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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2008;28:932.)
© 2008 American Heart Association, Inc.

Cell Biology and Signaling

Induction of CXCR2 Receptor by Peroxisome Proliferator-Activated Receptor in Human Macrophages

Elena Rigamonti; Coralie Fontaine; Bruno Lefebvre; Christian Duhem; Philippe Lefebvre; Nikolaus Marx; Bart Staels; Giulia Chinetti-Gbaguidi

From the Institut Pasteur de Lille (E.R., C.F., B.L., C.D., P.L., B.S., G.C.-G.), Lille, F-59019 France; Inserm (E.R., C.F., B.L., C.D., P.L., B.S., G.C.-G.), U545, Lille, France; Université de Lille 2 (E.R., C.F., B.L., C.D., P.L., B.S., G.C.-G.), Faculté des Sciences Pharmaceutiques et Biologiques et Faculté de Médecine, Lille, France; and the Department of Internal Medicine II (N.M), Cardiology, University of Ulm, Germany.

Correspondence to Bart Staels, Inserm UR 545, Institut Pasteur de Lille, 1, rue du Professeur Calmette, BP 245, Lille 59019, France. E-mail bart.staels{at}pasteur-lille.fr

	Abstract

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Objective— Macrophages play a central role in the immune response against infectious organisms. Once activated, macrophages secrete proinflammatory cytokines and chemokines. Interleukin (IL)-8 and related CXC chemokines play a role in the recruitment and activation of phagocytes acting through CXCR1 and CXCR2 receptors. The nuclear receptor peroxisome proliferator-activated receptor (PPAR) exerts antiinflammatory properties in macrophages, by inhibiting cytokine and CC chemokine production. In this study, we investigated whether PPAR- also plays a role in the regulation of the CXC chemokine pathway.

Methods and Results— Synthetic PPAR- ligands increase CXCR2 but not CXCR1 gene expression in a PPAR--dependent manner in primary human macrophages in vitro and in atherosclerotic plaques in vivo. The increase of CXCR2 mRNA was paralleled by an increase in membrane protein expression. EMSA, ChIP, and transient transfection assays indicate that PPAR- activates the CXCR2 promoter by binding to a PPAR response element (PPRE). Finally, human macrophages acquire responsiveness to the CXCR2 ligands (IL-8 and Groβ), as measured by superoxide anion production, after induction of CXCR2 expression by PPAR- ligands.

Conclusions— Our results provide a novel mechanism via which PPAR- can enhance the immune response in human macrophages.

IL-8 recruits and activates phagocytes acting through CXCR1 and CXCR2 receptors. Regulation of these genes by PPAR- was studied in macrophages. PPAR- induces CXCR2 expression, leading to an acquired induction of ROS production in response to IL-8 and Groβ. These results provide a novel role for PPAR- in macrophage immune response.

Key Words: nuclear receptors • chemokines • gene regulation • immune response • macrophage

	Introduction

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Chemokines are involved in a variety of immune and inflammatory responses, acting primarily as chemoattractants and activators of specific leukocytes.¹ IL-8 is one of the best-characterized CXC chemokines, which stimulates chemotaxis, degranulation, and production of oxygen radicals in phagocytes. In humans, IL-8 is primarily expressed during the inflammatory response to bacterial and viral infections.² IL-8 is secreted by many cell types in response to proinflammatory stimuli such as IL-1, tumor necrosis factor (TNF), and lipopolysaccharide (LPS) and mediates its activities by binding to the G protein-coupled transmembrane receptors CXCR1 and CXCR2.³ Whereas CXCR1 binds only IL-8, CXCR2 also binds with high affinity other CXC chemokines such as growth-regulated oncogene (Gro)/β/, neutrophil activating peptide (NAP)-2, epithelial-cell derived neutrophil activating protein (ENA)-78.⁴ More recently, the cytokine macrophage migration inhibitory factor (MIF) has been identified as a noncognate CXCR2 ligand, suggesting a role of CXCR2 in the recruitment of atherogenic and inflammatory monocytes.⁵

