P-Selectin Glycoprotein Ligand-1 Is Highly Expressed on Ly-6Chi Monocytes and a Major Determinant for Ly-6Chi Monocyte Recruitment to Sites of Atherosclerosis in Mice
Background— Ly-6Chi monocytes are key contributors to atherosclerosis in mice. However, the manner in which Ly-6Chi monocytes selectively accumulate in atherosclerotic lesions is largely unknown. Monocyte homing to sites of atherosclerosis is primarily initiated by rolling on P- and E-selectin expressed on endothelium. We hypothesize that P-selectin glycoprotein ligand-1 (PSGL-1), the common ligand of P- and E-selectin on leukocytes, contributes to the preferential homing of Ly-6Chi monocytes to atherosclerotic lesions.
Methods and Results— To test this hypothesis, we examined the expression and function of PSGL-1 on Ly-6Chi and Ly-6Clo monocytes from wild-type mice, ApoE−/− mice, and mice lacking both ApoE and PSGL-1 genes (ApoE−/−/PSGL-1−/−). We found that Ly-6Chi monocytes expressed a higher level of PSGL-1 and had enhanced binding to fluid-phase P- and E-selectin compared with Ly-6Clo monocytes. Under in vitro flow conditions, more Ly-6Chi monocytes rolled on P-, E-, and L-selectin at slower velocities than Ly-6Clo cells. In an ex vivo perfused carotid artery model, Ly-6Chi monocytes interacted preferentially with atherosclerotic endothelium compared with Ly-6Clo monocytes in a PSGL-1–dependent manner. In vivo, ApoE−/− mice lacking PSGL-1 had impaired Ly-6Chi monocyte recruitment to atherosclerotic lesions. Moreover, ApoE−/−/PSGL-1−/− mice exhibited significantly reduced monocyte infiltration in wire injury–induced neointima and in atherosclerotic lesions. ApoE−/−/PSGL-1−/− mice also developed smaller neointima and atherosclerotic plaques.
Conclusions— These data indicate that PSGL-1 is a new marker for Ly-6Chi monocytes and a major determinant for Ly-6Chi cell recruitment to sites of atherosclerosis in mice.
Received February 3, 2008; accepted April 14, 2008.
Atherosclerosis is characterized by inflammatory cell infiltration of the arterial wall.1,2 Early atherosclerotic lesions are largely composed of lipid-laden macrophages known as foam cells, which are derived from circulating monocytes.1,2 Thus, the recruitment of monocytes into the arterial wall plays decisive roles in the initiation and progression of atherosclerosis.
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In mice, monocytes are divided into Ly-6Chi and Ly-6Clo subsets. Ly-6Chi monocytes are considered short-lived “inflammatory” cells that are actively recruited to inflamed tissues, whereas Ly-6Clo monocytes have a longer half-life and home to noninflamed tissues to differentiate into resident macrophages.3 Ly-6Chi monocytes are preferentially recruited to atherosclerotic plagues and give rise to lipid-laden macrophages in apolipoprotein E–deficient (ApoE−/−) mice.4 Ly-6Chi monocytes are recognized as the key monocyte subset in the development of atherosclerosis,4,5 but the manner in which these monocytes selectively accumulate in atherosclerotic lesions is largely unknown.
