Upregulation of expression of the chemokine receptor CCR5 by hydrogen peroxide in human monocytes.

The objective of this study was to test the hypothesis that an oxidative stress can serve as a signal to regulate the expression of CCR5. When human monocytes were exposed to graded concentration of hydrogen peroxide (H(2)O(2)), CCR5 mRNA levels increased maximally at 4 h of exposure to 200 microM of H(2)O(2) and decreased by 24 h of treatment. Pretreatment of monocytes with the NF-kappaB inhibitor BAY 11-8072 blocked the H(2)O(2)-induced augmentation of CCR5 mRNA expression, suggesting a role for this transcription factor in the regulation of CCR5 expression. CCR5 protein expression on the plasma membrane was also increased by treatment with H(2)O(2,) as assessed by flow cytometry. This was accompanied by enhanced responsiveness of H(2)O(2)-pretreated monocytes to the CCR5 ligand MIP-1beta in terms of chemotaxis and c-fos gene activation. Our results suggest that oxidative stress may indeed modulate the expression of chemokine receptors and thus contribute to regulation of the inflammatory process.


Introduction
Recruitment of inflammatory cells to a tissue site is a complex process that depends in part on the local expression of appropriate chemoattractant proteins termed chemokines. The interaction of chemotactic factors with their corresponding receptors on leukocytes constitutes the basis for chemotaxis to inflammatory sites and cell activation. In addition to the production of chemotactic factors, the level of expression of their receptors on target cells will determine the magnitude of the response. CCR5 is a G-protein-coupled receptor that binds the chemokines monocyte inflammatory protein (MIP)-1a, MIP-1b and RANTES. 1,2 CCR5 is expressed on monocytes/macrophages, activated T cells and dendritic cells in lymphoid tissues, the ovaries and the lungs. 3 It has been proposed to be implicated in several inflammatory diseases, such as rheumatoid arthritis, asthma and glomerulonephritis. 4 Á 7 Inflammatory cells, such as macrophages and neutrophils, play an important role in host defense, but may also contribute to tissue injury through the release of reactive oxygen species (ROS). 8 Á 10 Increased production of ROS is now believed to be implicated in the pathogenesis of inflammatory disease such as acute respiratory distress syndrome, asthma, rheumatoid arthritis and AIDS. 11 Á 14 In addition, ROS are recognized as mediators of cell apoptosis. 15 Á 19 The role of the second messenger in signal transduction requires small, non-toxic concentrations of ROS that can be produced by every cell type. ROS have been shown to affect the production of the chemokine interleukin (IL)-8 in dendritic cells and monocytes, 20,21 monocyte chemotactic protein (MCP)-1 in monocytes and MIP-1a in the alveolar macrophages cell line NR8383. 22 These effects may be mediated in part through the action of ROS on redox-sensitive proteins, such as the transcription factor nuclear factor (NF)-kB, 23 Á 29 or some upstream protein kinases. 30 NF-kB regulates many of the genes involved in the immune response, including IL-2, IL-6, IL-8, interferon-g, tumour necrosis factor (TNF)-a, vascular cell adhesion molecule-1 and intracelular adhesion molecule-1. 31 It is a heterodimer of 50 kDa (p50) and 65 kDa (p65) subunits and is found in the cytoplasm in an inactive form, bound to the inhibitory protein IkB through the p65 moiety. 32,33 In response to various stimuli, including proinflammatory cytokines, IkB-a is first phosphorylated by IKK and then rapidly degraded by the proteasome, allowing translocation of NF-kB to the nucleus and subsequent gene activation. 34,35 The CCR5 gene promoter contains consensus binding sequences for the transcriptional activating factor NF-kB. 36 In this study, we investigated the hypothesis that ROS can increase the expression of the chemokine receptor CCR5 in human monocytes and thus contribute in the inflammatory process. We show here that hydrogen peroxide (H 2 O 2 ), a cell membrane-permeable ROS, can indeed upregulate CCR5 expression in monocytes and increase their responsiveness to the CCR5 ligand MIP-1b.

Materials and methods
Chemical reagents H 2 O 2 and dimethyl-sulfoxide were from Fisher Scientific (Fair Lawn, NJ, USA). MIP-1b was obtained from Pepro Tech (Rocky Hill, NJ, USA) and actinomycin D was from Merck Sharp & Dohme International (Rahway, NJ, USA). BAY 11-7082 was from Calbiochem (San Diego, CA, USA) and catalase was from Sigma-Aldrich Chemie (Steinheim, Germany).

