Ca2+-independent Protein Kinase Cs Mediate Heterologous Desensitization of Leukocyte Chemokine Receptors by Opioid Receptors
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m300430200
ISSN1083-351X
AutoresNing Zhang, Dave Hodge, Thomas J. Rogers, Joost J. Oppenheim,
Tópico(s)Neuropeptides and Animal Physiology
ResumoHeterologous desensitization of chemokine receptors by opioids has been considered to contribute to their immunosuppressive effects. Previous studies show that Met-enkephalin, an endogenous opioid, down-regulates chemotaxis of selected chemokine receptors via phosphorylation. In the present study, we further investigated the molecular mechanism of such cross-regulation. Our data showed that preincubation with Met-enkephalin inhibited both MIP-1α-mediated chemotaxis and Ca2+ flux of monocytes in a dose-dependent manner. The inhibitory effects were maximal using nanomolar concentrations of activating chemokines, a concentration found in physiological conditions. A decrease both in chemokine receptor affinity and in coupling efficiency between receptors and G protein were observed, which directly contributed to the desensitization effects. However, comparing with chemokines such as MIP-1α and MCP-1, opioids did not elicit a calcium flux, failed to induce MIP-1α receptors internalization, and mediated a less potent heterologous desensitization. We hypothesized that these differences might originate from the involvement of different protein kinase C (PKC) isotypes. In our studies, opioid-mediated down-regulation of MIP-1α receptors could be blocked by the general PKC inhibitor calphostin C, but not by the calcium-dependent classic PKC inhibitor Go6976. Western blotting analysis and immunofluorescent staining further showed that only calcium-independent PKCs were activated upon opioid stimulation. Thus, opioids achieve desensitization of chemokine receptors via a unique pathway, involving only calcium-independent PKC isotypes. Heterologous desensitization of chemokine receptors by opioids has been considered to contribute to their immunosuppressive effects. Previous studies show that Met-enkephalin, an endogenous opioid, down-regulates chemotaxis of selected chemokine receptors via phosphorylation. In the present study, we further investigated the molecular mechanism of such cross-regulation. Our data showed that preincubation with Met-enkephalin inhibited both MIP-1α-mediated chemotaxis and Ca2+ flux of monocytes in a dose-dependent manner. The inhibitory effects were maximal using nanomolar concentrations of activating chemokines, a concentration found in physiological conditions. A decrease both in chemokine receptor affinity and in coupling efficiency between receptors and G protein were observed, which directly contributed to the desensitization effects. However, comparing with chemokines such as MIP-1α and MCP-1, opioids did not elicit a calcium flux, failed to induce MIP-1α receptors internalization, and mediated a less potent heterologous desensitization. We hypothesized that these differences might originate from the involvement of different protein kinase C (PKC) isotypes. In our studies, opioid-mediated down-regulation of MIP-1α receptors could be blocked by the general PKC inhibitor calphostin C, but not by the calcium-dependent classic PKC inhibitor Go6976. Western blotting analysis and immunofluorescent staining further showed that only calcium-independent PKCs were activated upon opioid stimulation. Thus, opioids achieve desensitization of chemokine receptors via a unique pathway, involving only calcium-independent PKC isotypes. G protein-coupled receptor protein kinase C diacylglycerol classical PKC novel PKC phosphate-buffered saline Dulbecco's modified Eagle's medium guanosine 5′-3-O-(thio)triphosphate Cys2, Tyr3, Orn5, Pen7 amide green fluorescent protein Long term opioid usage induces immunosuppression by modulating a spectrum of immune activities, such as inhibition of lymphocyte proliferation, decreased production of interferon γ, interleukin 2, and interleukin 4 by activated lymphocytes, enhanced synthesis of tumor necrosis factor α and interleukin 1β in activated macrophage, enhanced production of MCP-1, RANTES (regulated on activation normal T-cell expressed and secreted), and IP-10 in peripheral blood mononuclear cells, and reduction in antibody production (1Novick D.M.O.M. Ghali V. Croxson T.S. Mercer W.D. Chiorazzi N. Kreek M.J. J. Pharmacol. Exp. Ther. 1989; 250: 606-610PubMed Google Scholar, 2Taub D.D., E.T. Geller E.B. Adler M.W. Rogers T.