Published online before print October 1, 2007, doi: 10.1161/CIRCULATIONAHA.106.678474
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(Circulation. 2007;116:1801-1811.)
© 2007 American Heart Association, Inc.
Vascular Medicine |
CXCR6 Promotes Atherosclerosis by Supporting T-Cell Homing, Interferon-
Production, and Macrophage Accumulation in the Aortic Wall
From the Departments of Biomedical Engineering (E.G., B.L.H., K.L.), Molecular Physiology and Biological Physics (K.L.), and Robert M. Berne Cardiovascular Research Center (E.G., A.B., J.M.S., K.L.), University of Virginia, Health Sciences Center, Charlottesville; and Institute of Molecular Cardiovascular Research, Aachen, Germany (A.L., E.A.L., C.W.).
Correspondence to Elena Galkina, PhD, Robert M. Berne Cardiovascular Research Center, University of Virginia, PO Box 801394, Charlottesville, VA 22908. E-mail evg7c{at}virginia.edu
Received November 27, 2006; accepted August 3, 2007.
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Abstract |
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Background— T lymphocytes are thought to be important in atherosclerosis, but very little is known about the mechanisms of lymphocyte recruitment into atherosclerosis-prone aortas. In this study we tested the hypothesis that CXCR6, a chemokine receptor that is expressed on a subset of CD4+ T helper 1 cells and natural killer T cells, is involved in lymphocyte homing into the aortic wall and modulates the development and progression of atherosclerosis.
Methods and Results— To investigate the role of CXCR6 in the development and progression of atherosclerosis, we bred CXCR6-deficient (CXCR6GFP/GFP) mice with apolipoprotein E–deficient (ApoE–/–) mice. We found that CXCR6GFP/GFP/ApoE–/– mice fed a Western diet for 17 weeks or a chow diet for 56 weeks had decreased atherosclerosis compared with ApoE–/– controls. Flow cytometry analysis of the aortas from CXCR6GFP/GFP/ApoE–/– mice showed that the reduction of atherosclerosis was accompanied by a decreased percentage of CXCR6+ T cells within the aortas. Short-term homing experiments demonstrated that CXCR6 is involved in the recruitment of CXCR6+ leukocytes into the atherosclerosis-prone aortic wall. The reduced percentage of CXCR6+ T cells within the aortas resulted in significantly diminished production of interferon-
and reduction of CD11b+/CD68+ macrophages in the aorta.
Conclusions— These data provide evidence for a proatherosclerotic role of CXCR6. Absence of CXCR6 alters the recruitment of CXCR6+ leukocytes and modulates the local immune response within the aortic wall.
Key Words: atherosclerosis • immune system • leukocytes • lymphocytes • vessels
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Introduction |
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Atherosclerotic lesions are characterized by lipid accumulation, cell death, fibrosis, and chronic vascular inflammation.1 The body of evidence is growing that T and B lymphocytes, dendritic cells, and macrophages reside within the noninflamed aortic wall, and a significant influx of macrophages and T cells correlates with the development and progression of atherosclerosis.2–4 The molecular mechanisms, kinetics of trafficking, and retention of different types of immune cells within the noninflamed and atherosclerosis-prone aortic wall are not well defined. Most of the focus to date has been on the ability of monocytes to migrate to the atherosclerotic wall.5 P-selectin, vascular cell adhesion molecule-1 (VCAM-1), P-selectin glycoprotein ligand-1 (PSGL-1), and
4ß1 integrin (VLA-4) are all involved in monocyte recruitment into atherosclerotic plaques,6 and the chemokines CCL5 (regulated on activation, normal T cell expressed and secreted [RANTES]) and CXCL1 (keratinocyte-derived chemokine [KC]) are responsible for triggering monocyte adhesion on the atherosclerotic endothelium.7–9 Lymphocyte trafficking into normal and atherosclerotic aortas is partially L-selectin dependent,2 but little is known about other potential molecular mechanisms of lymphocyte recruitment.