Preparation of monocytes
Human venous blood from healthy medication-free volunteers was collected on citrate/dextrose/adenine. The peripheral blood mononuclear leukocytes were enriched by dextran sedimentation, layered over a Ficoll Á/Hypaque cushion and centrifuged at 400 )/g for 20 min. Mononuclear leukocytes were collected at the interface and washed twice with phosphate-buffered saline (PBS) and resuspended in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum (FBS). Monocytes were then purified by adherence (60 min, 378C) to the surface of plastic petri dishes coated with defibrinized autologous serum and removed with ethylenediamine tetraacetic acid (EDTA) (0.01 M) in RPMI 1640, 10% FBS. This was effective in enriching the cell population to greater than 90% monocytes, with a viability greater than 98% as assessed by Wright Á/Giemsa staining and trypan blue exclusion, respectively. Cells were resuspended in RPMI 1640, 10% FBS, at a final concentration of 2 )/10 6 cells/ml. Monocytes were left to rest overnight in polypropylene tubes to allow them to return to baseline status following initial activation by adherence. The medium was then removed by pipetting and replaced by fresh RPMI 1640 without FBS 1 h before stimulation with hydrogen peroxide. The 30% H 2 O 2 stock was diluted in RPMI immediately before treatment. Cell viability after treatment was measured by trypan blue exclusion.

mRNA studies
After appropriate treatment, cells were pelleted in 15 ml polypropylene tubes and the total cellular RNA was isolated by acid guanidium thiocyanate Á/ phenolÁ/chloroform extraction according to Chomczynski and Sacchi. 37 RNA (10 mg) was separated by electrophoresis on 1% agarose and transferred onto a Hybond-N (Amersham, Arlington Heights, IL, USA) membrane for northern analysis. The cDNA corre-sponding to the whole coding sequence of human CCR5 was obtained as previously described 38 from genomic DNA of Raji cells using the primers 5?-GCTCTAGAGATTATCAAGTGTC-3? (sense), and 5?-GGGGTACCTCACAAGCCCACAGATATTTCCTGCTC-CCC-3? (antisense). The c-fos cDNA probe was obtained from the American Type Culture Collection (Rockville, MD, USA). Control hybridizations were performed with the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe obtained from the American Type Culture Collection. The probes were labelled with a multiprime DNA labeling system (Amersham) using [ 32 P]dCTP (specific activity /3000 Ci/mmol; Amersham). Membranes were prehybridized for 4 h in a mixture containing 120 mM of Tris, 600 mM of NaCl, 8 mM of EDTA, 0.1% sodium pyrophosphate, 0.2% sodium dodecyl sulphate, and 100 mg/ml of heparin. Hybridization was performed overnight at 688C in the same mixture in which the concentration of heparin was increased to 625 mg/ml and dextran sulfate at 10% was added. The membranes were then washed once at room temperature for 20 min in 2)/SSC (1 )/SSC: 0.15 M of NaCl, 0.15 M of sodium citrate, pH 7); once with 0.1 x SSC, 0.1% sodium dodecyl sulphate at 688C for 60 min, and then rinsed at room temperature with 0.1 )/ SSC. The membranes were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY, USA) with intensifying screens for 24 h at (/808C. Signal intensity was quantitated by densitometry using a UMAX PowerLook II scanner with the UMAX Magic Scan DA3.1 software and analysed with the NIH Image 1.61 software (Bethesda, MD, USA) on a Macintosh 7300/200 computer. Densitometric values are expressed as ratios of receptor/GAPDH or receptor/28s RNA densitometry quantification and normalized for control 0/1.

Flow cytometry
The expression of CCR5 on the surface of monocytes was assessed with the monoclonal anti-CCR5 antibody (2D7) (PharMingen, Mississauga, Ontario, Canada) and anti-IgG2a antibody (PharMingen) at 2.5 mg/ml, as previously described. 38 In brief, 3)/10 5 treated cells were washed twice with PBS and labelled for 30 min at 48C with anti-CCR5, washed with cold PBS and incubated for 30 min at 48C with FITC-conjugated goat anti-mouse IgG (Bio/Can Scientific, Mississauga, Ontario, Canada). Finally, cells were washed and resuspended in PBS before flow cytometry analysis with a FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA). Results are expressed as the mean peak channel fluorescence for each treatment.

