Glucocorticoid hormones (GC) are potent antiinflammatory agents widely used in the treatment of diverse human diseases. The present study was aimed at assessing the effect of GC on chemokine receptor expression in human monocytes. Dexamethasone (Dex) up-regulated mRNA expression of the monocyte chemotactic protein (MCP-1, CCL2) chemokine receptor CCR2. The effect was selective in that other chemokine receptors were not substantially affected. Stimulation by Dex was observed after 4 h of exposure at concentrations of 10−7 to 10−5 M. Steroids devoid of GC activity were inactive, and the GC receptor antagonist, RU486, inhibited stimulation. Dex did not affect the rate of nuclear transcription, but augmented the CCR2 mRNA half-life. Augmentation of CCR2 expression by Dex was associated with increased chemotaxis. Finally, Dex treatment induced productive replication of the HIV strain 89.6, which utilizes CCR2 as entry coreceptor, in freshly isolated monocytes. Together with previous findings, these results indicate that at least certain pro- and antiinflammatory molecules have reciprocal and divergent effects on expression of a major monocyte chemoattractant, MCP-1, and of its receptor (CCR2). Augmentation of monocyte CCR2 expression may underlie unexplained in vivo effects of GC as well as some of their actions on HIV infection.
Chemokines are a growing superfamily of low m.w. chemotactic proteins that can be divided into four branches according to the position of the first cysteine pair (C-X-C, C-C, and C-X3-C) or the lack of two of the four cysteines (C) (1, 2, 3, 4, 5). C-X-C chemokines, of which IL-8 is the prototype, are mainly active on neutrophils, T, and B lymphocytes. C-C chemokines have a wider spectrum of action being active on monocytes, granulocytes, T, and B lymphocytes, NK cells, and dendritic cells (1, 2, 3, 4, 6). Lymphotactin, the only C chemokine so far described, is active on T lymphocytes and NK cells (7, 8). Chemokines are produced by multiple cell types, including monocytes/macrophages, endothelial cells, mesothelial cells, fibroblasts, keratinocytes, and lymphocytes, and bind to seven transmembrane domain G protein-coupled receptors (1, 2, 3, 9, 10). Five receptors for C-X-C chemokines (CXCR1–5) and eight for C-C chemokines (CCR1–8) were recently cloned. These receptors show a promiscuous pattern of ligand recognition and are differentially expressed and regulated in leukocytes (10, 11, 12, 13, 14, 15, 16, 17, 18, 19).
Regulation of chemokine receptor expression has been studied less extensively than agonist production. IL-2 was recently shown to augment expression of CCR2 in T cells, NK cells, and monocytes (14, 15, 19). Recently, we found that LPS, a prototypic proinflammatory molecule, which induces monocyte chemotactic protein-1 (MCP-1)4 production in mononuclear phagocytes (20), causes a rapid, drastic, and selective down-regulation of CCR2 mRNA and surface expression in human monocytes (15). Similar results were obtained with other proinflammatory molecules (15, 21). It was therefore of interest to investigate whether and how antiinflammatory agents affect C-C chemokine receptor expression in human monocytes. In this regard, we recently reported that the anti-inflammatory cytokine IL-10 enhances expression of certain CC chemokine receptors in monocytes (22).
Glucocorticoid hormones (GC) are prototypic antiinflammatory/immunosuppressive agents widely used in the treatment of diverse human diseases. They exert complex effects on immunocompetent cells, including induction of the type II “decoy” receptor, which represents a unique pathway of negative regulation of the IL-1 system (23, 24). They inhibit production of chemokines in a variety of cell types (25, 26, 27, 28, 29, 30), including monocytes, and cause alterations in monocyte trafficking, some which are poorly understood (31, 32, 33, 34).
The present study was aimed at assessing whether and how GC affect CC chemokine receptor expression in human monocytes. Perhaps unexpectedly, GC selectively up-regulated CCR2 receptor expression by stabilizing mRNA transcripts. Thus, GC have divergent effects on agonist (MCP-1) production and receptor expression in monocytes. These observations provide an explanation for some of the in vivo effects of GC, as well as for some of their actions in HIV infection. More in general, they add to an emerging picture of prototypic pro- and antiinflammatory agents having reciprocal and divergent effects on expression in mononuclear phagocytes of a major monocyte attractant, MCP-1, and of its receptor, CCR2.
