Heterogeneity of human macrophages in culture and in atherosclerotic plaques

Abstract

Research suggests that monocytes differentiate into unique lineage-determined macrophage subpopulations in response to the local cytokine environment. The present study evaluated the atherogenic potential of two divergent lineage-determined human monocyte-derived macrophage subpopulations. Monocytes were differentiated for 7 days in the presence of alternative macrophage development cytokines: granulocyte-macrophage colony-stimulating factor to produce granulocyte-macrophage-CSF macrophages (GM-Mac), or macrophage colony-stimulating factor (M-CSF) to produce M-Mac. Gene chip analyses of three monocyte donors demonstrated differential expression of inflammatory and cholesterol homeostasis genes in the macrophage subpopulations. Quantitative PCR confirmed a fivefold elevation in the expression of genes that promote reverse cholesterol transport (PPAR-gamma, LXR-alpha, and ABCG1) and macrophage emigration from lesions (CCR7) in GM-Mac compared to that in M-Mac. Immunocytochemistry confirmed enhanced expression of the proinflammatory marker CD14 in M-Mac relative to GM-Mac. M-Mac spontaneously accumulated cholesterol when incubated with unmodified low-density lipoprotein whereas GM-Mac only accumulated similar levels of cholesterol after protein kinase C activation. Immunostained human coronary arteries showed that macrophages with similar antigen expression to that of M-Mac (CD68(+)/CD14(+)) were predominant within atherosclerotic lesions whereas macrophages with antigen expression similar to GM-Mac (CD68(+)/CD14(-)) were predominant in areas devoid of disease. The identification of macrophage subpopulations with different gene expression patterns and, thus, different potentials for promoting atherosclerosis has important experimental and clinical implications and could prove to be a valuable finding in developing therapeutic interventions in diseases dependent on macrophage function.

Figure 1

Morphology of differentiated macrophages. A: Human monocytes cultured for 2 hours were differentiated for 7 days in the presence of M-CSF or GM-CSF cytokines. B: Macrophages differentiated with GM-CSF maintained a spherical shape during differentiation. C: Macrophages differentiated with M-CSF were elongated with numerous vacuoles. Similar results were obtained with more than 10 monocyte donors. Scale bar = 10 μm (applies to all).

Figure 2

Differential expression of macrophage markers. Human monocytes were differentiated for 7 days in the presence of M-CSF or GM-CSF. Then, macrophage cultures were immunostained for the macrophage marker CD68 (green) and then stained for CD14 (red). Dual labeling demonstrated the presence of CD14 (B) on M-Mac without similar staining on GM-Mac (A). Similar results were obtained with two other monocyte donors. Scale bar = 10 μm (applies also to B).

Figure 3

Differential expression of macrophage marker 25F9. Human monocytes were differentiated for 7 days in the presence of GM-CSF (A and B) or M-CSF (B and D). Macrophage cultures were then immunostained for the macrophage marker 25F9 (green). A and C show fluorescence whereas B and D show phase microscopic images of the macrophages. Similar results were obtained with another monocyte donor. Scale bar = 50 μm (applies to all).

Figure 4

Cholesterol accumulation of differentiated macrophages. Human monocytes were differentiated for 7 days in the presence of M-CSF or GM-CSF. Macrophage cultures were subsequently incubated in triplicate with 0 or 2 mg/ml LDL for 24 hours and their cholesterol and protein contents were determined. Shown is the net cholesterol accumulation of macrophages incubated with LDL compared with macrophages incubated without LDL for the indicated conditions. M-Mac spontaneously accumulated significant quantities of LDL-derived cholesterol relative to GM-Mac (P < 0.05). Activation of the subpopulations with PMA (1 μg/ml) increased cholesterol accumulation in GM-Mac without a change in M-Mac. Data are presented as the mean ± SD for three monocyte donors.

Figure 5

Analysis of macrophage phenotype in coronary artery atherosclerotic lesions. Fluorescence immunostaining of CD68 and CD14 in an atherosclerotic lesion is shown. Cells were visualized by DAPI nuclear staining (blue fluorescence) and immunolabeled for CD 68 (red fluorescence; A, C, and E) and CD14 (green fluorescence; B, D, and F). C and D: Higher magnification (top left-hand boxed region in A and B) demonstrates CD68⁺/CD14⁺ macrophages. E and F: Higher magnification (bottom right-hand boxed region in A and B) demonstrates CD68⁺/CD14⁻ macrophages. Scale bars: 10 μm (B, and also applies to A); 2.5 μm (F, and also applies to C–E).

Figure 6

Analysis of 25F9 macrophage immunolabeling in coronary artery atherosclerotic lesions. Fluorescence immunostaining of 25F9 and CD14 in an atherosclerotic lesion is shown. Cells were visualized by DAPI nuclear staining (blue fluorescence) and immunolabeled for 25F9 (red fluorescence) and CD14 (green fluorescence). Separate clusters of 25F9⁺/CD14⁻ (bottom right, arrows) and 25F9⁻/CD14⁺ macrophages (top left, arrowheads) are shown in A. Arrowheads in B indicate minor cell subpopulation showing both 25F9 (red fluorescence) and CD14 (green fluorescence) immunolabeling. Scale bars = 50 μm.

Figure 7

Co-localization of GM-Mac with lipid. Co-localization of fluorescence oil-red-O staining of lipid (A and D) and fluorescence immunostaining of CD68 (red fluorescence, B and E) and CD14 (green fluorescence, C and F) in an advanced atherosclerotic lesion is shown. Cells were visualized by DAPI nuclear staining (blue fluorescence). The boxed region is shown at higher magnification at the bottom and demonstrates lipid-containing CD68⁺/CD14⁻ macrophages (ie, GM-CSF phenotype). Scale bars: 20 μm (C; also applies to A and B); 5 μm (F; also applies to D and E).

Figure 8

Co-localization of M-Mac with lipid. Co-localization of fluorescence oil-red-O staining of lipid (A and D) and fluorescence immunostaining of CD68 (red fluorescence, B and E) and CD14 (green fluorescence, C and F) in an advanced atherosclerotic lesion is shown. Cells were visualized by DAPI nuclear staining (blue fluorescence). The boxed region is shown at higher magnification at the bottom and demonstrates lipid-containing CD68⁺/CD14⁺ macrophages (ie, M-CSF phenotype). Scale bars: 20 μm (C; also applies to A and B); 5 μm (F; also applies to D and E).