Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques

Abstract

Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2(+)CX3CR1(+)Ly-6C(hi) and CCR2(-)CX3CR1(++)Ly-6C(lo) monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X(3)-C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE(-/-) mice and skewed toward an increased frequency of CCR2(+)Ly-6C(hi) monocytes in apoE(-/-) mice fed a high-fat diet. CCR2(+)Ly-6C(hi) monocytes efficiently accumulated in plaques, whereas CCR2(-)Ly-6C(lo) monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2(-) monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE(-/-) mice. By comparison, CCR2(+)Ly-6C(hi) monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2(+) monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.

Figure 1. Characterization of blood monocytes in WT and apoE^–/– mice.

(A) The frequency of monocytes (monos) was determined in WT mice and in 16-week-old apoE^–/– mice fed a standard chow (Chow) or 12 weeks of a high-cholesterol/high-fat diet (Diet). Left: dot plots and histograms of stained monocytes; middle panel: bar graph of total monocyte frequency. The relative frequencies of the 2 subsets of blood monocytes (Ly-6C^hi and Ly-6C^lo) and the minor Ly-6C^int subset were determined. apoE^–/– mice kept on a high-fat diet had significantly more Ly-6C^hi and fewer Ly-6C^lo blood monocytes. Plots show the mean values (±SEM) for 12–16 mice in each condition. *Significantly different from WT, P < 0.001. (B) Histograms show CD11c expression of either CD115⁺Ly-6C^hi (gray) or CD115⁺Ly-6C^lo cells (black). Plots are representative of flow cytometric analyses for more than 10 animals studied per group. Cut-off for isotype-matched control mAb staining is shown by vertical lines in the plots.

Figure 2. Recruitment of labeled WT monocytes to peripheral sites of acute inflammation.

Ly-6C^lo or Ly-6C^hi monocytes in WT mice were labeled with latex (24). (A) The appearance of latex⁺ cells in the peritoneal cavity was monitored by flow cytometry using F4/80 and CD115 antigens to identify monocytes and macrophages. Macrophages are seen as the prominent F4/80^hi population in the noninflamed peritoneum (left dot plots). They become outnumbered by numerous infiltrating monocytes (inflamed plots; lower levels of F4/80 in lower right quadrants) and F4/80^–CD115^– neutrophils (inflamed plots; lower left quadrants). Note that Ly-6C^hi labeling of monocytes leads to both latex⁺F4/80⁺ monocytes (inflamed plots; upper right quadrants) and latex⁺F4/80^– neutrophils (inflamed plots; upper left quadrants) appearing in the peritoneum, since some neutrophils transiently carry latex in this labeling protocol (24). In the inflamed peritoneum, latex⁺Ly-6C^hi monocytes outnumbered latex⁺ neutrophils 4:1, even though neutrophils dominate in acute peritoneal inflammation, in contrast to what occurs in atherosclerosis. (B) The graph shows the actual frequency of latex⁺ Ly-6C^lo or Ly-6C^hi monocytes in the inflamed peritoneum (gray bars), determined by flow cytometry, compared with the expected/known frequency (white bars) at which they would enter the peritoneum as unlabeled cells (“expected” data are derived from previously published calculations [ref. 16], as described in Methods). n = 4 mice per group. Differences between the actual and expected frequencies were not significant. (C) Levels of plasma cytokines (± SD) were measured at baseline or 2, 12, and 24 hours after administration of latex in the Ly-6C^hi or Ly-6C^lo monocyte labeling protocol. n = 3 mice per time point.

Figure 3. Labeling of monocyte subsets in apoE^–/– mice.

(A) Blood analysis after i.v. injection of latex beads in apoE^–/– mice. Representative plots (≥5 mice per time-point) at days 1–5 gated on all cells, monocytes, or latex⁺ cells. (B) Blood apoE^–/– monocyte subsets 4, 8, or 24 hours after injection of latex: gating on all monocytes (All monos) or only latex⁺ monocytes (Latex⁺ monos); n ≥ 4 mice per time point. (C) Schematic of injection protocols for labeling monocyte subsets. Injection of latex labeled Ly-6C^lo monocytes; injection of clodronate-loaded liposomes (clo-lip) followed by latex 18 hours later labeled Ly-6C^hi monocytes. (D) Blood analyses trace the depletion, reappearance, and subset of apoE^–/– monocytes over time after i.v. injection of clo-lip followed by latex (n ≥ 5 per time point). (E) Latex⁺ apoE^–/– monocytes (%) that remained Ly-6C^hi or Ly-6C^lo after clo-lip/latex or latex only injection, respectively. Reductions in frequency of Ly-6C^hi monocytes among circulating latex⁺ cells reflected the extent of their conversion to Ly-6C^lo monocytes. (F and G) Summary of the labeling efficiency using the 2 labeling techniques in apoE^–/– mice (shown for 7-month-old mice on chow diet), presented as (F) percentage of latex⁺ cells among all blood monocytes or (G) normalized to account for the absolute frequency of apoE^–/– monocytes after injection of latex i.v. (Ly-6C^lo monocyte labeling) or latex i.v. following monocyte depletion (Ly-6C^hi monocyte labeling). Normalized data were calculated by multiplying total frequency of the traced monocyte subset in the PBMC population by frequency of latex labeling within that subset.