Materials and Methods
Human monocytes were separated from peripheral blood of human healthy donors by Percoll gradient centrifugation (20, 21). Briefly, whole blood was fractionated by Ficoll gradient centrifugation (Seromed-Biochem KG, Berlin, Germany) and monocytes collected from the ring, layered on top of 46% Percoll (Pharmacia, Uppsala, Sweden) after a centrifugation at 2000 rpm for 30 min at room temperature. Monocytes (>98% pure, as assessed by morphology) were resuspended at 5 × 106 cells/ml in RPMI 1640 (Seromed) with 10% FCS (HyClone, Logan, UT). All reagents contained <0.125 EU/ml of endotoxin, as checked by Limulus amebocyte lysate assay (Microbiological Associates, Rockville, MD). THP-1 cell line was obtained from the American Type Culture Collection ( Manassas, VA) and cultivated in RPMI 1640 medium containing 10% FCS with 2 mM glutamine. MonoMac 6 cell line (gift of Dr. L. Ziegler Heitbrock, Radiologische Klinik, Universitat Müncher, Munich, Germany) was cultured in RPMI 1640 with 20% FCS, 2 mM glutamine, 1 mM sodium pyruvate (Seromed), 1 mM oxaloacetic acid (Sigma, St. Louis, MO), and 1× nonessential amino acids (Seromed).
Stimulation conditions and reagents
Monocytes, THP-1, and MonoMac 6 cell lines were incubated in endotoxin-free RPMI 1640 with 10% FCS at 5 × 106 cells/ml, with or without stimuli for the indicated times at 37°C in the presence of 5% CO2. Dexamethasone (Dex; Merck, Rahway, NJ) was used at 10−7 M, unless otherwise specified. Actinomycin D (Act D; Sigma) was used at 1 μg/ml. 5,6-Dichloro-1-β-d-ribofuranosylbenzimidazole (DRB; Sigma) was used at 20 μg/ml. Cycloheximide (Sigma) was used at 10 μg/ml. Cortexolone (17-hydroxy-11-deoxycorticosterone; Sigma) and progesterone (Sigma) were used at 10−5 M and 10−7 M, respectively; RU486 (Roussel Uclaf, Romainville, France) at 10−6 M, as previously described (24). For phenotype analysis, indirect immunofluorescence was performed with the human anti-CCR2 Ab 132.1D9 (G. LaRosa, data not shown) and PE-labeled goat anti-mouse Ig (Jackson ImmunoResearch West Grove, PA) using a FACStar (Becton Dickinson, Mountain View, CA)
Northern blot and run off analyses
Total RNA was isolated by the guanidium isothiocyanate method, as previously reported (35). A total of 10 μg of total RNA was analyzed by electrophoresis through 1% agarose/formaldehyde gels, followed by Northern blot transfer to Gene Screen Plus membranes (New England Nuclear, Boston, MA). Probes were labeled by the Megaprime DNA labeling system (Amersham, Little Chalfont, U.K.) with [α-32P]dCTP of 3000 Ci/mmol sp. act. (Amersham). CCR2-B cDNA was obtained by PCR amplification of the reported sequences (19, 36), and the CCR1, CCR4, and CCR5 cDNAs were obtained from Dr. T. Wells (Glaxo Wellcome Research and Development S.A., Geneva, Switzerland).
Nuclear run-off experiments were performed essentially as described (35) using nuclei from 20–30 × 106 cells isolated after 4 h of stimulation.
Cell migration was evaluated using a chemotaxis microchamber technique (37). Then, 27 μl of chemoattractant solution or control medium (RPMI 1640 with 1% FCS) was added to the lower wells of a chemotaxis chamber (Neuroprobe, Pleasanton, CA). A polycarbonate filter (5-μm pore size; Neuroprobe) was layered onto the wells and covered with a silicon gasket and with the top plate. A total of 50 μl of the cell suspension (1.5 × 106/ml monocytes in PBMC) was seeded in the upper chamber. The chamber was incubated at 37°C in air with 5% CO2 for 90 min. At the end of the incubation, filters were removed and stained with Diff-Quik (Baxter, Rome, Italy), and five high-power oil-immersion fields were counted.
Percoll-derived monocytes were resuspended in DMEM (BioWhittaker, Verviers, Belgium) supplemented with 10% FCS (HyClone Europe, Oud-Beijerland, The Netherlands) and 5% AB human serum, and seeded in 24-well plates (Falcon; Becton Dickinson Labware, Lincoln Park, NY) at 0.5 × 106 cell/ml. Monocytes were either left untreated or were treated with Dex (10−7 M) 30 min before infection with the 89.6 HIV strain (kind gift of Prof. Ronald G. Collman, University of Pennsylvania, Philadelphia, PA) at the fixed concentration of 105 cpm of RT activity per 0.5 × 106 cells. Fifty percent of culture media was replaced with fresh media twice a week. Aliquots of culture supernatants were harvested at the indicated times points and stored at −80°C until simultaneously tested for RT activity contents.