Chemotaxis assay
Monocyte chemotactic activity was performed in modified single-well Boyden chambers separated by 5 mm pore size polycarbonate filters (Neuroprobe, Cabin John, MD, USA), as previously described. 38 MIP-1b or medium was added to the lower chamber and 200 ml of monocytes (6 )/10 5 ) in Gey's BSS (Gibco-BRL, Burlington, ON, Canada), supplemented with 2% bovine serum albumin, were added to the upper chamber. In chemokinesis assays, both chambers contained an equal concentration of MIP-1b. After incubation for 2 h at 378C, the filters were disassembled and the upper side of the filters were scraped free of cells. Cells on the lower side were removed with 5 mM of EDTA, centrifuged and counted.

Statistical analysis
Data were analysed for statistical significance using analysis of variance or Student's t -test for paired values, as appropriate. Differences were considered significant at p B/0.05. Results are expressed, when relevant, as means9/SEM.

Results
Monocytes isolated from different blood donors were treated with graded concentrations of H 2 O 2 for 4 h and mRNA levels encoding CCR5 were measured by northern blot analysis. As illustrated in Fig. 1, H 2 O 2 induced a concentration-dependent augmentation of CCR5 mRNA expression in human monocytes with a maximum induction at 200 mM of H 2 O 2 . Parallel experiments were performed to evaluate the effect of H 2 O 2 on the expression of the chemokine receptor CXCR4 and the receptor for the lipid mediator, platelet-activating factor (PAFR). As illustrated in Fig. 2A, H 2 O 2 also induced an increase in CXCR4 mRNA expression, albeit of a lesser magnitude, but had no effect on PAFR mRNA expression (Fig. 2B). In a time-course study, illustrated in Fig. 3, the effect of H 2 O 2 on CCR5 mRNA levels was detectable as early as at 2 h of treatment, was maximal by 4 h and decreased by 24 h.
Increased mRNA expression is commonly controlled by a transcriptional or post-transcriptional mechanism. To determine whether H 2 O 2 elevated CCR5 mRNA levels through transcriptional activation, monocytes were pretreated with Actinomycin D, a transcriptional inhibitor, for 15 min. The cells were then treated with medium or 200 mM of H 2 O 2 for 3 h. Total cellular RNA was then extracted and northern blot analysis was performed. As illustrated in Fig. 4, this treatment blocked the H 2 O 2 -induced accumulation of CCR5 mRNA, suggesting that H 2 O 2 -regulated CCR5 expression involves a transcriptional mechanism.
Since the transcription factor NF-kB has been shown to be affected by H 2 O 2 in some cell lines and in T lymphocytes, 29,39 and because the CCR5 gene promoter contains consensus binding sequences for this transcription factor, 36 we examined We then investigated whether H 2 O 2 -induced accumulation of CCR5 mRNA was associated with an increased expression of CCR5 protein on the cell surface. Monocytes were treated for 4 h with either medium or 200 mM H 2 O 2 . Supernatants were then replaced with new medium and cells were incubated for 16 h. Flow cytometry studies were then performed using the 2D7 monoclonal anti-CCR5 antibody. As illustrated in Fig. 6, H 2 O 2 treatment of monocytes induced a marked increase in membrane expression of CCR5, as compared with untreated cells (H 2 O 2 -treated monocytes, 6.119/1.7 peak channel fluorescence versus control cells, 2.449/0.6; pB/ 0.05).
To assess the specificity of action of H 2 O 2 , monocytes were treated with H 2 O 2 in the presence or absence of catalase, which degrades H 2 O 2 to H 2 O and O 2 . As expected, the H 2 O 2 -induced upregulation of CCR5 expression was prevented by catalase both at the mRNA and protein levels (Fig. 7A,B). Interestingly, however, catalase itself was found to increase the expression of CCR5.
We next investigated whether the H 2 O 2 -induced upregulation of CCR5 expression was associated with augmented responsiveness of treated monocytes to the CCR5 ligand MIP-1b. Receptor function was first evaluated for MIP-1b-induced chemotaxis; as shown in Fig. 8, monocytes pretreated with H 2 O 2 showed enhanced responsiveness to the chemotactic action of MIP-1b. Chemokinetic activity in response to MIP-1b was not affected, however (data not shown). extracted for northern blot analysis. As illustrated in Fig. 9, c-fos mRNA expression was markedly increased in H 2 O 2 -pretreated monocytes following exposure with MIP-1b, compared with untreated cells. These observations suggest that the increased expression of CCR5 following H 2 O 2 treatment is associated with enhanced functional activities of the receptor.