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 360-364Crossref PubMed Scopus (169) Google Scholar, 3Wang J Charboneau R. Balasubramanian S. Barke R.A. Loh H.H. Roy S. J. Leukocyte Biol. 2002; 71: 782-790PubMed Google Scholar, 4McCarthy L. Szabo I. Nitsche J.F. Pintar J.E. Rogers T.J. J. Neuroimmunol. 2001; 114: 173-180Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 5McCarthy L, W.M. Sliker J.K. Eisenstein T.K. Rogers T.J. Drug Alcohol Depend. 2001; 62: 111-123Crossref PubMed Scopus (337) Google Scholar). The fact that accelerated human immunodeficiency virus pathogenesis occurred in patients who abuse heroin is consistent with the immunosuppressive activity of the opioids (6Donahoe R.M. Falek A. Adv. Biochem. Psychopharmacol. 1988; 44: 145-158PubMed Google Scholar). Although previous studies indicate that opioids may regulate immune responses through their action on central nervous system or sympathetic nervous system, the discovery of opioid receptors on peripheral blood mononuclear cells suggested that these opioid receptors could directly modify the response of proinflammatory receptors (2Taub D.D., E.T. Geller E.B. Adler M.W. Rogers T.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 360-364Crossref PubMed Scopus (169) Google Scholar, 3Wang J Charboneau R. Balasubramanian S. Barke R.A. Loh H.H. Roy S. J. Leukocyte Biol. 2002; 71: 782-790PubMed Google Scholar, 4McCarthy L. Szabo I. Nitsche J.F. Pintar J.E. Rogers T.J. J. Neuroimmunol. 2001; 114: 173-180Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 7Grimm M.C. Ben-Baruch A. Taub D.D. Howard O.M. Resau J.H. Wang J.M. Ali H. Richardson R. Snyderman R. Oppenheim J.J. J. Exp. Med. 1998; 188: 317-325Crossref PubMed Scopus (188) Google Scholar). Data from knock-out mice and in vitro studies suggest that such suppression is mediated largely by μ, δ, and κ opioid receptors. Opioids receptors, members of the seven-transmembrane receptor family, perform their function by coupling to heterotrimeric Gi/o proteins. Their activation leads to inhibition of adenylyl cyclase by Gα, inhibition of voltage-dependent calcium channels, and activation of G protein-coupled inwardly rectifying K+channels by Gβγ. In cells expressing multiple G protein-coupled receptors (GPCRs),1 prolonged activation of one receptor has been shown to result in the down-regulation of other GPCR through a process called heterologous desensitization. Accumulating data have demonstrated that heterologous down-regulation of GPCR is mediated by protein kinase A and protein kinase C (PKC) (8Ali H. Richardson R.M. Haribabu B. Snyderman R. J. Biol. Chem. 1999; 274: 6027-6030Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 9Richmond A, M.S. White J.R. Schraw W. Methods Enzymol. 1997; 288: 3-15Crossref PubMed Scopus (7) Google Scholar, 11Lefkowitz R. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). Calcium flux, a potent activator of PKC, has been considered essential for heterologous desensitization of chemokine receptors in peripheral blood mononuclear cells (8Ali H. Richardson R.M. Haribabu B. Snyderman R. J. Biol. Chem. 1999; 274: 6027-6030Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 9Richmond A, M.S. White J.R. Schraw W. Methods Enzymol. 1997; 288: 3-15Crossref PubMed Scopus (7) Google Scholar, 10Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1995; 270: 10439-10448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Interaction of chemokine receptors with their ligands activates Giproteins, induces the production of DAG, and releases Ca2+, followed by activation and translocation of PKC to the plasma membrane. This process is associated with desensitization of other GPCRs in the same cell by phosphorylation of their consensus sites (10Mueller S.G. Schraw W.P. Richmond A. J. Biol. Chem. 1995; 270: 10439-10448Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 13Mueller S.G. White J.R. Schraw W.P. Lam V. Richmond A. J. Biol. Chem. 1997; 272: 8207-8214Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The family of PKC consists of more than 12 isozymes, and each of them exhibits a unique pattern of tissue distribution, subcellular translocation, and function (14Mochly-Rosen D. Science. 