HIV-RT activity assay
Selective up-regulation of CCR2 by Dex
CCR1, CCR2, and CCR5 mRNAs were present at high levels in untreated human monocytes (Fig. 1), whereas the CCR4 transcript was less expressed under the same experimental conditions, with considerable donor-to-donor variation. In a series of 10 experiments with 10 donors, Dex (10−7 M for 4 h) caused a median 30-fold increase in CCR2 transcript levels (range 6- to 80-fold), as assessed by densitometry. CCR1, CCR4, and CCR5, as well as CXCR2 and CXCR4, were not substantially affected under the same conditions (Fig. 1, and data not shown). Therefore, subsequent analysis focused on CCR2.
The effect of Dex was dose-dependent, with augmentation at concentrations between 10−7 and 10−5 M (Fig. 2,A, representative of two donors). Peak induction of CCR2 mRNA occurred at 4 h and decreased between 8 and 20 h of stimulation (Fig. 2,B, two donors). To establish whether the increase in CCR2 mRNA was specific for Dex, we stimulated monocytes with different types of steroids (Fig. 2,C, two donors). Cortexolone and progesterone were inactive, while Dex was able to augment CCR2. We also treated monocytes with RU486, a GC-receptor antagonist, (Fig. 2 D, two donors), and we found that RU486 blocked by 75% the increase induced by Dex. Thus, the action of Dex on CCR2 mRNA is mediated by GC receptors.
To assess whether the effect of Dex was related to cellular differentiation, we evaluated the CCR2 mRNA expression in monocyte-derived macrophages after 5 days of culture, THP-1 and MonoMac 6 cell lines (Fig. 3). Also in these cells, Dex up-regulated CCR2 transcripts, though with lower efficiency compared with fresh monocytes. For monocyte-derived macrophages the increase was 5-fold (two donors), for the MonoMac 6 cell line it was 2-fold (two experiments), and for the THP-1 cell line it was only 25% (two experiments).
Mechanism of action
The mechanisms involved in Dex augmentation of CCR2 steady state transcripts in monocytes were studied. We evaluated its effects on both mRNA stability and gene transcription. Act D (1 μg/ml) and DRB (20 μg/ml) were added to human monocytes in the presence or absence of 10−7 M Dex, and total RNA was extracted at different times as indicated (Fig. 4). In the presence of Act D, the estimated half-life of the transcript was about 1.5 h, in agreement with recent results (15, 39). Treatment with Act D plus Dex increased the CCR2 mRNA half-life to 2.3 h (Fig. 4,A). Similar increases were observed when DRB was used instead of Act D (Fig. 4,B). To explore the effects of Dex on gene transcription, we performed a nuclear run-off analysis (Fig. 4 C). Dex did not result in variations of the transcriptional rate of the CCR2 gene, but, in contrast, it reduced MCP-1 gene transcription, as expected.
Up-regulation of CCR2 surface expression and chemotactic responsiveness
To assess whether the up-regulation that we observed at the mRNA level correlated with protein expression, monocytes were exposed to Dex (10−7 M) for 8 h, and surface CCR2 was examined by flow cytometry. As shown in Fig. 5, Dex augmented surface expression of CCR2 The mean channel of fluorescence was 340 and 396 for control and Dex-treated cells, respectively, with 46 and 70% of positive cells, respectively (Fig. 5). In an effort to assess the functional relevance of CCR2 up-regulation, monocytes were incubated with Dex (10−7 M) for 10 h, washed, and cultured for an additional 2 h before examining their chemotactic responsiveness (Fig. 6, representative of two experiments). This experimental protocol was designed to avoid any confounding direct influence of Dex on the locomotory capacity of monocytes, which has been the object of conflicting results (40, 41). As shown in Fig. 6,A, Dex pretreated monocytes showed stronger responsiveness to MCP-1, better evident at suboptimal agonist concentrations. As expected on the basis of the lack of substantial effects on CCR5 (Fig. 1), the responsiveness to the CCR5 agonist macrophage-inflammatory protein-1β was little or not affected by Dex (Fig. 6 B). In one experiment (data not shown), binding of radiolabeled MCP-1 was also increased in Dex-treated monocytes.
Dex enhances the replication of a CCR2-using HIV strain in monocytes
Freshly purified monocytes were infected with the 89.6 strain of HIV, known to utilize CCR2b as well as other chemokine receptors in order to enter CD4+ cells (42). As expected, no replication was observed in monocytes that were left untreated, whereas a sustained production of HIV was consistently detected in cultures of different donors that were stimulated with Dex (Fig. 7). Consistent with the observed pattern of modulation of chemokine receptor, Dex did not enhance the replication of the CCR5-dependent BaL strain of HIV-1 (data not shown).
