[Abstract]
  47. Sozzani, S., S. Ghezzi, G. Iannolo, W. Luini, A. Borsatti, N. Polentarutti, A. Sica, M. Locati, C. Mackay, T. N. C. Wells, et al 1998. Interleukin-10 increases CCR5 expression and HIV infection in human monocytes. J. Exp. Med. 187:439.[Abstract/Free Full Text]
  48. Mennicken, F., R. Maki, E. B. de Souza, R. Quirion. 1999. Chemokines and chemokine receptors in the CNS: a possible role in neuroinflammation and patterning. Trends Pharmacological Sci. 20:73.[Medline]
  49. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, M. Parmentier, R. G. Collman, R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the ß-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149.[Medline]
  50. Smith, M. W., M. Dean, M. Carrington, C. Winkler, G. A. Huttley, D. A. Lomb, J. J. Goedert, T. R. O’Brien, L. P. Jacobson, R. Kaslow, et al 1997. Contrasting genetic influence of CCR2 and CCR5 variants on HIV-1 infection and disease progression. Science 15:959.
  51. Chao, C. C., G. Gekker, S. Hu, W. S. Sheng, P. S. Portoghese, P. K. Peterson. 1995. Upregulation of HIV-1 expression in cocultures of chronically infected promonocytes and human brain cells by dynorphin. Biochem. Pharmacol. 50:715.[Medline]
  52. Chao, C. C., G. Gekker, S. Hu, W. S. Sheng, K. B. Shark, D.-F. Bu, S. Archer, J. M. Bidlack, P. K. Peterson. 1996. {kappa} opioid receptors in human microglia downregulate human immunodeficiency virus 1 expression. Proc. Natl. Acad. Sci. USA 93:8051.[Abstract/Free Full Text]



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