During inflammation, monocyte recruitment follows the well-defined leukocyte trafficking cascade.6–8 Circulating leukocytes first tether to and roll on the activated endothelium, then firmly adhere and eventually transmigrate into the underlying tissues. These steps are regulated by adhesion molecules and chemokines. Murine Ly-6Chi and Ly-6Clo monocytes differ with regard to their expression of chemokine receptors and adhesion molecules. Ly-6Chi monocytes are CCR2+, CX3CR1lo, L-selectin+, CD44+, LFA1+, and VLA4+, whereas Ly-6Clo cells are CCR2−, CX3CR1hi, L-selectin−, CD44+, LFA12+, and VLA4+.3 Interestingly, only CCR2 and L-selectin are differentially expressed on Ly-6Chi cells.3,4 Although CCR2 is important for monocytes to enter atherosclerotic lesions,5,9 it does not mediate the early adhesion steps such as tethering and initial rolling.5,10 In blood flow conditions, especially arterial flow conditions, the initial steps serve as an anchoring system to capture flowing leukocytes and initiate rolling on the endothelium for subsequent firm adhesion and transmigration. The initial steps are primarily mediated by P-, E-, and L-selectin and their common ligand on leukocytes, P-selectin glycoprotein ligand-1 (PSGL-1).7 E-selectin and P-selectin are expressed on activated endothelial cells and/or platelets, whereas L-selectin is expressed on leukocytes. PSGL-1 binds to selectins with exceptionally high on- and off-rates, which favors interactions under flow. Moreover, shear stress actually strengthens the interactions of PSGL-1 with P- and L-selectin.7,11 Ly-6Chi cells exhibit an advantage over the Ly-6Clo subset with regard to homing to atherosclerotic plagues.4,5 This preferential homing could be a result of differential expression or function of PSGL-1. In addition, L-selectin–mediated rolling may also contribute to this difference because L-selectin is expressed on Ly-6Chi but not on Ly-6Clo monocytes.
To test this hypothesis, we examined the surface expression and function of PSGL-1 on Ly-6Chi and Ly-6Clo monocytes from wild-type (WT) and ApoE−/− mice. We also investigated PSGL-1–dependent interactions of Ly-6Chi or Ly-6Clo monocytes with P-, E-, and L-selectin under in vitro flow conditions and with early atherosclerotic endothelium using ex vivo perfusion of carotid arteries. To evaluate the role of PSGL-1 in the development of atherosclerosis, we studied atherosclerosis and wire injury–induced neointimal formation in the carotid artery in ApoE−/− mice and in mice lacking both ApoE and PSGL-1 genes (ApoE−/−/PSGL-1−/−). Our results demonstrate that PSGL-1 is highly expressed on Ly-6Chi monocytes and is a major determinant for Ly-6Chi monocyte recruitment to atherosclerotic lesions.
A full description of all methods can be found in the online-only Data Supplement.
ApoE−/−/PSGL-1−/−-deficient mice (C57BL/6J background) were generated after crossing PSGL-1−/− mice12 with ApoE−/− mice. WT C57BL/6J and ApoE−/− mice were from the Jackson Laboratory (Bar Harbor, Me). Mice were kept in a specific pathogen-free facility. All mouse experiments were approved by the institutional animal care and use committees of the Oklahoma Medical Research Foundation and the University of Minnesota.
All antibodies were obtained from BD Biosciences (San Diego, Calif) unless specified otherwise. Mouse peripheral blood was used for monocyte analysis. Leukocytes that were positive for myeloid marker CD11b but negative for the rest of the lineage markers were defined as monocytes.4 Anti-Ly-6C monoclonal antibody (mAb) (AL-21) was used to classify monocytes into subsets with either high expression of Ly-6C (Ly-6Chi) or low expression of Ly-6C (Ly-6Clo).4 A rat anti-mouse PSGL-1 mAb (4RA10) was used to measure PSGL-1 expression.
The chemokine receptor CX3CR1 was originally used to classify monocytes.3 In mice, the CX3CR1loCD11b+ monocyte subset is the equivalent of Ly-6Chi monocytes.3 To examine PSGL-1 expression and P- and E-selectin binding of CX3CR1loCD11b+ monocytes, C57BL/6 CX3CR1GFP/+ mice were used. In CX3CR1GFP/+ mice, 1 CX3CR1 allele was replaced with the gene encoding green fluorescent protein (GFP).13
Human monocytes have CD14+CD16− and CD14−CD16+ subsets, which resemble murine Ly-6ChiCX3CR1lo and Ly-6CloCX3CR1hi subsets, respectively.3 Human leukocytes that were HLA-DR and CD14 double-positive or HLA-DR and CD16 double-positive were analyzed for PSGL-1 expression with a mAb to human PSGL-1 (KPL1). The protocol was approved by the institutional review board committee of the University of Minnesota.