CXCR2 is expressed in neutrophils, but also in fibroblasts, circulating endothelial progenitor cells (EPCs), monocytes, macrophages, and macrophage foam cells.^1,6,7 Circulating human monocytes express low levels of surface IL-8 receptors.⁸ Although IL-8 and Groβ can weakly induce calcium flux and trigger a weak respiratory burst in concanavalin A-primed monocytes,⁹ these chemokines are generally not considered as activators of monocyte functions. Although human monocytes do not respond to IL-8 and Groβ in terms of chemotaxis and release of oxygen radicals, this response can be readily induced after exposure to IL-4 and IL-13 and subsequent upregulation of CXCR1 and CXCR2 expression. A similar IL-13-induced CXCR1 and CXCR2 upregulation has also been observed in monocyte-derived macrophages and dendritic cells.¹⁰

The peroxisome proliferator-activated receptor (PPAR-) is a ligand-activated nuclear receptor, which on heterodimerization with the retinoic X receptor (RXR) binds to specific DNA sequences called PPAR response elements (PPREs), thus regulating target gene expression. PPAR- is activated by natural and synthetic substances, including 15-deoxy-D12;14-prostaglandin (PG) J2 (15 days-PGJ2),¹¹ the GW1929 compound and the antidiabetic thiazolidinediones (TZD), such as Rosiglitazone.¹² PPAR- expression is very low in human monocytes but is induced early on differentiation into macrophages.¹³ Various leukocyte populations, including lymphocytes and dendritic cells, also express PPAR-, suggesting a role for this receptor in the regulation of immune response.¹⁴ In these cells, PPAR- generally inhibits inflammatory response genes by negatively interfering with the NF-B, STAT, and AP-1 signaling pathways in a DNA-binding independent manner.¹⁵

Indeed, PPAR- modulates chemokine gene expression by inhibiting expression of monocyte chemoattractant protein (MCP)-1 and its receptor CCR2 in monocyte/macrophages.^16,17 However, it is currently unknown whether PPAR- also plays a role in the regulation of CXC chemokine pathway. Here we show that PPAR- activation increases CXCR2 expression. CXCR2 upregulation by PPAR- leads to the acquisition of IL-8/Groβ responsiveness, as measured by enhanced reactive oxygen species (ROS) formation. Our results identify a novel mechanism via which PPAR- could modulate the immune response in human macrophages.

	Methods

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Cell Culture
Mononuclear cells were isolated by Ficoll gradient centrifugation.¹³ Mature monocyte-derived macrophages were used for experiments after 10 days of differentiation. Human atherosclerotic plaques were obtained from 15 nondiabetic patients with symptomatic carotid artery stenosis randomized to receive 4 weeks placebo or Rosiglitazone before surgery¹⁸ (see supplemental data, available online at http://atvb.ahajournals.org).

RNA Extraction and Analysis
Total RNA isolated from human carotid plaques and macrophages using Trizol (Life Technologies, France) were reverse transcribed. cDNA were quantified by quantitative polymerase chain reaction (PCR) (QPCR) (see supplemental data).

Adenovirus Preparation and Cell Infection
The recombinant adenovirus (Ad)-GFP and Ad-PPAR- were obtained by homologous recombination. Primary human macrophages were incubated with viral particles for 12 hours and subsequently treated for 24 hour with Rosiglitazone (100nmol/L) (see supplemental data).

Protein Extraction and Western Blot Analysis
Membrane proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with monoclonal anti-CXCR2 (R&D systems) and polyclonal anti-β-actin (Santacruz Biotechnology) antibodies (see supplemental data).

Flow Cytometry Analysis of CXCR2 Expression
Primary human macrophages were incubated with GW1929 (600 nmol/L) or Rosiglitazone (100 nmol/L) for 24 hours and subsequently labeled with mouse antihuman CXCR2 (R&D systems) or isotype matched control followed by incubation with PE polyclonal anti mouse IgG (Becton Dickinson). Fluorescence analysis was performed by a FACStra^plus.

Electrophoretic Mobility Shift Assays
PPAR- and RXR- were in vitro transcribed from the pSG5-hPPAR- and pSG5-hRXR- plasmids and subsequently translated. For supershift assays, polyclonal anti-PPAR- antibody (Santacruz Biotechnology) was used. DNA/protein complexes were resolved by 6% nondenaturing polyacrylamide gel electrophoresis (see supplemental data).