Clinical Perspective p 1811
CXCR6 is a chemokine receptor expressed on some T helper 1 (Th1) and natural killer T (NKT) cells found in rheumatoid joints and inflamed livers.10 CXCR6, also known as Bonzo/STRL33/TYMSTR,11 is expressed on subpopulations of CD4+ effector memory T cells,10 on V 24+ NKT cells,12,13 a Forkhead Box P3+ (Foxp3) subset (CD69+/CD45RO+) of regulatory T cells in the tonsils,14 bone marrow plasma cells, and myeloma cells.15 In mice, interstitial intraepithelial lymphocytes, NKT cells,16 and memory subsets of CD8+ and CD4+ T cells express CXCR6, which is upregulated by interleukin-2 and interleukin-15 during short-term culture.17 Recently, lipopolysaccharide-induced toll-like receptor 4–dependent CXCR6 expression has been shown in aortic smooth muscle cells.18
CXCL16 is the only known ligand for CXCR6 and is expressed by dendritic cells,16 macrophages,19 T cells,20 and cytokine-stimulated smooth muscle and endothelial cells.21,22 The disintegrin and metalloproteinase ADAM10 is involved in CXCL16 cleavage from the cell membrane,21 leading to the release of a soluble protein that functions as a chemoattractant for responsive leukocytes.
In addition to its properties as a chemokine, CXCL16 acts also as a scavenger receptor for apoptotic cells, phosphatidylserine, and oxidized low-density lipoprotein.16,19 Expression of CXCL16 has been reported in atherosclerosis-prone vessels from apolipoprotein-E–deficient (ApoE–/–) mice23 and human atherosclerotic plaques.24 Absence of CXCL16 has been shown to accelerate atherosclerosis, enhance macrophage recruitment, and elevate mRNA levels for CCL2 (monocyte chemotactic protein-1) and tumor necrosis factor-.25 Because the absence of CXCL16 results in the loss of scavenger function as well as chemotactic activity, inactivating the CXCL16 gene does not separate the roles of these 2 functions in atherosclerosis.
To investigate the involvement of the chemokine receptor CXCR6 in atherosclerosis, we crossed CXCR6-GFP knockin mice17 to atherosclerosis-prone ApoE–/– mice. Because Th1 cells are thought to participate in the development of atherosclerosis,26 we hypothesized that CXCR6, a receptor highly expressed on these cells, might be relevant for their trafficking into atherosclerotic vessel wall. In mice carrying 1 functional copy of CXCR6 (CXCR6GFP/+/ApoE–/–) and mice that lack functional CXCR6 (CXCR6GFP/GFP/ApoE–/–), GFP-expressing cells were tracked by flow cytometry. Lesion size and composition were assessed by en face staining and aortic root histology. Homing of CXCR6+ leukocytes was determined by flow cytometry. Functional consequences of CXCR6 cell recruitment were assessed as cytokine production and macrophage infiltration.
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Methods |
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Mice
CXCR6GFP/+ and CXCR6GFP/GFP mice (backcrossed onto C57BL/6 >8 generations, 98.18% of C57BL/6 [microsatellite marker screening, Charles River Lab]) were a kind gift of Dr Littman (Howard Hughes Medical Institute, New York University). ApoE–/– mice (C57BL/6 background) were purchased from Jackson Laboratories (Bar Harbor, Maine) and crossed with CXCR6 knockin mice to obtain CXCR6GFP/+/ApoE–/– and CXCR6GFP/GFP/ApoE–/– mice. Mice were bred and maintained under specific pathogen-free conditions in the barrier of the University of Virginia, Charlottesville. Mice appeared healthy and were used at ages 8 to 60 weeks. All animal experiments were approved by the institutional Animal Care and Use Committee.