Flow cytometry was performed on a FACSCalibur (BD Biosciences). Data were analyzed with Summit Software v4.3 (Dako, Carpinteria, Calif).
In Vitro Flow Chamber Assay
To obtain sufficient cells, Ly-6Chi and Ly-6Clo monocytes from murine spleens were used. The spleen-derived monocytes are surrogates for circulating monocytes.4 Ly-6Chi and Ly-6Clo monocytes were sorted from the monocyte population with the use of the inFlux V-GS Cytometer Work Bench (Cytopeia, Seattle, Wash).
Flow chamber experiments were performed as described.11 Briefly, murine P-selectin-IgM, E-selectin-IgM, control CD45-IgM, biotinylated 2-glycosulfopeptide-6 (2-GSP-6; gift from Dr Richard Cummings, Emory University), or human L-selectin IgG chimera was captured on the dishes. 2-GSP-6 is modeled after the NH2-terminal selectin-binding region of PSGL-1.14 Human L-selectin is the equivalent of murine L-selectin because they share the same binding activity to murine PSGL-1. Sorted Ly-6Chi or Ly-6Clo monocytes (0.5×106/mL in Hanks’ balanced salt solution with 0.5% human serum albumin) were perfused over P-selectin, E-selectin, control CD45-IgM, 2-GSP-6, or L-selectin in dishes mounted in a parallel-plate flow chamber. Rolling cells were analyzed with the use of a Silicon Graphics workstation (Silicon Graphics, Sunnyvale, Calif).
Ex Vivo Perfusion of Murine Carotid Arteries
WT or PSGL-1−/− Ly-6Chi or Ly-6Clo monocytes were labeled with calcein AM (Molecular Probes, Eugene, Ore) and infused into the ApoE−/− carotid arteries at a rate of 10 mL/min (1×106 cells/mL), resulting in a wall shear stress of 3.0±0.1 dyne/cm2.10 Cell rolling and adhesion were recorded on videotape by stroboscopic epifluorescence illumination with an intravital microscope (Axioskop, ×10 water immersion objective, NA 0.5, Carl Zeiss, Thornwood, NY).
Atherosclerosis and Carotid Artery Wire Injury Models
To induce atherosclerosis, ApoE−/−/PSGL-1−/− or ApoE−/− mice at 5 weeks of age were fed the Western diet for 12 weeks.5 Atherosclerotic lesions stained with Oil Red O were quantified by en face analysis of the whole aorta and by cross-sectional analysis of the proximal aorta.15 For the carotid artery wire injury models, 8-week-old male ApoE−/− mice were fed the Western diet for 2 weeks, and carotid artery wire injury was performed.16 For quantification of neointimas, ten 5-μm aortic sections that were stained with Movat pentachrome (Sigma, St Louis, Mo) were analyzed for each mouse. Images were analyzed with the use of NIH Image software.10
Cryosections of aortas from ApoE−/− and ApoE−/−/PSGL-1−/− mice were stained with anti-F4/80 mAb (MOMA-2) (Accurate Chemical, Westbury, NY).17 Eight sections every 40 μm from each aorta root were examined. Digital images were used to quantify macrophages with NIH Image J software.
Ly-6Chi monocytes in cryosections of ApoE−/− and ApoE−/−/PSGL-1−/− carotid arteries were examined with an FITC-conjugated anti-mouse Ly-6C mAb (AL-21, 1:100 dilution, BD Biosciences) as described.4 Peripheral Ly-6Chi or Ly-6Clo monocytes, which were cytocentrifuged on glass slides, were stained with the same fluorescein isothiocyanate–conjugated mAb and used as controls for the identification of Ly-6Chi cells. Fluorescence was detected with a fluorescence microscope (×4 objective, NA 0.3, Olympus America, Center Valley, Pa).