Chromatin Immunoprecipitation Assays
Primary human macrophages cell lysate was immunoprecipitated with either a nonspecific IgG or the PPAR- antibody (Santacruz Biotechnology). Immunoprecipitated DNA was PCR amplified using primers covering either the –502 to –303 region of the human CXCR2 promoter containing the (–408/–396)CXCR2-PPRE or part of the β-actin gene (see online supplementary data).

Plasmid Cloning and Transient Transfection Experiments
Cos-7 cells and primary human macrophages were transfected with reporter plasmids and expression vectors (pSG5-empty or pSG5-hPPAR-) and subsequently incubated with GW1929 (600nmol/L) or DMSO for 24 hours. At the end, luciferase and β-galactosidase assays were performed (see supplemental data).

ROS Measurement
Human macrophages were incubated for 24 hours with GW1929 (600 nmol/L) or Rosiglitazone (100 nmol/L). In some experiments, cells were preincubated for 30 minutes with GW9662 (10 µmol/L) before PPAR- activation. Cells were then stimulated with IL-8, Groβ (1 µg/mL; R&D systems), or PMA (100 ng/mL; Sigma), incubated with dichlorofluoresceine diacetate (DCFH-DA, 10µmol/L) and DCF fluorescence was read (see supplemental data).

Statistical Analysis
Statistical differences between groups were analyzed by Mann-Whitney test with Bonferroni correction and were considered significant when P0.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
PPAR- Activation Increases CXCR2 Gene Expression in a PPAR--Dependent Manner
To investigate whether PPAR- regulates CXC receptor gene expression, Q-PCR analysis was performed in primary human macrophages treated with PPAR- ligands for 24 hours. GW1929 (600nmol/L) or Rosiglitazone (100nmol/L) significantly increased CXCR2 mRNA levels (Figure 1A). By contrast, PPAR- activation did not affect CXCR1 expression (data not shown). Interestingly, CXCR2 regulation by PPAR- was also observed in macrophage foam cells (Figure 1B) and in atherosclerotic plaques from Rosiglitazone compared to placebo-treated patients (Figure 1C), indicating in vivo conservation of CXCR2 regulation.

View larger version (19K): [in this window] [in a new window]   Figure 1. PPAR- activation increases CXCR2 gene expression in a PPAR--dependent manner. Primary human macrophages incubated with GW1929 (600nmol/L), Rosiglitazone (100nmol/L) (A) or AcLDL (B); atherosclerotic lesions from placebo- and rosiglitazone-treated subjects (C); human macrophages treated with Rosiglitazone for the indicated time points (D), with Rosiglitazone (100 nmol/L), GW9662 (10 µmol/L), or both (E), or infected with Ad-GFP or Ad-PPAR--adenovirus and treated with Rosiglitazone (100 nmol/L; F). For the detailed figure legends please see supplemental materials.

Time-course experiments in primary human macrophages showed that CXCR2 gene induction is already observed after 12 hours of stimulation with Rosiglitazone and becomes maximal at 24 hours (Figure 1D). Moreover, CXCR2 regulation by Rosiglitazone was concentration-dependent (supplemental Figure I). Expression of CD36, a known PPAR- target gene,¹⁹ was also induced in a dose-dependent manner (supplemental Figure I).

To determine whether PPAR- agonists upregulate CXCR2 expression in a PPAR--dependent manner, experiments were performed in the presence or absence of GW9662, an irreversible PPAR- antagonist.²⁰ GW9662 clearly abolished Rosiglitazone-induced CXCR2 mRNA (Figure 1E). Moreover, infection with a PPAR--expressing adenovirus increased CXCR2 expression approximately 2-fold compared with Ad-GFP-infected cells, and addition of Rosiglitazone resulted in a further 2.5-fold increase (Figure 1F).

