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

Abstract

Background: Ly-6C(hi) monocytes are key contributors to atherosclerosis in mice. However, the manner in which Ly-6C(hi) 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-6C(hi) monocytes to atherosclerotic lesions.

Methods and results: To test this hypothesis, we examined the expression and function of PSGL-1 on Ly-6C(hi) and Ly-6C(lo) monocytes from wild-type mice, ApoE(-/-) mice, and mice lacking both ApoE and PSGL-1 genes (ApoE(-/-)/PSGL-1(-/-)). We found that Ly-6C(hi) monocytes expressed a higher level of PSGL-1 and had enhanced binding to fluid-phase P- and E-selectin compared with Ly-6C(lo) monocytes. Under in vitro flow conditions, more Ly-6C(hi) monocytes rolled on P-, E-, and L-selectin at slower velocities than Ly-6C(lo) cells. In an ex vivo perfused carotid artery model, Ly-6C(hi) monocytes interacted preferentially with atherosclerotic endothelium compared with Ly-6C(lo) monocytes in a PSGL-1-dependent manner. In vivo, ApoE(-/-) mice lacking PSGL-1 had impaired Ly-6C(hi) 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-6C(hi) monocytes and a major determinant for Ly-6C(hi) cell recruitment to sites of atherosclerosis in mice.

Figure 1

Ly-6C^hi monocytes have significantly increased PSGL-1 expression. A. Peripheral murine leukocytes were stained with mAbs to CD90, B220, CD49b, NK1.1, Ly-6G, and CD11b. Cells in gate M were defined as monocytes. B. Representative dot plots showing expression of Ly-6C and PSGL-1 in the total monocytes (gate M). Percentages in the upper right panels represent Ly-6C^hiPSGL-1^hi monocytes. C. Comparisons of the number of Ly-6C^hi monocytes in ApoE^-/- and ApoE^-/-/PSGL-1^-/- mice on the Western diet with ApoE^-/- mice fed the chow diet. D. Representative PSGL-1 expression on Ly-6C^hi and Ly-6C^lo monocytes from an ApoE^-/- mouse fed the chow diet. E. Quantification of PSGL-1 expression (MFI, mean fluorescence intensity) on Ly-6C^hi and Ly-6C^lo monocytes from WT and ApoE^-/- mice. F. Real-time PCR quantification (fold changes) of PSGL-1 transcripts of Ly-6C^hi and Ly-6C^lo monocyte subsets from WT mice. The fluorescent intensities of both axes (A and B) or x axis (D) are on a log scale. Data are means ± SEM (A-E: n = 17 mice/group; F, n = 9 mice/group).

Figure 2

PSGL-1 is highly expressed on murine CX3CR1^loCD11b⁺ monocytes as well as on human CD14⁺CD16^- monocytes. A. Representative dot plot showing CX3CR1^loCD11b⁺ (R1 region, red) and CX3CR1^hiCD11b⁺ (R2 region, blue) monocytes. Cells in the gated R1 and R2 regions were examined for expression of PSGL-1 (B) and for interactions with P-selectin (C) and E-selectin (D). Labels on both axes are on a log scale. The data represent at least three experiments. E. Representative histogram showing PSGL-1 expression on human CD14⁺CD16^- monocytes. Labels on x axis is on a log scale. Shown are the results of three independent experiments.

Figure 3

Ly-6C^hi monocytes exhibit enhanced rolling on P-, E- and L-selectin under flow, and increased PSGL-1-dependent interactions with atherosclerotic endothelium. A and B. Representative histograms compare PSGL-1-dependent P-selectin and E-selectin binding activities of Ly-6C^hi and Ly-6C^lo monocytes from WT mice fed chow. 4RA10 is a blocking mAb to PSGL-1. Shaded histograms represent isotype controls. The fluorescent intensity of x axis is on a log scale. C, D and E. Accumulation of rolling Ly-6C^hi and Ly-6C^lo monocytes over P-selectin (P-sel) or E-selectin (E-sel), or L-selectin (L-sel). Shown are the mean ± SEM of three independent experiments. F and G. Rolling and adhesion of WT Ly-6C^hi and Ly-6C^lo as well as PSGL-1^-/- Ly-6C^hi monocytes on atherosclerotic endothelium in an ex vivo carotid artery model. Shown are the mean ± SEM of three independent experiments.

Figure 4

The Western diet does not change the expression profiles of other adhesion molecules or the chemokine receptor CCR2 on Ly-6C^hi and Ly-6C^lo monocytes. Representative histograms compare expression of L-selectin, CD44, CD18, CD49d, and CCR2 on both Ly-6C^hi and Ly-6C^lo monocytes from WT or ApoE^-/- mice. Shaded histograms represent isotype controls. The fluorescent intensity of x axis of each histogram is on a log scale. Shown are the results of three independent experiments.

Figure 5

ApoE^-/-/PSGL-1^-/- mice have reduced macrophage accumulation in atherosclerotic lesions and develop smaller atherosclerotic plaques. A. Macrophage staining (light brown color) in aortic root cross-sections of atherosclerotic lesions in ApoE^-/- and ApoE^-/-/PSGL-1^-/- mice that were fed the Western diet for 12 weeks. B. Quantitative analysis of the macrophages (n = 8 mice/group). Data are means ± SEM. C and D. Oil-Red O-stained (red color) en face preparations of aortas (C), and aortic root cross-sections (D) from atherosclerotic lesions in ApoE^-/- and ApoE^-/-/PSGL-1^-/- mice that were fed the Western diet for 12 weeks. E and F. Quantitative analysis of atherosclerotic lesion area in the entire aorta (E, n = 10 mice/group) and aortic root cross-sections (F, 8 sections/mouse, n = 12 mice/group) using NIH Image J software. Data are means ± SEM. Scale bars: A and D = 100 mm; C = 1 cm.

Figure 6

Lack of PSGL-1 results in reduced monocyte infiltration in neointima, impaired Ly-6C-positive monocyte recruitment to injured arterial walls and reduced formation of neointimal lesions. A. Macrophage staining (dark brown color) in neointima of carotid arteries of ApoE^-/- and ApoE^-/-/PSGL-1^-/- mice after wire-induced injury. B. Quantification of the macrophage staining of neointima. Data are means ± SEM (n = 8 mice/group). C and D. Representative immunofluorescence staining of Ly-6C-positive monocytes (arrows) of ApoE^-/- (C) or ApoE^-/-/PSGL-1^-/- (D) carotid arterial cryosections after wire-induced injury. Arrowheads indicate autofluorescence of vascular elastic membranes. E. Movat stained neointimas in ApoE^-/- and ApoE^-/-/PSGL-1^-/- carotid arteries after wire injury. F. Quantification of the sizes of neointima and media (n = 8). Scale bars: A, C, and E = 100 mm.

Figure 7

Model for PSGL-1-mediated selective homing of Ly-6C^hi monocytes. A. Ly-6C^hi monocytes that are also PSGL-1^hi and L-selectin⁺ preferentially interact with P- and E-selectin on activated endothelium/adherent platelets (primary tethering) or with L-selectin on a rolling/adherent leukocyte or a leukocyte fragment (a neutrophil in this case, secondary tethering) under flow. B. Ly-6C^lo monocytes that are also PSGL-1^lo and L-selectin^- cannot efficiently interact with activated endothelium under flow because of low-level surface expression of PSGL-1, and, possibly, lack of L-selectin.