The results presented here show that Dex up-regulates expression of CCR2 in human monocytes. A similar effect, though less marked, was observed with monocyte-derived macrophages and two myelomonocytic cell lines. The action of Dex was selective in that other C-C and C-X-C chemokine receptors (CXCR2 and CXCR4) expressed in monocytes were unaffected. Dex mediated CCR2 up-regulation by interacting with GC receptors and prolonging the half-life of CCR2 transcripts.
GC are potent immunosuppressive/antiinflammatory molecules (32, 33, 34, 43) whose mode of action has not been fully defined. GC inhibit expression of a variety of cytokines (44), including chemokines (26, 27, 28, 29, 30, 31). GC also affect cytokine receptor expression, though frequently do they do so in concert with other agents (45, 46).
The type II IL-1 decoy receptor is up-regulated by GC in monocytes and PMN, which release this molecule in increased amounts (24, 47). GC-augmented IL-1 decoy receptor expression and release may contribute to the antiinflammatory activity of these molecules. Interestingly, as for CCR2, this effect was mediated by stabilization of receptor transcripts (24, 47). The 3′-untranslated region of the mRNAs of several rhodopsin family members, contains AU-rich elements, which correlate with highly regulated short- lived mRNAs (48, 49)
GC have profound effects on the number of circulating monocytes and on their recruitment at sites of inflammation and tumor growth (32, 33, 34, 50). GC cause profound monocytopenia, which, at least initially in humans, is caused by redistribution of circulating monocytes (32, 34, 51). It is tempting to speculate that up-regulation of CCR2 may render circulating monocytes more sensitive to tissue-derived chemoattractants (MCP-1, -2, and -3) and thus contribute to this unexplained in vivo action of GC. In the same perspective, acute stress augments Ag-specific cell-mediated immunity, at least in part via GC (52). Again, up-regulation of CCR2 in monocytes and possibly in T and NK cells, could underlie this phenomenon, which may represent a means to mobilize defenses under extreme acute conditions. Under conditions of chronic exposure to elevated GC, we speculate that inhibition of cytokine and chemokine production prevails and results in inhibition of recruitment. Finally, sustained up-regulation of CCR2 may play a role in the rebound of inflammatory reactions and symptoms that often follow chronic GC therapy.
Chemokine receptors, including CCR2-B, act as fusion coreceptors for HIV (42, 53). Elevated levels of GC are found in the blood and saliva of HIV-infected patients (54). GC have complex actions on HIV infection in in vitro system (38, 55, 56, 57). In particular, GC have been shown to enhance HIV replication during viral isolation from activated PBMC of HIV-positive individuals (57). In addition, Dex and related molecules enhanced the transcriptional activation of HIV in latently infected U1 cells costimulated with TNF-α (38, 56). Monocytes, unlike tissue macrophages, are not efficient targets for HIV replication in vitro infection in vitro (22, 58) and in vivo (59). However, they express both CD4 and some chemokine receptors, potentially acting as entry coreceptor for HIV. The results presented here show that GC can substantially enhance the levels of HIV replication in mononuclear phagocytes infected by CCR2-using viral strain, such as 89.6 (42), but not by a more common CCR5-dependent virus BaL. By an analogous mechanism, we have recently shown that another antiinflammatory molecule, IL-10, could enhance the replication of HIV-1 BaL via selective up-regulation of CCR5 (22).
Thus, GC are potent inhibitors of chemokine production (26, 27, 28, 29, 30, 31), MCP-1 in particular (31), yet they up-regulate the CCR2 receptor. This dual action of GC mirrors the effects of at least some proinflammatory molecules (15). LPS and other proinflammatory signals, potent inducers of MCP-1 and MCP-3 in mononuclear phagocytes (20), were found to selectively destabilize CCR2 mRNA in monocytes (15, 21, 60). Hence, major pro- and antiinflammatory molecules have reciprocal and opposing effects on agonist production and CCR2 receptor expression in mononuclear phagocytes. These opposing and reciprocal influences may serve to attenuate the major net pro- or antiinflammatory action of these molecules. These results also suggest that regulation of CCR2 receptor mRNA stability may represent an interesting novel target for therapeutic intervention.
We thank Dr. P. Allavena and Prof. S. Garattini for invaluable discussions and critical review of the paper.
This work was supported by Project AIDS from Istituto Superiore di Sanità and by 40% fund from Ministero dell’Universitá e della Ricerca Scientifica e Technologica (MURST; Italy). The generous contribution of the Italian Association for Cancer Research (Milan, Italy) is gratefully acknowledged. This work was conducted under a research contract with Consorzio Autoimmunità Tardiva (Pomezia, Italy) within the “Programma Nazionale Farmaci-seconda fase” of MURST.
Abbreviations used in this paper: MCP-1, monocyte chemotactic protein-1; GC, glucocorticoid hormones; Act D, actinomycin D; DRB, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole; Dex, dexamethasone.