PPAR- Activation Increases CXCR2 Membrane Protein Expression
To determine whether CXCR2 gene induction by PPAR- ligands leads to an increase of protein levels, flow cytometry analysis was performed in primary human macrophages incubated with PPAR- ligands for 24 hours. Under basal conditions, 13% of cells stained positively for CXCR2. GW1929 (600 nmol/L) and Rosiglitazone (100 nmol/L) enhanced the number of cells expressing CXCR2x2.4- and 1.9-fold, respectively (Figure 2A and 2B). Western blot analysis of human macrophages showed that PPAR- activation significantly increases CXCR2 membrane protein levels (Figure 2C and 2D).

View larger version (17K): [in this window] [in a new window]   Figure 2. PPAR- activation increases CXCR2 membrane protein levels. A and B, Flow cytometry analysis of CXCR2 in primary human macrophages incubated with GW1929 (600 nmol/L) or Rosiglitazone (100 nmol/L). C and D, Western Blot analysis of CXCR2 expression in cell membranes from primary human macrophages treated with GW1929 (600 nmol/L) or Rosiglitazone (100 nmol/L). For the detailed figure legends please see supplemental materials.

PPAR- Regulates CXCR2 Gene Expression at the Transcriptional Level
To determine whether CXCR2 is a direct PPAR- target gene, we examined the CXCR2 promoter by bio-informatic analysis. A putative PPRE was identified at position –409/–396. EMSA were performed to examine whether the PPAR--RXR heterodimer binds to this putative CXCR2-PPRE. Incubation of this labeled-PPRE oligonucleotide with in vitro translated PPAR- and RXR- resulted in the formation of a retarded complex (Figure 3A, lane 4). The binding-specificity of PPAR- to this CXCR2-PPRE site was demonstrated by the competitive inhibition by 100-, 200-, and 500-fold excess of unlabeled wild type (Figure 3A, lanes 5 to 7) but not mutated (Figure 3A, lanes 8 to 10) CXCR2-PPRE probe as well as by the supershift with a anti-PPAR- antibody (Figure 3A, lane 11).

View larger version (19K): [in this window] [in a new window]   Figure 3. PPAR- regulates CXCR2 gene expression at the transcriptional level. A, EMSA were performed using the indicated end-labeled oligonucleotides. Supershift assays were performed using an anti-PPAR- antibody. B, Chromatin immunoprecipitation assays were performed using chromatin isolated from primary human macrophages treated or not with Rosiglitazone (100 nmol/L). Control PCRs were done with nonimmunoprecipitated genomic DNA (input). For the detailed figure legends please see supplemental materials.

To evaluate whether PPAR- binds to the native CXCR2 promoter in vivo, chromatin immunoprecipitation assay was performed in primary human macrophages. The genomic DNA region encompassing the PPRE of the CXCR2 gene was immunoprecipitated with a polyclonal anti-PPAR- antibody demonstrating PPAR- binding. Interestingly, as previously shown for other target genes,²¹ PPAR- binding was already observed in the absence of ligand (Figure 3B). PCR amplification with specific primers for the β-actin gene did not result in any significant signal, thus demonstrating the specificity of immunoprecipitation and PCR reactions. Thus, in human macrophages, PPAR- binds to the PPRE sequence of the CXCR2 gene.

PPAR- Increases CXCR2 Promoter Activity via the CXCR2-PPRE
To test whether PPAR- activates transcription from the (–409/–396)CXCR2-PPRE site, 3 copies of this element were cloned in front of the herpes simplex virus thymidine kinase promoter to obtain the (CXCR2-PPRE)3x-Tk-Luc luciferase reporter vector. Cotransfection of the pSG5-PPAR- expression vector with the (CXCR2-PPREwt)3x reporter vector in Cos-7 cells and in primary human macrophages led to a significant induction of transcriptional activity, enhanced by GW1929 (600 nmol/L; Figure 4A and 4D). The mutated PPRE site (CXCR2-PPREmut) was not responsive to PPAR- (Figure 4A and 4D). To determine whether the CXCR2-PPRE site is functional in the context of the natural promoter, cotransfection assays were carried out using luciferase reporter constructs driven by the natural CXCR2 promoter of 510 bp or by the CXCR2 promoter fragment that contains 401 bp and lacks the CXCR2-PPRE. CXCR2–510 bp, but not CXCR2–401 bp promoter activity was induced by PPAR- transfection and treatment with GW1929 (600 nmol/L; Figure 4B). In addition, specific mutations of the PPRE in the context of the CXCR2–510 bp promoter abolished induction of the CXCR2 promoter by PPAR- transfection in the absence or in the presence of GW1929 (600 nmol/L; Figure 4C). Taken together these results demonstrate that CXCR2 is a PPAR- target gene.

