Figure 5

Platelets induce differentiation of monocytes into CD163⁺ macrophages and foam cells through CXCR4–CXCR7. (a) Brightfield phase-contrast images from platelet-monocyte co-culture system showing the differentiation of monocytes into macrophages in the presence/absence of neutralizing antibody against platelet-derived-CXCL12 (10 μg/ml) or blocking antibody against CXCR4–CXCR7 (10 μg/ml) with respect to control IgG. Bar=5 μm. Right, Bar diagram representing the number of macrophages (mØ) differentiated from a co-culture of platelets–monocytes. The presence of a neutralizing antibody against CXCL12 (***P<0.001 as compared with IgG) in the culture system or blocking antibody against CXCR4–CXCR7 significantly (***P<0.001 as compared with IgG) reduced the number of differentiated macrophages. (b) (i) Data represent phenotypic characterization of platelet-monocyte co-culture-derived macrophages by flow cytometry. Flow-cytometric histogram overlay for the surface expression of CD86 and CD163 (in red and blue lines as indicated) as overlayed and compared with respective isotype controls (gray fill) showing prominent surface expression of CD163 but not of CD86 in platelet–monocyte co-culture-derived macrophages as compared with culture-derived M2 macrophages (cultured in the presence of M-CSF followed by IL4) which were positive for CD163 but not for CD86 and culture-derived M1 macrophages (cultured in the presence of GM-CSF followed by IFN-γ and LPS) which were positive for CD86 but not for CD163. (ii) Bar diagram representing phenotypic characterization of platelet–monocyte culture-derived macrophages in terms of CD86, CD163 surface expression which shows a predominantly (***P<0.001 as compared with CD86) CD163 phenotype. Below, Bar diagram representing a trend of decrease in the relative % of CD163 macrophages generated in the platelet–monocyte co-culture in presence of a neutralizing antibody against CXCL12. P=0.17 as compared with IgG control. Data are mean±S.E.M. from five independent platelet–monocyte co-culture experiments. (c) (i) Density plots of cells derived from monocyte–platelet co-culture showing the relative abundance of CD163⁺, CD86⁺, CD209⁺, CD68⁺, CD36⁺, CD14⁺, CD11b⁺, CD11c⁺ cells. (ii) Bar diagram showing the relative percentage of CD163⁺, CD86⁺, CD209⁺, CD68⁺, CD36⁺, CD14⁺, CD11b⁺, CD11c⁺ cells from platelet– monocyte co-culture. Data are mean±S.E.M. from four independent platelet-monocyte co-culture sets. (d) (i) Representative images for Oil-red staining of foam cells formed in the co-culture of platelets and monocytes. Bar=5 μm. (ii) Bar diagram representing the relative percentage of foam cells generated in platelet–monocyte co-culture, which was significantly (**P<0001 as compared with IgG control) reduced in the presence of blocking antibody against CXCR4–CXCR7 and neutralizing anti-CXCL12 antibody (***P<0001 as compared with IgG control) in culture. Data are mean±S.E.M. from three independent platelet–monocyte co-culture systems