Direct contact with the endothelium is required for the formation and maintenance of macrophage colonies. (A) Schematic representation of the colonies as they take residence under the endothelial monolayer. (B) Confocal 3D reconstruction of the DsRed colonies (arrows) growing underneath the IMEC layer. Composite x-y sections and a single z-section are shown. Scale bar, 50 μm. (C) Ultrastructural analysis of cells cocultured for 28 days by transmission electron microscopy. (Bottom) Magnified micrograph reveals direct contact (arrows) between the 2 cell types. EC indicates endothelial cell; and Mφ, macrophage-like cell. Scale bar, 4 μm. (D) Colonies disaggregate (arrow) after the removal of IMEC layer for 1 day, depicted by phase contrast and fluorescent micrographs (left). Colonies with IMECs were maintained (right). Scale bar, 200 μm. (E) Colonies failed to form via transwells. (Left) Cocultures of HCs and IMECs separated by the transwell (0.4-μm pore size). (Middle) Cocultures through direct contact. Arrows indicate colonies. (Right) Quantification of colony number at day 10 of coculture (n = 3). Scale bar, 200 μm. (F) Real time PCR quantification of ICAM1, VCAM1, and VE-Cadherin in colonies. Ctrl indicates bone marrow–derived macrophages with 5 ng/mL IL-4 stimulated for 24 hours (n = 6). (G) Corresponding cell adhesion molecules (CAMs) expression in IMECs. Ctrl indicates IMECs in the absence of coculture (n = 6; *P < .05; **P < .01; ***P < .001; unpaired Student t test).