1995; 268: 247-251Crossref PubMed Scopus (835) Google Scholar). For instance, PKCζ is essential in mediating neutrophil chemotaxis and in regulating the polarity of astrocytes during wound healing process (15Laudanna C. Mochly-Rosen D. Liron T. Constantin G. Butcher E.C. J. Biol. Chem. 1998; 273: 30306-30315Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 16Etienne-Manneville S. Hall A. Cell. 2001; 106: 489-498Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar). PKCθ is predominantly expressed in lymphocytes and is recruited to the membrane during antigen presentation to T-cells (17Meller N. Elitzur Y. Isakov N. Cell. Immunol. 1999; 193: 185-193Crossref PubMed Scopus (57) Google Scholar). The 12 PKC isozymes can be divided into three subfamilies: classical PKCs (cPKCs), such as α, βI, βII, and γ, require both Ca2+ and DAG for activation; novel PKCs (nPKCs), such as δ, ε, θ, and η, are DAG-dependent but Ca2+-independent; and atypical PKCs, such as ζ and λ, require neither Ca2+ or DAG. Eight PKC isozymes, α, β1, β2, δ, ε, η, μ, and ζ, have been identified in human blood monocytes (18Monick M.M. Carter A.B. Gudmundsson G. Geist L.J. Hunninghake G.W. Am. J. Physiol. 1998; 275: L389-L397Crossref PubMed Google Scholar). However, their contribution to heterologous desensitization of chemokine receptors has not been defined. Preincubation with μ- or δ-opioid agonists has been shown to inhibit MIP-1α-mediated chemotaxis of monocytes and neutrophils (7Grimm M.C. Ben-Baruch A. Taub D.D. Howard O.M. Resau J.H. Wang J.M. Ali H. Richardson R. Snyderman R. Oppenheim J.J. J. Exp. Med. 1998; 188: 317-325Crossref PubMed Scopus (188) Google Scholar,21Grimm M.C. Ben-Baruch A. Taub D.D. Howard O.M. Wang J.M. Oppenheim J.J. Ann. N. Y. Acad. Sci. 1998; 840: 9-20Crossref PubMed Scopus (75) Google Scholar). Such inhibition can be blocked by the nonselective opioid antagonist naxolone or the μ- or δ-selective antagonists CTOP and naltrindole (7Grimm M.C. Ben-Baruch A. Taub D.D. Howard O.M. Resau J.H. Wang J.M. Ali H. Richardson R. Snyderman R. Oppenheim J.J. J. Exp. Med. 1998; 188: 317-325Crossref PubMed Scopus (188) Google Scholar). Preincubation with opioids has been shown to enhance phosphorylation of CXCR1, correlating with modest impairment of chemotaxis. Compared with other chemokines, opioids are less capable of mediating heterologous desensitization. Heterologous desensitization of chemokine receptors seems to follow a hierarchy; certain receptors are more potent in desensitizing than others (19Tomhave E.D. Richardson R.M. Didsbury J.R. Menard L. Snyderman R. Ali H. J. Immunol. 1994; 153: 3267-3275PubMed Google Scholar,20Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The capacity of a receptor to cross-desensitize GPCRs has been proposed to correlate with its ability to induce a greater phosphoinositide hydrolysis (8Ali H. Richardson R.M. Haribabu B. Snyderman R. J. Biol. Chem. 1999; 274: 6027-6030Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). We set out to investigate the possibility that the hierarchy may be related to the PKC isotypes. In this study, we first show that, in monocytes, opioid-mediated heterologous desensitization inhibits not only chemokine-induced chemotaxis but also Ca2+ mobilization in a dose-dependent manner. Such inhibitory effects result from a decrease in chemokine receptor affinity and their coupling efficiency to G protein. Furthermore, we find that, unlike chemokine receptors, opioid receptors fail to elicit a calcium flux and only activate novel and atypical PKC, resulting in modest suppression of chemokine receptor function. Our data suggest that the relative potency of G protein-coupled