Mean and SEM values are reported where appropriate. Data were analyzed by the Student t test. P<0.05 was considered significant.
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.
Murine Ly-6ChiCX3CR1lo Monocytes and Human CD14+CD16− Monocytes Express High Levels of PSGL-1
We examined PSGL-1 expression on mouse Ly-6Chi and Ly-6Clo monocytes. We defined monocytes as CD11b+ CD90loB220loCD49bloNK1.1loLy-6Glo cells (Figure 1A).4 Within this monocyte population, cells were classified into Ly-6Chi and Ly-6Clo subsets on the basis of their differential expression of Ly-6C (Figure 1B). The overall monocytes were not significantly different among ApoE−/− and ApoE−/−/ PSGL-1−/− mice fed the Western diet for 12 weeks and ApoE−/− mice fed a standard chow (Figure 1A and data not shown). However, ApoE−/− mice fed the Western diet had an elevated number of Ly-6Chi monocytes compared with the number of Ly-6Chi monocytes from ApoE−/− mice fed chow (Figure 1B and 1C), which is consistent with published results.4,5 The percentage of Ly-6Chi monocytes was similar in ApoE−/− and in ApoE−/−/PSGL-1−/− mice fed the high-fat diet (Figure 1C). Remarkably, PSGL-1 was expressed at a much higher level on Ly-6Chi than on Ly-6Clo monocytes from ApoE−/− mice fed either chow or the Western diet (Figure 1D and 1E). The increase in PSGL-1 expression was not caused by the ApoE deficiency because Ly-6Chi monocytes from WT mice also expressed more PSGL-1 (Figure 1E). Real-time polymerase chain reaction demonstrated that PSGL-1 mRNA transcripts in Ly-6Chi monocytes were also expressed at a higher level than the transcripts in Ly-6Clo monocytes (Figure 1F).
We confirmed that PSGL-1 was also highly expressed on CX3CR1loCD11b+ monocytes compared with its expression on CX3CR1hiCD11b+ monocytes (Figure 2A and 2B). In addition, CX3CR1loCD11b+ monocytes exhibited greater binding to P- and E-selectin (Figure 2C and 2D) compared with CX3CR1hiCD11b+ monocytes.
We also investigated PSGL-1 level on human monocytes. Interestingly, consistent with the expression patterns of PSGL-1 on murine monocytes, PSGL-1 level was also higher on human CD14+CD16− monocytes than on CD14−CD16+ monocytes (Figure 2E; n=3; P<0.01).
Ly-6Chi Monocytes Interact Preferentially With P-, E-, and L-Selectin as Well as 2-GSP-6 Under Flow
To evaluate the function of PSGL-1 on Ly-6Chi monocytes, we first tested fluid-phase binding of P- and E-selectin to WT monocytes using flow cytometry. Compared with Ly-6Clo monocytes, Ly-6Chi monocytes had greater binding to P- and E-selectin (Figure 3A and 3B). The interaction between PSGL-1 and P-selectin was PSGL-1 dependent because 4RA10, a mAb that blocks PSGL-1 function, eliminated this interaction (Figure 3A). 4RA10 substantially reduced but did not abolish the PSGL-1 interaction with E-selectin (Figure 3B), which reflects E-selectin ligand activities other than PSGL-1 on Ly-6Chi monocytes.18 Ly-6Chi monocytes from ApoE−/− mice fed the Western diet for 12 weeks had similar P- and E-selectin binding profiles (data not shown), suggesting that the 12-week high-fat diet did not significantly alter selectin ligand activities on monocytes. Similar levels of mRNAs of α-1,3-fucosyltransferase VII (FTVII) and core 2 β1,6-glucosaminyltransferase-I (C2GlcNAcT-I), the protein products of which are essential for PSGL-1 function, were detected in Ly-6Chi and Ly-6Clo monocytes (Figure I in the online-only Data Supplement).