View larger version (19K): [in this window] [in a new window]   Figure 4. PPAR- increases CXCR2 promoter activity via a PPRE. Cos-7 cells (A, B, C) and primary human macrophages (D) were transfected with wild-type or mutated human CXCR2-PPRE sites (A and D) and a reporter driven by the indicated human CXCR2 promoter constructs (B and C), in the presence of pSG5-PPAR- or pSG5 empty vector and treated with GW1929 (600 nmol/L) or DMSO. For the detailed figure legends please see supplemental materials.

PPAR- Ligands Induce Responsiveness of Primary Human Macrophages to IL-8 and Gro-β
To investigate the functional relevance of the CXCR2 induction by PPAR- stimulation in human macrophages, ROS production was measured in the presence of IL-8 and the selective CXCR2 ligand Gro-β. Primary human macrophages were incubated with GW1929 (600 nmol/L) or Rosiglitazone (100 nmol/L) for 24 hours and then tested for their ability to produce oxygen free radicals after IL-8 (1 µg/mL) or Gro-β (1 µg/mL) stimulation. IL-8 or Gro-β did not induce ROS production in untreated macrophages (Figure 5A and 5B). As reported previously, PPAR- activation did not affect ROS production in human macrophages.²² However, IL-8 or Gro-β induced a respiratory burst in human macrophages only after treatment with PPAR- ligands (Figure 5A and 5B), suggesting that induction of CXCR2 expression mediates the enhanced response of PPAR--treated macrophages. Under similar experimental conditions, GW1929 and Rosiglitazone did not modify the response of macrophages to PMA, a well-known activator of oxidative burst in macrophages (Figure 5C). Finally, Rosiglitazone effect on IL-8-induced ROS production was blocked by the PPAR- antagonist GW9662 (Figure 5D).

View larger version (46K): [in this window] [in a new window]   Figure 5. PPAR- ligands enhance the response of primary human macrophages to IL-8 and Groβ. A, B, and C, Primary human macrophages were treated with GW1929 (600 nmol/L) or Rosiglitazone (100 nmol/L) and then incubated for 10 minutes with IL-8 (1 µg/mL), Groβ (1 µg/mL), or PMA (100 ng/mL), and DCFH-DA (10 µmol/L) was subsequently added. D, Primary macrophages were incubated with PPAR- antagonist before PPAR- activation. For the detailed figure legends please see supplemental materials.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Regulation of chemokine receptor expression has recently emerged as a key checkpoint of chemokine action. Specifically, LPS, TNF- and interferon (INF)- downregulate the expression of CCR2 in monocytes and CXCR1 and CXCR2 in neutrophils,^23,24 leading to a reduction of chemotactic responsiveness and ability to deal with an infectious insult. Of note, CXCR2 expression is downregulated in neutrophils from patients with sepsis,²⁵ and both IL-8 receptors are also downregulated in neutrophils from patients with HIV and pulmonary tuberculosis.²⁶ In this study, we demonstrate that PPAR- agonists upregulate the expression of CXCR2 but not CXCR1 in primary human macrophages. PPAR- activates CXCR2 transcription via a PPRE located in the proximal CXCR2 promoter. Increased expression of CXCR2 resulted in an acquired responsiveness of macrophages to IL-8 and Groβ. Both CXC chemokines were able to induce the respiratory burst in human macrophages pretreated with PPAR- agonists, at concentrations similar to those active on leukocytes.²⁷ In these conditions, PPAR- agonists did not induce a general activation of macrophages, but act specifically to induce functional CXCR2 receptor. This conclusion is based on the following observations: (1) CXCR2 induction by PPAR- ligands does not occur at short incubation times generally required for macrophage activation,²⁸ but requires at least 12 hours; (2) the PPAR- effect is specific for CXCR2 because GW1929 and Rosiglitazone treatment did not modify the response of macrophages to PMA; and (3) we reported previously²² and confirmed here that PPAR- activation per se does not affect ROS production in human macrophages (Figure 5).