receptors in heterologous desensitization is dependent on which subfamily of PKC is activated. A robust heterologous desensitization requires both calcium-dependent and -independent kinase C. Opioids are unique and only utilize a calcium-independent PKC pathway to phosphorylate and inactivate heterologous receptors. MCP-1 and MIP-1α were obtained from Pepro Tech (Rocky Hill, NJ); I125-MIP-1α,3H-DAMGO, and Taq polymerase were from PerkinElmer Life Sciences; Met-enkephalin was from Peninsula Laboratories, Inc. (San Carlos, CA); DAMGO was from Sigma; calphostin C and Go6976 were from Alexis Biochemicals (San Diego, CA); chemotaxis chamber and membrane were from Neuroprobe (Gaithersburg, MD); polyclonal anti-PKCζ (sc-216), anti-PKCα, βI, βII (c-20), and anti-PKCδ (c-17) antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and ECL reagents were from Pierce. All of the other reagents were reagent grade and were obtained from standard suppliers. Human peripheral monocytes were obtained from healthy donor blood packs and isolated from Buffy coats (Transfusion Medicine Department, National Institutes of Health Clinical Center, Bethesda, MD) by iso-osmotic Percoll gradient. The monocytes were >90% pure by nonspecific esterase staining or by morphological analysis. Freshly isolated monocytes were suspended in ice-cold PBS and used for experiments on the same day. HEK293, vector/HEK293, and μ-opioid receptor/HEK293 cells were grown in DMEM (Biowhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 1 mm glutamine (Invitrogen), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). CCR1/HEK293, μ-receptor/CCR1/HEK293, and vector/CCR1/HEK293 cells were grown in the same buffer with 400 μg/ml Geneticin (Invitrogen). Macrophages were generated in vitro by incubating freshly isolated monocytes at 1 × 106/ml in RPMI 1640 medium and 10% fetal bovine serum in the presence of recombinant human macrophage colony-stimulating factor (50 ng/ml) at 37 °C in a humidified CO2 (5%) incubator for 7 days with the addition of fresh recombinant human macrophage colony-stimulating factor-containing medium every 2–3 days (22Yang D. Chen Q. Le Y. Wang J.M. Oppenheim J.J. J. Immunol. 2001; 166: 4092-4098Crossref PubMed Scopus (73) Google Scholar). PCR fragments of full-length CCR1 were inserted into pcDNA 3.1 to make pCCR1. pCCR1 was linearized with ScaI and electroporated into HEK293 cells. After selection with 800 μg/ml Geneticin, the CCR1 level was screened by Western blotting, and a single colony with high expression of CCR1 was selected to generate CCR1/HEK293. Full-length μ-opiate receptor was inserted into pLZRS-IRES-EGFP retroviral vector to generate pμ-opioid receptor. The empty vector was used as a control. Phoenix-Amphotrophic Retroviral Packaging cells were plated at 1.5–2 million cells/60-mm plate in DMEM with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% glutamine, 18–24 h prior to transfection (23Kinsella T.M. Nolan G.P. Hum. Gene Ther. 1996; 7: 1405-1413Crossref PubMed Scopus (672) Google Scholar). Approximately 2 μg of each plasmid containing the desired inserts (pLZRS-IRES-EGFP) were prepared for transfection into cells by using FuGENE 6 Transfection Reagent (Roche Diagnostics Corporation, Indianapolis, IN) according to the manufacturer's instructions (23Kinsella T.M. Nolan G.P. Hum. Gene Ther. 1996; 7: 1405-1413Crossref PubMed Scopus (672) Google Scholar, 24Heemskerk M.H.H.E. Ruizendaal J.J. van der Weide M.M. Kueter E. Bakker A.Q. Schumacher T.N. Spits H. Cell. Immunol. 1999; 195: 10-17Crossref PubMed Scopus (59) Google Scholar). The LZRS vector replicates episomally via use of the EBNA-1 protein and also contains a puromycin resistance gene. Finally, the inclusion of the IRES-EGFP expression cassette allows for sorting of infected target cells by flow cytometry. After transfection, the Phoenix-Ampho cells were selected with 2 μg of puromycin for 7 days, at which time the population consisted of virtually 100% EGFP-expressing cells. Infectious virus-containing supernatants were prepared by growing packaging cell lines infected with recombinant retroviral vectors as described above, in T75 flasks to ∼80% confluency and then overlaying this culture with 10 ml of complete medium to allow cells to produce virus overnight. At the same time, target cell populations were prepared by plating cells into suitable tissue culture vessels and overlaying the target culture with 0.22 μm of filtered, recombinant retrovirus-infected supernatants the following morning. Infectious supernatants were supplemented with 5 μg/ml polybrene to assist in virus attachment. 