Figure 4. Tracing recruitment of monocyte subsets in atherosclerotic plaques.

(A) Representative photomicrograph of a lesional section from a 7-month-old apoE^–/– mouse where latex⁺Ly-6C^hi monocyte-derived cells were observed. Red: CD68⁺ cells; blue: DAPI-stained nuclei; green: latex beads (internal elastic lamina is also green due to autofluorescence). Arrows indicate the presence of latex⁺ cells within the lesion section. lu, lumen of aorta. Original magnification, ×260. (B) Quantification of the recruitment of apoE^–/– Ly-6C^hi and Ly-6C^lo monocyte subsets into lesions 1, 3, or 5 days after subset labeling in age-matched apoE^–/– mice (7 months) maintained on a chow diet. Mean number of latex⁺ cells per lesion section is shown on the left, and normalized data that accounts for the differences in frequency of latex⁺ monocyte subsets over time are shown on the right. (C) The effect of feeding a high-fat/high-cholesterol diet on the accumulation of apoE^–/– monocyte subsets into plaques 5 days after latex labeling. Mean number of latex⁺ cells per lesion section is shown on the left, and normalized data that accounts for the differences in frequency of latex⁺ monocyte subsets in response to diet are shown on the right. For all data points, 5–8 mice were studied.

Figure 5. Phenotype of invading monocytes within atherosclerotic lesions of apoE^–/– mice.

(A and B) CD11c⁺ cells (red) in lesions (A, from Ly-6C^lo labeling; original magnification, ×260; B, from Ly-6C^hi labeling; original magnification, ×130). In areas where plaques were more advanced, CD11c⁺ cells typically surrounded and coalesced with necrotic cores that lacked intact nuclei (identified by DAPI staining in blue). Some CD11c⁺ cells were multinucleated. Cells bearing latex beads (green) are indicated by arrows. (C) The fraction of each latex⁺ monocyte subset that colocalized with CD11c in lesion sections after monocyte labeling. For each time point and condition, 3 or more mice were individually analyzed, and approximately 50 sections per animal were scored.

Figure 6. Chemokine receptor utilization for entry of monocytes into atherosclerotic lesions.

Atherosclerotic aortic arches from apoE^–/– mice were surgically transplanted into WT, CCR2^–/–, or CX3CR1^–/– mice 1 day after Ly-6C^hi or Ly-6C^lo blood monocytes had been labeled, respectively. Recruitment of latex⁺ monocytes into the aortic graft was analyzed 3 days later. (A) Ly-6C^hi and (B) Ly-6C^lo monocyte trafficking. Plots show the number of latex⁺ cells in atherosclerotic lesion sections (left; mean ± SEM shown for each animal studied) or the normalized mean values that take into account frequency of circulating latex⁺ monocytes (right; values from all animals are averaged) after Ly-6C^hi or Ly-6C^lo monocyte labeling in mice that received transplants of apoE^–/– aortic arches containing atherosclerotic lesions. (C) Immunostaining of lesion section to identify CD31⁺ endothelial cells (green) and CX3CL1 (red). Original magnification, ×130.

Figure 7. Role of CCR5 in monocyte migration into plaques.

(A) Blood monocyte subsets were stained for CCR5 expression in apoE^–/– mice. Bold line, staining with anti-CCR5 mAb; thin line, isotype control staining. (B) apoE^–/– mice were treated i.v. with latex to label Ly-6C^lo monocytes. The mean number of latex⁺ cells in lesion sections collected along the entire aortic arch of apoE^–/– mice treated with control mAb or anti-CCR5 was determined by an observer blinded to the experimental protocol. Each data point represents analysis of an individual mouse. Differences between control and anti-CCR5 were significant; P < 0.01. (C) An analysis similar to that represented in B, except apoE^–/– mice were treated so that Ly-6C^hi monocytes were latex⁺ during the assay. Differences between control and anti-CCR5 were significant; P < 0.04.