Macrophages are an essential first line of innate defense against microbial pathogens. Once a pathogen has been engulfed by a phagocyte, it is killed in the phagolysosome by the activity of digestive enzymes or by the release of free radicals. Through its ability to upregulate expression of the scavenger receptor CD36, PPAR- has been found to enhance the phagocytosis of malarian parasites by human macrophages.²⁹ In the present study we demonstrate that PPAR--activated human macrophages acquire induction of ROS production in response to IL-8 and Groβ. As such, PPAR- may contribute to pathogen elimination through a combination of enhanced phagocytosis and induction of antimicrobial ROS.

Inflammation is one of the first responses of the immune system to infection. Recently, PPAR- has been shown to enhance phagocytosis of apoptotic neutrophils by alveolar macrophages thus promoting the resolution of lung inflammation.³⁰ Moreover, PPAR- ligands repress the expression of several inflammatory genes, including inducible nitric oxide synthase (iNOS), matrix metalloproteinase (MMP)-9, TNF-, IL-1β, and chemokines in macrophages.^31,32 Of note, PPAR- inhibits MCP-1 expression and secretion³³ but fails to modulate IL-8 levels both in THP-1 cells³⁴ and in primary human macrophages as confirmed by our results (data not shown).

Inflammation plays also a crucial role in the progression of chronic diseases such as atherosclerosis, a main cause of cardiovascular complications. Recently,^35,36 results from several meta-analysis reported that Rosiglitazone administration may be associated with an increased risk of myocardial infarction. Although intermediary safety analysis of the Rosigiltazone Evaluated for Cardiac Outcomes and Regulation of Glycemia in Diabetes (RECORD) study reported nonsignificant changes in cardiovascular morbidity and mortality after Rosiglitazone treatment,³⁷ it remains puzzling why Rosiglitazone does not decrease the risk of CVD in light of its many reported pleiotropic antiinflammatory actions. Interestingly, our results show that PPAR- activation increases CXCR2 expression both in macrophage foam cells and in atherosclerotic plaques. Notably, CXCR2 plays a crucial role in macrophage accumulation and atherosclerotic lesion progression, as evidenced by the reduced susceptibility to atherosclerosis in mice reconstituted with CXCR2^–/– bone marrow.^38,39 Moreover, CXCR2 appears critical in controlling adhesion and influx of EPCs. During early atherosclerosis, CXCR2 may be beneficial for improving endothelial recovery or regeneration by promoting EPC homing.⁴¹ However, a recent study revealed that transfer of spleen-derived EPCs in ApoE^–/– mice can destabilize atherosclerotic plaques, implying proatherogenic features of EPCs, possibly recruited via CXCR2, during advanced atherosclerosis.^40,41 Interestingly, TZDs have been shown to increase the number and functions of EPCs in patients with coronary heart disease.⁴² Therefore, it is tempting to speculate that the CXCR2 induction by PPAR- ligands in macrophages and subsequent increased production of ROS could be a mechanism which counteracts other beneficial effects of Rosiglitazone, such as the inhibition of the CCR2 pathway and its antiinflammatory properties.

In conclusion, our results identify a novel role for PPAR- in the regulation of the inflammatory and immune response in human macrophages via CXCR2 upregulation, thus resulting in an enhanced ROS production in the presence of CXCR2 ligands IL-8 and Groβ.

	Acknowledgments

 
We acknowledge J. Bertout and B. Derudas for technical help.

Sources of Funding

Grants from the Région Nord-Pas de Calais/FEDER, the Fondation Coeur et Artères, and the European Vascular Genomic Network (EVGN) are acknowledged.

Disclosures

None.

	Footnotes

 
E.R. and C.F. contributed equally to the work.

Original received October 5, 2007; final version accepted February 12, 2008.

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