3–5 days post-infection, the target cells were sorted by flow cytometry and analyzed as described under "Results." Chemotaxis was performed as described by the manufacturer (Neuroprobe). In brief, the monocytes were pretreated with PBS, MIP-1α, MCP-1, Met-enkephalin, or morphine for 30 min at 37 °C. The cells were then washed twice with binding medium (RPMI from Biowhittaker, 1% bovine serum albumin, and 20 mmHEPES) and loaded into the upper chemotaxis chambers. Chemokines, diluted to various concentrations were loaded into the lower chambers. The chambers were incubated at 37 °C, 100% humidity, and 5% CO2 for 1 h. The 5-μm filter between the chambers was then washed, fixed, and stained. The cells that migrating through the filter were counted by microscopy. The chemotaxis index was the ratio of chemotactic cell numbers in a chemokine gradientversus the cell numbers in a medium control. For HEK293 cells, the polycarbonate filter was pretreated with 50 μg/ml of collagen in binding medium at 4 °C overnight. Chemotaxis of HEK293 cells was assayed after 5 h. The statistical analysis of chemotactic responses was performed by PRISM3.0. Calcium flux was measured as described by Badolato et al. (25Badolato R, J.J. Wang J.M. McVicar D. Xu L.L. Oppenheim J.J. Kelvin D.J. J. Immunol. 1995; 155: 4004-4010PubMed Google Scholar). In brief, the cells were incubated at 107/ml for 30 min at room temperature in DMEM containing 1 μm Fura-2. The cells were then washed with DMEM once, washed with Hanks' balanced salt solution twice, and diluted into 2 × 106/ml. The cells were then loaded into a 2-ml cuvette at 37 °C, and the relative ratio of fluorescent emission at 510 nm when excited by 340 nm and 380 nm was recorded by a PerkinElmer Life Sciences luminescence spectrometer. For heteorologous desensitization experiments, the cells were first incubated with Met-enkephalin or chemokines at 37 °C for 30 min before adding Fura-2. The ligand binding assays were carried out as described by Grimm et al. (7Grimm M.C. Ben-Baruch A. Taub D.D. Howard O.M. Resau J.H. Wang J.M. Ali H. Richardson R. Snyderman R. Oppenheim J.J. J. Exp. Med. 1998; 188: 317-325Crossref PubMed Scopus (188) Google Scholar) with modifications. The cells were preincubated with MIP-1α, MCP-1, or Met-enkephalin for 30 min at 37 °C, washed extensively, and resuspended in binding medium at 107/ml. The binding assays were carried out on ice using 0.5 nmI125MIP-1α in the presence of increasing concentration of competing unlabelled-MIP-1α. The cells were incubated at 4 °C for 30 min, and unbound ligands were separated from cells by a 10% sucrose gradient. The level of binding was determined in a γ-counter. Nonlinear regression analysis of data was performed by a PRISM3.0 program by fitting the following equation.Total Binding=Bmax*[Hot]/([Hot]+[Cold]+Kd)Equation 1 +Nonspecific Binding The assay was performed as described by Richardson et al. (20Richardson R.M. Haribabu B. Ali H. Snyderman R. J. Biol. Chem. 1996; 271: 28717-28724Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) with minor modifications. The cells were pretreated with MIP-1a, MCP-1, Met-enkephalin, or binding medium for 45 min at 37 °C. After three washes, the cell membranes were isolated for binding assays. Assay of PKC translocation in monocytes were performed as described by Laudanna et al.