Preparation of Mouse Aortas and Quantification of Atherosclerosis
The aortas of mice were collected and stained with oil red O.27 Images were scanned, and the percent surface areas occupied by lesions were determined with Image-ProPlus (Media Cybernetics). From the point of the appearance of aortic valve leaflets, sequential 5-µm-thick sections were cut, and 4 sections >300 µm were collected and analyzed by Movat staining.28
Flow Cytometry Analysis of Immune Cells Within Murine Aorta
Anesthetized mice were perfused by cardiac puncture with PBS containing 20 U/mL of heparin. Harvested aortas were digested with 125 U/mL collagenase type XI, 60 U/mL hyaluronidase type I-s, 60 U/mL DNAse1, and 450 U/mL collagenase type I (all enzymes, Sigma, St Louis, Mo) in PBS containing 20 mmol/L HEPES at 37°C for 1 hour.2 A cell suspension was obtained by mashing the aorta through a 70-µm strainer. Cells were incubated with antibodies for 20 minutes at 4°C, washed twice, and incubated with secondary antibodies for an additional 20 minutes. After they were washed, immunofluorescence was detected by flow cytometry (FACSCalibur, BD Immunocytometry Systems, San Jose, Calif) or CyanADP (Dako, Colo), and data were analyzed with the use of FlowJO (Tree Star Inc, Ashland, Ore) software. PE-Texas Red-CD45, APC-Cy7 or Pe-Cy5-CD3, APC-TCRß, PE-I-Ab, NK1.1-APC, CD62L-APC, Ly6C-biotin antibodies, and streptavidin-Per-CP, APC-Cy7 were from BD Biosciences, San Jose, Calif; CD115-PE from eBioscience (San Diego, Calif); Pacific Blue-CD11b and streptavidin-PE-Texas Red from Caltag (Burlingame, Calif); and FITC-CD68 from Serotec, Raleigh, NC. Biotin-conjugated anti-rat and anti-hamster antibodies were from Vector Laboratories, Inc (Burlingame, Calif). CCR2 antibody was kindly provided by M. Mack (University of Regensburg, Germany). In some experiments, the aortas from 2 to 3 mice were pooled and analyzed.
In Vivo Trafficking Experiments
Splenocytes from CXCR6GFP/+/ApoE–/– mice were labeled with 7 µmol 5- (and 6)-([{4-chloromethyl}benzoyl]amino) tetramethylrhodamine (CMTMR) (Molecular Probes, Eugene, Ore) in RPMI at 37°C for 20 minutes. In some experiments, the cell suspension from CXCR6GFP/GFP/ApoE–/– mice was labeled instead. Cells from CXCR6GFP/+/ApoE–/– and CXCR6GFP/GFP/ApoE–/– mice were mixed 1:1, and 35x106 labeled cells of each population were injected intravenously into recipient mice. At 24 hours, aortas, spleens, and inguinal, axillary, and cervical lymph nodes (peripheral lymph nodes [PLN]) were harvested. Cell suspensions were stained for CD45 and analyzed for CMTMR and GFP expression by flow cytometry. Homing of CXCR6GFP/GFP/ApoE–/– lymphocytes was expressed relative to CXCR6GFP/+/ApoE–/– cells (=100%).
In separate experiments, splenocytes from CXCR6GFP/+/ApoE–/– mice (8 weeks old) were labeled with CMTMR and injected into atherosclerotic 30-week-old ApoE–/– or C57BL/6 recipient mice. PLN were collected 3 or 5 days later, and CMTMR+CD3+ cells were analyzed for GFP expression.
Detection of Cytokine Production Within the Aortic Wall
Dissected aortas were chopped into small pieces and placed in 100 µL of PBS at 37°C. After 10 minutes, supernatants were collected and cytokines were detected according to the BD Cytometric Bead Array protocol.
Immunohistochemistry
Paraformaldehyde-fixed cryosections of aortic root from ApoE–/– mice were incubated with a monoclonal antibody to CXCR6 (R&D, Minneapolis, Minn), antibody to CXCL16 (R&D), or respective isotype controls. Cell type–specific expression of the cryosections was characterized by costaining for Moma-2 (Serotec, Raleigh, NC) and -smooth muscle actin (Dako, Hamburg). Next, secondary antibodies with FITC and TRITC-coupled reagents (Sigma-Aldrich, St Louis, Mo) were used. Paraformaldehyde-fixed paraffin sections of human carotid endarterectomy specimens were incubated with a monoclonal antibody to CXCR6 or CXCL16 followed by FITC-coupled secondary antibodies and costained for F4/80 (Serotec), -smooth muscle actin (Dako, Hamburg), or CD3 (Serotec) with the use of TRITC-coupled reagents. Nuclei were counterstained with DAPI. Paraformaldehyde-fixed paraffin sections of murine aortic root were stained with Movat pentachrome28 or with monoclonal antibody to Mac-2 (Cedarlane Laboratory, Limited) and hematoxylin.