We then compared the rolling of Ly-6Chi and Ly-6Clo monocytes, respectively, on immobilized P-, E-, or L-selectin under flow conditions. Comparatively more Ly-6Chi than Ly-6Clo monocytes rolled on P-, E-, and L-selectin at low shear stress (0.5 to 1 dyne/cm2) (Figure 3C, 3D, 3E). Ly-6Chi cells rolled more stably on P- and L-selectin, as manifested by their slow rolling velocities (μm/s; 1.37±0.48 for Ly-6Chi versus 3.55±1.22 for Ly-6Clo on P-selectin; 8.9±3.07 for Ly-6Chi and 117.9±4.38 for Ly-6Clo on L-selectin, at 0.5 dyne/cm2; P<0.01; n=15). No significant difference was found in rolling velocities on E-selectin between Ly-6Chi and Ly-6Clo cells, which is consistent with previous studies showing that PSGL-1 is important for tethering to E-selectin and that other E-selectin ligand(s) such as CD44 other than PSGL-1 contributes to the slow rolling on E-selectin.12,18 Rolling was PSGL-1 and selectin dependent because mAbs to PSGL-1 and P-, E-, or L-selectin reduced the number of rolling cells to the basal levels observed on the surfaces coated with control reagents (data not shown). Significantly, only Ly-6Chi monocytes rolled on P-, E-, or L-selectin at relatively high shear stress (>2 dyne/cm2) (Figure 3C, 3D, 3E).
Because L-selectin was preferentially expressed on Ly-6Chi monocytes (Figure 4), we also compared rolling of Ly-6Chi and Ly-6Clo monocytes on immobilized 2-GSP-6, which is modeled after the NH2-terminal L-selectin–binding region of PSGL-1.13 At all shear stress levels tested, Ly-6Chi cells rolled on 2-GSP-6 (32±4 cells/mm2 at 1 dyne/cm2) (Figure IIA in the online-only Data Supplement). Rolling was PSGL-1 dependent as it was blocked by mAb 4RA10 to PSGL-1 (Figure IIA in the online-only Data Supplement). In contrast, almost no Ly-6Clo rolling on 2-GSP-6 was observed (Figure IIA and IIB in the online-only Data Supplement). This experiment suggests that L-selectin may contribute to the selective homing of Ly-6Chi cells to atherosclerotic lesions.
Ly-6Chi Monocytes Roll Preferentially on Early Atherosclerotic Endothelium in a PSGL-1–Dependent Manner
To test whether Ly-6Chi monocytes have increased capacity to interact with early atherosclerotic endothelium, we used the ex vivo perfusion of ApoE−/− carotid artery model.10 In this model, rolling of monocytes on early atherosclerotic endothelium of ApoE−/− mice is mediated primarily by P-selectin and vascular cell adhesion molecule-1.10,19 Compared with Ly-6Clo monocytes, more Ly-6Chi monocytes rolled on atherosclerotic endothelium at 3 dyne/cm2 (Figure 3F). Consequently, the number of adherent Ly-6Chi monocytes was increased at all time points compared with Ly-6Clo monocytes (Figure 3G). PSGL-1−/− Ly-6Chi monocytes had dramatically reduced rolling and adhesion on atherosclerotic endothelium, indicating primarily PSGL-1–dependent interactions. The observed residual rolling of PSGL-1−/− Ly-6Chi monocytes on endothelium may be from interactions between integrin VLA4 and vascular cell adhesion molecule-1.20
High-Fat Diet Does Not Change the Expression Profile of Other Adhesion Molecules or the Chemokine Receptor CCR2 on Ly-6Chi Monocytes
To exclude the possibility that the increased recruitment of Ly-6Chi monocytes into atherosclerotic lesions is a result of altered expression of other molecules resulting from consumption of the Western diet, we compared the expression of L-selectin, CD44, CD18 (β2 integrin), CD49d (VLA4), and CCR2 on both Ly-6Chi and Ly-6Clo monocytes. Consistent with previous reports,3,5 expression of L-selectin and CCR2 was higher on Ly-6Chi than on Ly-6Clo monocytes, whereas CD18 and CD49d were lower on Ly-6Chi than on Ly-6Clo monocytes (Figure 4). Both Ly-6Chi and Ly-6Clo cells expressed a similar level of CD44 (Figure 4). Ly-6Chi or Ly-6Clo monocytes from WT or ApoE−/− mice fed either chow or the Western diet for 12 weeks express similar levels of these molecules (Figure 4), suggesting that diet does not affect the expression of these molecules.