(15Laudanna C. Mochly-Rosen D. Liron T. Constantin G. Butcher E.C. J. Biol. Chem. 1998; 273: 30306-30315Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) with minor modifications. In brief, fresh monocytes were stored in ice-cold PBS for 90 min to decrease the membrane-bound PKC before stimulation. Cocktails of protease inhibitors were added. The cells were then stimulated at 37 °C, stopped by ice-cold PBS, homogenized by sonication, and underwent ultra-centrifugation at 100,000 ×g for 1 h. The supernatant was kept as the cytosolic fraction. The precipitates were sonicated in one half volume of PBS with 1% Triton X-100 and centrifuged at 100,000 × gagain for 30 min. The solublized precipitates were kept as the membrane fraction. The samples were then loaded on 10% SDS-PAGE, followed by Western blotting analysis. Confocal microscopic analysis of immunofluorescent staining of PKC isotypes was carried out as described by Etienne-Manneville (16Etienne-Manneville S. Hall A. Cell. 2001; 106: 489-498Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar). The cells were pretreated with 100 ng/ml MCP-1 or 10−7m Met-enkephalin for 10 min, then fixed, permeablized, and stained by rabbit antibody to PKC isotypes followed by fluorescein isothiocyanate-labeled goat anti-rabbit antibody. The cells were visualized using a Zeiss inverted fluorescent confocal microscope. First, we compared the degree of Met-enkephalin-mediated chemotaxis of freshly isolated monocytes to those induced by conventional chemokines, MIP-1α and MCP-1. As shown in Fig.1A, MCP-1, presumably by activating chemokine receptor, CCR2, induced a robust chemotaxis response, which peaked at 3 × 10−10m. MIP-1α, the endogenous ligand for receptor CCR1 and 5, was also a potent chemoattractant of monocytes. However, the chemotactic response of Met-enkephalin on monocytes, although significant, was less potent, with an index ranging from 2 to 4.5. Morphine was also a weaker stimulus of monocyte chemotaxis (data not shown). The lower chemotactic activity of opioids may be due to the lower receptor expression on cell surface or perhaps due to inefficient coupling between opioid receptors and Gi proteins. Furthermore, although MCP-1 and MIP-1α induced monocyte chemokinesis, the background level of monocyte motility was unchanged when treated with opiates over a wide concentration ranging from 10−11 to 10−6m (data not shown). Because opioid receptors couple to Gi proteins and induce chemotaxis, we determined to assess their capacity to elicit a concomitant Ca2+ response. As shown in Fig. 1B, both MCP-1 and MIP-1α rapidly induced a potent Ca2+ flux in monocytes, confirming that the inositol 1,4,5-trisphosphate-induced Ca2+ flux is functional (26Locati M. Murphy P.M. Annu. Rev. Med. 1999; 50: 425-440Crossref PubMed Scopus (253) Google Scholar). However, Met-enkephalin, at concentrations from 10−10 to 10−4m, failed to initiate a detectable Ca2+response in monocytes (Fig. 1B and data not shown). Morphine also did not induce any measurable Ca2+ mobilization (data not shown). The deficiency of opioid-induced Ca2+mobilization is likely due to insufficient production of inositol 1,4,5-trisphosphate by activated Gi-coupled PLCβ. These data further suggest that the μ- and/or δ-opioid receptors are less potent in inducing chemotactic Gi signaling in monocytes than chemokine receptors. To evaluate the potency of opioid-induced desensitization, the inhibitory effects of Met-enkephalin on MIP-1α-induced chemotaxis were compared with the desensitizing effects of MIP-1α and MCP-1 on chemokine receptors. Freshly isolated monocytes pretreated with 100 ng/ml MIP-1α exhibited a reduction of more than 85% of the chemotactic response to MIP-1α, because of homologous desensitization (Fig.2A). This homologous desensitization was dose-dependent (Fig. 2B). Monocytes pretreated with 100 ng/ml MCP-1 for 30 min also showed a marked decrease in their chemotactic response to MIP-1α, because of heterologous desensitization of chemokine receptors (Fig. 2,A and B). Pretreatment with 10−8m of Met-enkephalin significantly impaired MIP-1α-mediated chemotaxis but to a lesser degree (Fig.2A). Met-enkephalin exhibited dose-dependent heterologous desensitization effects with optimal inhibitory activity of 50% at 10−6m (Fig. 2B). To determine the effect of opioid mediated heterologous desensitization on Ca2+ flux, we compared the dose dependence of the MIP-1α-elicited Ca2+ response of