Statistical Analysis
Data are represented as mean±SE. Comparisons were made with the use of the unpaired Student test or Mann-Whitney test as appropriate.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
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Results |
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CXCR6 Deficiency Diminishes the Development of Atherosclerosis in CXCR6GFP/GFP/ApoE–/– Mice
At 54 to 56 weeks of age, CXCR6GFP/GFP/ApoE–/– and ApoE–/– mice on a chow diet had comparable body weight and levels of total plasma cholesterol, high-density lipoproteins, low-density lipoproteins, and triglycerides (Table). Other groups of mice were fed a Western diet starting at 8 to 11 weeks for 17 weeks. This caused further elevation of total cholesterol levels but no significant difference in body weight (data not shown).
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In mice placed on a Western diet for 17 weeks, CXCR6GFP/GFP/ApoE–/– mice developed 39.5% smaller lesions throughout the aortas in comparison to those in ApoE–/– mice (Figure 1A and 1B). At 56 weeks on a chow diet, the average lesion size was 55% smaller in CXCR6GFP/GFP/ApoE–/– mice than in ApoE–/– mice (lesions: 5.0±0.5% and 11.3±1.9%, respectively; Figure 1C). The percentage of plaque area within the aortic roots of 41-week-old mice fed a chow diet was decreased by 22% in CXCR6GFP/GFP/ApoE–/– mice compared with ApoE–/– mice (Figure 1D and 1E). We also found decreased macrophage content within the aortic root of CXCR6GFP/GFP/ApoE–/– mice (Figure 1D). Partial deficiency of CXCR6 in CXCR6GFP/+/ApoE–/– mice had no effect on disease progression because the average lesion size in CXCR6GFP/+/ApoE–/– mice on a Western diet was not different from that in ApoE–/– mice (en face lesions: 13.5±1.7%). Taken together, these results suggest that CXCR6 is proatherogenic.
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Reduced Number of CXCR6-Expressing T Lymphocytes in CXCR6-Knockin Mice on ApoE–/– Background
To investigate the influence of an atherosclerotic environment on CXCR6 expression, we compared GFP expression in PLN, spleen, and blood of CXCR6GFP/GFP and CXCR6GFP/+ mice with CXCR6GFP/GFP/ApoE–/– and CXCR6GFP/+/ApoE–/– mice (Figure 2). To draw a gate for GFP-positive cells, C57BL/6 leukocytes (no GFP expression) and fluorescence-minus-one controls were used (Figure 2A). The gate for CD3 and other antibody staining was set up according to isotype and appropriate fluorescence-minus-one controls. Most GFP+ cells analyzed in the organs of CXCR6GFP mice as well as CXCR6GFP/ApoE–/– mice were CD3+ or CD3interm lymphocytes with a broad range of GFP expression (Figure 2B). The percentage of GFP+ cells was comparable in the PLN, spleen, and blood between CXCR6GFP/GFP/ApoE–/– and CXCR6GFP/+/ApoE–/– mice, but the intensity of GFP and the fraction of GFP+ cells were significantly reduced compared with the same organs of CXCR6GFP/GFP or CXCR6GFP/+ mice (Figure 2B and 2C). To address whether the inflammatory environment of ApoE–/– mice leads to CXCR6 downregulation, we performed adoptive transfer of CMTMR-labeled CXCR6GFP/+/ApoE–/– splenocytes into either C57BL/6 mice or ApoE–/– mice and collected PLN from the recipients 3 or 5 days later. Adoptively transferred T cells (CMTMR+/CD3+) recovered from ApoE–/– recipient mice expressed lower levels of GFP compared with C57BL/6 recipient mice (Figure 2D). Thus, the expression of CXCR6 is downregulated under the atherosclerotic conditions in ApoE–/– mice.