ApoE−/−/PSGL-1−/− Mice Have Reduced Monocyte/Macrophage Accumulation in Atherosclerotic Lesions and Develop Smaller Atherosclerotic Plaques
Ly-6Chi monocytes are key contributors to atherosclerosis.4 To examine whether PSGL-1 contributes to monocyte recruitment in atherosclerotic lesions and to the development of atherosclerosis, ApoE−/− and ApoE−/−/PSGL-1−/− mice were fed the Western diet for 12 weeks. Compared with ApoE−/− mice, ApoE−/−/PSGL-1−/− mice exhibited less monocyte/macrophage infiltration in the lesions (Figure 5A and 5B). In addition, the lesions in ApoE−/−/PSGL-1−/− aortas were significantly smaller than lesions in ApoE−/− aortas (Figure 5C, 5D, 5E, 5F). No significant difference was found between ApoE−/− and ApoE−/−/PSGL-1−/− mice in their plasma lipid levels (Figure III in the online-only Data Supplement). These data support the important role of PSGL-1 in mediating Ly-6Chi monocyte recruitment into atherosclerotic lesions and in the development of early atherosclerotic plaques.
Lack of PSGL-1 Results in Impaired Ly-6Chi Monocyte Recruitment to Injured Arterial Walls and Reduces Formation of Neointimal Lesions
Monocytes are critical in arterial neointimal formation.21 We examined whether PSGL-1 deficiency reduces monocyte infiltration in neointima and the size of the lesions. Our experiments showed that ApoE−/−/PSGL-1−/− mice had less monocyte/macrophage infiltration in the neointima of carotid arteries after wire injury than ApoE−/− mice (Figure 6A and 6B). To examine Ly-6Chi monocyte homing to injured arteries, cryosections of ApoE−/− and ApoE−/−/PSGL-1−/− carotid arteries, which were harvested 7 days after wire-induced injury, were stained with an anti-murine Ly-6C mAb as described.4 In this model, P-selectin expressed by regenerating endothelial cells and adherent platelets contributes to the monocyte recruitment into injured carotid arterial wall.22 We found that ApoE−/− carotid arteries had substantial accumulation of Ly-6Chi monocytes after wire-induced injury (Figure 6C). In contrast, Ly-6Chi monocytes were rarely observed in wire-injured ApoE−/−/PSGL-1−/− carotid arterial sections (Figure 6D). These data suggest the critical contribution of PSGL-1 to the recruitment of Ly-6Chi monocytes to neointimal lesions.
We also used this model to investigate the role of PSGL-1 in neointima formation. The average size of neointima in the carotid arteries of ApoE−/−/PSGL-1−/− mice after wire injury was dramatically reduced compared with that in ApoE−/− mice (Figure 6E and 6F), indicating an important role for PSGL-1 in this pathology.
Our results demonstrate that PSGL-1 is highly expressed on both murine Ly-6Chi and human CD14+CD16− monocytes and therefore is a new marker for this subset of inflammatory monocytes. PSGL-1 on Ly-6Chi monocytes interacts with all 3 selectins and contributes significantly to the selective homing of Ly-6Chi monocytes to atherosclerotic lesions.