monocytes, which were pretreated cells with 1000 ng/ml of MIP-1α, MCP-1, or 10−7m Met-enkephalin. MIP-1α, at 0, 5, 15, 50, 150, and 500 ng/ml, induced Ca2+ flux in monocytes in a dose-dependent manner (Fig. 2C and data not shown). To directly compare homologous and heterologous desensitization, the maximal value of each Ca2+ flux was used to plot the dose response of the stimulus (Fig. 2D). Monocytes pretreated with 1000 ng/ml MIP-1α for 30 min followed by three washes failed to exhibit a detectable response to 5 and 15 ng/ml MIP-1α (Fig. 2D). Homologous desensitization also decreased the response to MIP-1α at 150 and 500 ng/ml. MCP-1 pretreatment severely reduced the MIP-1α-induced Ca2+response, but the inhibitory effects decreased as the concentration of stimulus was increased and became undetectable at 150 ng/ml of MIP-1α (Fig. 2D). Pretreatment with Met-enkephalin resulted in moderate inhibition of Ca2+ flux (Fig. 2D). Monocytes pretreated with 10−7mMet-enkephalin lost 70% of the Ca2+ response to 5 ng/ml MIP-1α. Met-enkephalin-mediated inhibitory effects were maximal when chemokine concentration was low and decreased as the chemokine concentration was increased (Fig. 2D). Both chemotaxis and calcium flux data show that the heterologous desensitization of the MIP-1α response by Met-enkephalin was significant but less potent than either homologous or heterologous desensitization of chemokine receptors by chemokines. We further examined the effect on MIP-1α-mediated chemotaxis inin vitro macrophage colony-stimulating factor-stimulated macrophages. As shown in Fig. 3, although opioids also did not induce calcium flux in activated macrophage, they induced similar heterologous desensitization of chemokine receptors in activated macrophages. Desensitization of seven-transmembrane receptors may involve internalization of receptors, decrease in ligand binding affinity, or impaired interaction with Gi proteins (11Lefkowitz R. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar, 12Ceder G.J.A. Clin. Chem. 2001; 47: 1980-1984Crossref PubMed Scopus (68) Google Scholar, 13Mueller S.G. White J.R. Schraw W.P. Lam V. 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Homologous competitive binding analyses showed a 3-fold decrease in MIP-1α binding affinity after Met-enkephalin pretreatment, whereas the total number of binding sites was unchanged (Fig. 4, A and B). In contrast, preincubation with MIP-1α reduced the number of surface binding sites by over 70%, presumably because of receptor internalization during homologous desensitization (Fig.4C). Activation of MCP-1 receptors also decreased the number of MIP-1α receptors by more than 35% (Fig. 4C). In contrast with our results, previously reported binding analysis, carried out at room temperature, did not reveal any detectable affinity change (7Grimm M.C. Ben-Baruch A. Taub D.D. Howard O.M. Resau J.H. Wang J.M. Ali H. Richardson R. Snyderman R. Oppenheim J.J. J. Exp. Med. 1998; 188: 317-325Crossref PubMed Scopus (188) Google Scholar). Our binding assays were performed at 4 °C to prevent MIP-1α-induced homologous desensitization. Upon ligand binding, chemokine receptors activate downstream heterotrimeric G proteins by enhancing the exchange of bound GDP to GTP. A desensitized GPCR shows a decrease in its capability to induce the binding of [35S]GTPγS to membrane G proteins. Pretreatment with Met-enkephalin for 30 min resulted in a 34% loss of MIP-1α-stimulated [35S]GTPγS binding on the membrane, indicating an impairment of the coupling efficiency between chemokine receptors and downstream G protein (Fig. 4D). In contrast, the capability of MIP-1α receptors to enhance membrane [35S]GTPγS binding was severely inhibited (up to 62–70%) after pretreatment with MCP-1 or MIP-1α (Fig.4D). The decrease in both receptor affinity and coupling efficiency to G protein may directly contribute to the desensitization of MIP-1α-mediated chemotaxis, calcium flux, and other signals. The expression level of opioid receptors on monocytes and macrophages is very low relative to that of chemokine receptors. Le
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