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Decreased Accumulation of CXCR6+ Cells Within the Aorta of CXCR6GFP/GFP/ApoE–/– Mice
We hypothesized that the absence of CXCR6 might alter the immune cell composition of aortas. Because no reduction in the size of aortic plaque burden was detected in heterozygous CXCR6GFP/+/ApoE–/– mice compared with ApoE–/– mice, heterozygous CXCR6GFP/+/ApoE–/– knockin mice served as a control for CXCR6-deficient CXCR6GFP/GFP/ApoE–/– mice. These flow cytometry data reflect the distribution of immune cells within the aortic wall including atherosclerotic plaques and the surrounding adventitia. Consistent with previous findings, aortas contained CD45+ leukocytes (Figure 3A). The total number of CD45+ leukocytes (Figure 3A) was similar in aortas of CXCR6GFP/GFP/ApoE–/– and CXCR6GFP/+/ApoE–/– mice (0.68±0.11x106 and 0.65±0.16x106 per aorta, respectively). GFP+ leukocytes were found in the aortas of both heterozygous and CXCR6-deficient knockin mice (Figure 3A). However, the percentage of GFP+ leukocytes was significantly decreased in the aortas of CXCR6GFP/GFP/ApoE–/– mice compared with the CXCR6GFP/+/ApoE–/– controls (Figure 3A; P<0.01). Most GFP+ cells within the aortas of CXCR6GFP/+/ApoE–/– animals were CD3+ or CD3inter lymphocytes (Figure 3A). Flow cytometry analysis also demonstrated small populations of CD3inter/NK1.1+ NKT-GFP+ cells at similar abundance in the aortas of CXCR6GFP/GFP/ApoE–/– and CXCR6GFP/+/ApoE–/– mice (Figure 3A, 3B). Among the CD3+ population in the aortas, we observed a 30% decrease in the percentage of CD3+/GFP+ leukocytes in the aortas of CXCR6GFP/GFP/ApoE–/– mice compared with CXCR6GFP/+/ApoE–/– mice (Figure 3A, 3B). Immunofluorescence of frozen sections of murine aortas showed colocalization of CXCR6+ with CD3+ T cells (Figure 3C) and with some Moma-2+ macrophages (Figure 3C). No colocalization of CXCR6 with smooth muscle -actin was present (Figure 3C). As expected, the CXCR6 ligand CXCL16 was expressed in most macrophages (Figure 3C). Confocal microscopy of murine atherosclerotic aortas confirmed colocalization of some Moma-2+ cells with CXCR6 (Figure 3D). Flow cytometry analysis of the aortas of CXCR6GFP/GFP/ApoE–/– and CXCR6+/GFP/ApoE–/– mice showed that 
10% to 20% of CD115+/CD11b+ leukocytes expressed CXCR6 (Figure 3E).
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Decreased Trafficking of CXCR6GFP/GFP/ApoE–/– Lymphocytes Into the Aortic Wall
Reduced percentages of GFP+/CD3+ lymphocytes within the aortas of CXCR6GFP/GFP/ApoE–/– mice suggested that CXCR6 might play a role in lymphocyte trafficking, survival, or proliferation. To test CXCR6+ T cell recruitment into the aortas, we performed short-term homing assay of CXCR6GFP/+/ApoE–/– and CXCR6GFP/GFP/ApoE–/– lymphocytes into the aortas using CMTMR labeling (Figure 4A). CXCR6GFP/GFP/ApoE–/– (GFP+/CMTMR+) lymphocytes showed impaired migration into the aortic wall compared with CXCR6GFP/+/ApoE–/– (GFP+/CMTMR–) lymphocytes (0.22±0.03% versus 0.55±0.12%, respectively; n=5) (Figure 4B and 4E). Although the absence of CXCR6 significantly reduced lymphocyte homing into the aortas, it did not change lymphocyte trafficking into the spleen (0.41±0.11% versus 0.39±0.10%; n=4) and PLN (0.16±0.05% versus 0.15±0.05%; n=4) for CXCR6GFP/+/ApoE–/– and CXCR6GFP/GFP/ApoE–/–, respectively (Figure 4).
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To provide further support for the role of CXCR6-expressing T lymphocytes in regulating the development and persistence of atherosclerosis, we evaluated the production of the Th1 cytokine interferon (IFN)- and the Th2 cytokine interleukin-4 within the aortas. We found a significant reduction in IFN- but not interleukin-4 production within the CXCR6GFP/GFP/ApoE–/– aortas in comparison with the aortas of CXCR6GFP/+/ApoE–/– mice (Figure 5; n=8; P<0.05 by t test with Bonferroni correction for multiple comparison).