Selective leukocyte homing to a particular tissue is primarily regulated by unique combinations of adhesion molecules and chemokines.6,23 Regulated expression of functional PSGL-1 has been recognized as a primary mechanism for the tissue-specific homing of activated T cells.23 All T cells express a similar level of PSGL-1, yet naive T cells do not interact with P-selectin because their PSGL-1 is not appropriately glycosylated and thus not functional. During activation, T cells acquire functional PSGL-1 on induced expression of glycosyltransferases FTVII and C2GlcNAcT-I, which add selectin-interacting carbohydrate groups to PSGL-1.7,23–26 We sought to examine whether a similar mechanism regulates selective homing of Ly-6Chi monocytes. Surprisingly, Ly-6Chi monocytes expressed a higher level of PSGL-1, which interacted with all 3 selectins, compared with Ly-6Clo monocytes. However, no difference was found in the level of FTVII and C2GlcNAcT-I transcripts in both Ly-6Chi and Ly-6Clo monocytes. Thus, unlike activated T cells that gain function of PSGL-1 as a result of upregulation of glycosyltransferases, Ly-6Chi monocytes express a higher level of fully modified and functional PSGL-1 than Ly-6Clo monocytes, which provides a molecular basis for their preferential homing to atherosclerotic plaques.
PSGL-1 is critical for leukocyte homing; it is a well-characterized ligand for all 3 selectins and enables circulating leukocytes to roll on activated endothelium under physiological flow conditions.7 In humans, P- and E-selectin have been detected on the luminal surface of arteries with nascent or established atherosclerotic lesions.27 In mouse models, P- and E-selectin are closely correlated with monocyte/macrophage infiltration and lesion formation.27,28 Leukocytes can initiate rolling on endothelial cells in 3 ways.7,8,23 First, leukocytes can directly contact the endothelium when entering venules from capillaries. Alternatively, leukocytes can be captured by the endothelium through PSGL-1–dependent tethering to P- and E-selectins, a process called primary tethering (Figure 7A, left panel).6–8 Primary tethering is considered particularly important for leukocytes to initiate rolling in larger venules and arterial vessels because these vessels do not have upstream capillaries. The recruitment of Ly-6Chi monocytes occurs in arteries during early atherosclerosis. Thus, PSGL-1–mediated primary tethering may be the key to Ly-6Chi monocyte homing. PSGL-1 on Ly-6Chi monocytes may interact with P-selectin on either activated endothelial cells or activated platelets adhered to injured arterial wall.27 PSGL-1 may also mediate tethering to and rolling on E-selectin on activated endothelium, either independently or in cooperation with other leukocyte E-selectin ligand(s).7,12,18 Third, Ly-6Chi monocytes may interact with the endothelium through secondary tethering in which a freely flowing leukocyte transiently interacts with a rolling or adherent leukocyte or adherent leukocyte fragment and subsequently rolls on the endothelium (Figure 7A, right panel).29–31 Interestingly, Ly-6Chi monocytes express L-selectin, whereas Ly-6Clo monocytes do not. Ly-6Chi monocyte L-selectin interacted well with 2-GSP-6, the functional domain of PSGL-1, under flow conditions. Reciprocal interactions between PSGL-1 and L-selectin on leukocytes are important for leukocyte adhesion to atherosclerotic lesions.29 Limited by the availability of Ly-6Chi and Ly-6Clo monocytes, we were unable to analyze L-selectin–dependent secondary rolling. Nevertheless, secondary tethering may serve as another important mechanism for selective Ly-6Chi monocyte homing. In vivo, the secondary tethering of a flowing Ly-6Chi monocyte may occur at much higher frequencies on rolling or adherent neutrophils, which are known to interact with atherosclerotic lesions, than on rolling or adherent monocytes because of the dramatic difference in their number in circulation.29 Our data demonstrate that Ly-6Chi monocytes, which are PSGL-1hi and L-selectin+, use PSGL-1–dependent primary and possibly secondary capturing mechanisms to selectively home to atherosclerotic sites (Figure 7A), whereas Ly-6Clo monocytes, which are PSGL-1lo and L-selectin−, are impaired in these homing mechanisms (Figure 7B).