Discussion
It is now accepted that a wide variety of cell types can respond to oxidative stress by the induction of multiples genes controlled by redox-sensitive proteins. 40 Several investigators have demonstrated that ROS can modulate gene expression of chemokines such as IL-8, 41 MCP-1 21,42 and MIP-1a, 22 suggesting a role for ROS in the inflammation process. In this report, we demonstrated that externally applied H 2 O 2 , a cell-permeable oxidant, significantly increased mRNA expression of the chemokine receptors CCR5 and, to a lesser extent, CXCR4 in human monocytes. Similar findings were recently reported by Saccani et al., 43 who also found that the antioxidant pyrrolidinedithiocarbamate (PDTC) downregulated the expression of CCR5 and CXCR4. We further extended our studies by showing that the upregulation of CCR5 by H 2 O 2 was dependent on transcriptional activity and could be prevented by inhibition of NF-kB activation. Moreover, the H 2 O 2induced augmentation of CCR5 mRNA levels was associated with enhanced CCR5 expression at the cell surface. We further demonstrated that these newly induced receptors were functional since pretreatment with H 2 O 2 was associated with an augmented responsiveness of monocytes to MIP-1b in terms of cell migration as well as c-fos gene activation.
The transcription factor NF-kB is involved in the expression of a wide variety of genes controlling immune and inflammatory responses. Several lines of evidence suggest a role for ROS as a common and critical intermediate in NF-kB activating signals. For example, the antioxidant PDTC or the overexpression of superoxide dismutase have been shown to be inhibitory for TNF-induced activation of NF-kB in Jurkat T cells. 27,44 Á 47 It has also been shown that addition of H 2 O 2 to specific cell types results in the appearance of the phosphorylated form of IkB-a, whereas treatment with reducing agents such as dithiothreitol and b-mercaptoethanol enhances DNA-binding activity of NF-kB itself. 48 Consistant with these reports, treatment with antioxidants or overexpression of antioxidant enzymes can block IkB-a phosphorylation and degradation induced by lipopolysaccharide, TNF-a and phorbol-myristate acetate (PMA). 35 These observations suggest that redox regulation may affect more than one step in the NF-kB activating pathway. Although the molecular mechanism of the redox regulation of NF-kB is still unclear, signalling molecules such as protein kinases, which are involved in the dissociation of IkB from the NF-kB complex, may be targets of the redox regulation. Our study also suggests a role for the NF-kB pathway in the effect mediated by H 2 O 2 on CCR5 expression in monocytes. Since the preatreatment of cells with an IkB-a phosphorylation inhibitor resulted in a partial inhibition of the H 2 O 2 -mediated induction of CCR5 mRNA, we can suppose that IkB-a itself or some upstream factors, such as one or more protein kinases, could be the target for the oxidant. Moreover, the CCR5 gene promoter also contains consensus binding sequences for the transcriptional activating factor AP-1 and for such regulatory elements as STAT, 36 which have recently been shown to be redox sensitive. 30 These findings present the possibility that additional transcription factors might be implicated in the regulation of CCR5 expression by H 2 O 2 .
Regulation of chemokine receptors by reactive oxygen species may be important in the pathogenesis of several inflammatory diseases. ROS have been shown to be produced by immune cells after activation by proinflammatory cytokines, such as IL-1 and TNF-a. 49 ROS are also implicated in a number of inflammatory diseases where expression of CCR5 ligands is enhanced, such as in rheumatoid arthritis, sepsis and autoimmune encephalomyelitis. 50 Á 53 Understanding the effect of ROS in the direct environment of inflammatory cells could lead to a better understanding of the mechanisms implicated in the progression of these diseases.
In conclusion, our results demonstrate that ROS in the extracellular environment of human monocytes can increase the expression of the chemokine receptor CCR5 through redox-sensitive transcription factors, such as NF-kB. In an inflammatory disease context, activated phagocytic cells would release inflammatory mediators and ROS, such as H 2 O 2 . H 2 O 2 accumulation in the environment of immune cells would then promote an increase in CCR5 expression on the cell surface of monocytes, leading to a greater response to the chemokines MIP-1a, MIP-1b and RANTES. This increased response to chemokines would extend the inflammatory process and possibly facilitate a greater accumulation of activated monocytes at the inflammatory site.