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Reduction of Resident Aortic Macrophages in CXCR6GFP/GFP/ApoE–/– Aortas
To investigate whether CXCR6 absence might alter monocyte and macrophage distribution, monocytes in blood and macrophages in PLN and spleen (data not shown) were examined for Ly6C expression. We found no difference in the percentage of CD115+/CD11b+ monocytes in blood of CXCR6GFP/GFP/ApoE–/– and ApoE–/– mice (8.1±1.5% and 10.8±1.5%, respectively; n=11) (Figure 6A). A distinct population existed of Ly6Chigh blood monocytes in CXCR6GFP/GFP/ApoE–/– mice that was not different from the subset of Ly6high blood monocytes in ApoE–/– mice (Figure 6B and 6D). Because Ly6Chigh monocytes are typically CCR2+,29 we analyzed monocyte subsets for CCR2 expression. We found no difference in the percentage of CD115+/CCR2+/Ly6Chigh,inter monocytes in the blood of CXCR6GFP/GFP/ApoE–/– mice compared with ApoE–/– mice (71±5% and 64±7%, respectively; n=6). Further phenotypic characterization of Ly6Chigh monocytes showed a similar pattern of CD62L expression on CD115+/CD11b+ blood monocytes of CXCR6GFP/GFP/ApoE–/– and ApoE–/– mice (Figure 6C).
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Lack of CXCR6+ T lymphocytes may curb IFN- production within the aortas and thus may influence the recruitment or survival of monocytes. CD11b+/CD68+ macrophages were reduced by 90% (Figure 6E) in the aortas of CXCR6GFP/GFP/ApoE–/– mice compared with CXCR6GFP/+/ApoE–/– mice (P<0.0005; n=8). The percentage of Ly6Chigh macrophages was also reduced within the aortas of CXCR6GFP/GFP/ApoE–/– mice compared with ApoE–/– mice (6.8±1.5% versus 15.1±2.0%, respectively; P<0.05; n=4).
To address the potential importance of our findings for human atherosclerosis, the expression of CXCR6 and CXCL16 mRNA was investigated in carotid endarterectomy specimens versus nondiseased arteries by quantitative reverse transcription polymerase chain reaction, demonstrating 4-fold upregulation of both the chemokine and the receptor (data not shown). Sections from carotid endarterectomies were then stained for CXCR6 and CXCL16. Atherosclerotic plaques have few T cells, and most T cells reside under the media layer within tertiary lymphoid structures.2 Therefore, only few T cells were detected within the plaques. CXCR6 was highly expressed in some CD3+ T cells and F4/80+ macrophages but not in smooth muscle cells (Figure 7A). CXCL16 mainly colocalized with macrophages (Figure 7B). Taken together with the mouse data, this suggests that the CXCR6/CXCL16 interaction plays an important role in atherosclerosis.
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Discussion |
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This study reveals a significant proatherogenic role of CXCR6 in atherosclerosis. Absence of CXCR6 resulted in a
50% reduction in lesion formation as assessed by en face staining of the whole aorta and 22% of the lesion reduction within the aortic root. The decrease in lesion size was found in 56-week-old chow-fed mice and 25-week-old mice fed a Western diet. Removing 1 functional copy of CXCR6 had no effect. Flow cytometry analysis of aortas revealed a marked reduction in aortic GFP+/CD3+ but not NKT cells in CXCR6GFP/GFP/ApoE–/– mice that was likely due to defective trafficking into the aortic wall. The decrease in the number of CXCR6+ cells was accompanied by a reduction of aortic IFN- production, one of the most powerful inflammatory cytokines that initiates and supports inflammation in the aorta. Even more notably, macrophages were decreased up to 90% in the aorta of CXCR6GFP/GFP/ApoE–/– mice. Thus, CXCR6 is likely to be involved in atherosclerosis through the alteration of the homing of activated effector T cells into the aortas where those T cells produce proinflammatory cytokines, which determine macrophage recruitment. T cells are abundant in the aortic adventitia of normal mice.2,3,30 In atherosclerotic mice, some T cells are found in atherosclerotic plaque31 and more in atherosclerosis-induced adventitial lymphoid structures.2,30 In the present study we describe a T-cell population that expresses CXCR6 and resides within the aortas of mice fed with either a chow or Western diet. Interestingly, the levels of CXCR6 expression and the percentage of GFP+/CXCR6-expressing T cells from CXCR6GFP/GFP/ApoE–/– mice were lower than in CXCR6GFP/GFP mice. Adoptive transfer experiments suggest that the proinflammatory ApoE–/– environment reduces the levels of CXCR6 expression. CXCR6 expression is downregulated when T cells are stimulated via CD3/CD28 ligation.32 The exact mechanism of CXCR6 downregulation remains to be determined, but one of the possible mechanisms might be downregulation secondary to T-cell receptor engagement.