Ly-6Chi monocytes use the chemokine receptors CCR2 and CX3CR1 to enter atherosclerotic plaques.5 However, CCR2 was found not to be involved in the early steps of monocyte adhesion,10 and CX3CR1 is expressed at a lower level on Ly-6Chi monocytes than on Ly-6Clo monocytes.3,5 Therefore, these chemokine receptors are less likely to contribute to the homing advantage of Ly-6Chi monocytes. Other adhesion molecules such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 may not be key factors for this selective Ly-6Chi cell homing process because their ligands are expressed at lower levels on Ly-6Chi monocytes than on Ly-6Clo monocytes.3 Nevertheless, these chemokine receptors and adhesion molecules are important for later processes such as firm adhesion and transmigration once Ly-6Chi monocytes initiate rolling on the endothelium.5,8,20
In our study, Ly-6Chi monocytes exhibit PSGL-1–dependent rolling ability on selectins under relatively high shear stress (2 to 4 dyne/cm2), whereas almost no Ly-6Clo monocytes rolled under such conditions. Wall shear stress in vivo is higher in arteries than in veins. Atherosclerotic lesions are prone to develop at bifurcations, branchings, and curvatures where the shear stress ranges from 1 to 3 dyne/cm2.32,33 Neointimal lesions after wire injury develop at locations where shear stress can be much higher because of an angioplasty procedure.32,33 Thus, the high level of functional PSGL-1 may give Ly-6Chi monocytes a unique homing advantage under arterial flow conditions. Indeed, lack of PSGL-1 protected ApoE−/− mice from developing severe wire injury–induced neointimal and atherosclerotic plaques, which may be attributable to diminished Ly-6Chi monocyte recruitment. Significantly, lack of PSGL-1 provided ApoE−/− mice more protection from developing neointimas than atherosclerotic plaques, which supports the contention that PSGL-1 plays a more important role in the recruitment of monocytes in acute lesions than in chronic lesions. Our data indicate that PSGL-1 is also highly expressed on human CD14+CD16− monocytes. Thus, these results support the potential for PSGL-1 blockade as a therapeutic approach for the treatment of restenosis after angioplasty.
We thank Rodger McEver and Charles Esmon for critically reading the manuscript.
Sources of Funding
This study was supported by National Institutes of Health grants HL085607 and RR018758 to Dr Xia and American Heart Association grant 0430151N and National Institutes of Health grants HL78679 and HL080569 to Dr Huo.
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The recruitment of monocytes into the arterial wall plays decisive roles in the initiation and progression of atherosclerosis. In mice, monocytes are divided into Ly-6Chi and Ly-6Clo subsets. Ly-6Chi monocytes are recognized as the key monocyte subset in the development of atherosclerosis, but the mechanisms by which these monocytes selectively accumulate in atherosclerotic lesions are largely unknown. In the present study, we identified that Ly-6Chi monocytes expressed a higher level of P-selectin glycoprotein ligand-1 (PSGL-1) and had enhanced binding to P-, E-, and L-selectin compared with Ly-6Clo monocytes under physiological conditions. ApoE−/− mice lacking PSGL-1 (ApoE−/−/PSGL-1−/−) had impaired Ly-6Chi monocyte recruitment to arterial injuries and exhibited significantly reduced monocyte infiltration in wire injury–induced neointima and in atherosclerotic lesions. Moreover, ApoE−/−/PSGL-1−/− mice also developed smaller neointima and atherosclerotic plaques. These results indicate that PSGL-1 is a new marker for Ly-6Chi monocytes and a major determinant for Ly-6Chi cell recruitment to sites of atherosclerosis in mice.
↵*Drs An and Wang contributed equally to this work.
The online-only Data Supplement, which contains Methods and figures, is available with this article at http://circ.ahajournals.org/cgi/content/full/ CIRCULATIONAHA.108.771048/DC1.