Several studies have demonstrated an important role for chemokines and chemokine receptors in the recruitment of monocytes during atherosclerosis, but little is known about T-cell recruitment to the aortas. In the present study we provide evidence that CXCR6 is involved in the recruitment of Th1 cells into the aortic wall.
CXCL16 is the only known chemokine ligand for CXCR6 and plays a dual role as a chemokine and a scavenger receptor that binds oxidized low-density lipoprotein. Aslanian and Charo25 generated CXCL16–/– mice and showed that CXCL16 in vivo is atheroprotective. The present data suggest that this must be due to its scavenger receptor activity. Because CXCL16 deletion results in the abolishment of both CXCL16 functions (chemotaxis and scavenging), it is impossible to determine whether the chemokine or scavenger receptor function leads to the accelerated atherosclerosis in CXCL16–/– mice. Shedding-resistant CXCL16 mutant mice might be useful to resolve this question. An alternative explanation for our finding is that CXCR6 may have additional ligand(s) that remain to be discovered.
The presence of NKT cells within atherosclerotic aortas and their proatherogenic role have been reported.33 CXCR6 is broadly expressed on NKT cell populations in different organs such as the liver and spleen,34 but the percentage of NKT cells expressing GFP in CXCR6-deficient mice is not different compared with control mice. Not only T cells but also a subset of macrophages expresses CXCR6 in atherosclerotic aortas. Further studies will be needed to determine the role of CXCR6 on macrophages. Interestingly, the absence of CXCR6 had no effect on the subpopulation of Ly6Chigh monocytes in the blood of CXCR6GFP/GFP/ApoE–/– mice but resulted in the alteration of the number of Ly6Chigh CD11b+/CD68+ macrophages in the aorta, suggesting that the absence of CXCR6 alters the subsets of aortic macrophages residing within the aortic wall.
CXCR6+ T cells have a capacity to secrete a large amount of IFN- on activation.10,35 In this study we show that CXCR6-deficient aortas secrete less IFN-, likely because of the decreased number of CXCR6+ Th1 effector lymphocytes within the aortic wall. IFN- is one of the key proinflammatory cytokines that participates in atherosclerosis and increases inflammatory aspects of this disease through upregulation of proinflammatory genes.36 The decreased level of IFN- in aortas of CXCR6-deficient mice might reduce macrophage recruitment. Indeed, we observed up to a 90% decrease of CD11b+/CD68+ M
within the aortas of CXCR6-deficient mice. IFN- also regulates the expression of CXCL10, CCL2, and CX3CL1,36 which can further affect the recruitment of macrophages and T cells into the atherosclerotic aortic wall. It appears that CXCR6+ T cells are an important source of IFN- that regulates the cell content of atherosclerotic lesions.
In summary, the present study establishes CXCR6 as an important chemokine receptor in the development and progression of atherosclerosis through the recruitment of CXCR6+ effector T cells into atherosclerotic aortas. Our findings suggest a new pathway by which effector T cells may influence the development of atherosclerosis through the alteration of immune composition within the aortic wall.
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Acknowledgments |
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We thank D. Littman for his kind gift of CXCR6 mice and D. Varughese and R. Clohessy for technical help.
Sources of Funding
This work was supported by Deutsche Forschungsgemeinschaft grants Sonderforschungsbereich 542-C12/Z1 and Forschergruppe 809-TP4/5 (Dr Weber), National Institutes of Health grants HL 58108 and 55798 (Dr Ley), and American Heart Association Scientist Development Grant 0730234N (Dr Galkina).
Disclosures
None.
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