Entrapment of circulating apoptotic cells by MZ DCs.

Uptake of apoptotic cells by DCs was studied by 2-photon confocal microscopy on spleen blocks (A-C) and by immunofluorescence on spleen sections (D-F), 6 hours (A,D), 24 hours (B,E), and 48 hours (C,F) after intravenous injection of PKH67-labeled (green) BALB/c apoptotic splenocytes in B10 mice. Early entrapment of apoptotic cells (green) by MZ DCs (CD11c⁺ in red) at the periphery of splenic follicles 6 hours (A,D) and 24 hours (B,E) after intravenous injection is evident. Staining with Cy3–anti-CD11c shows DCs with internalized apoptotic cell fragments (DCs in red, apoptotic cell fragments in green, panels D-F and insets). After 48 hours, DCs with green apoptotic cells mobilized to the center of the follicle (C,F) close to the central arteriole of the T-cell area (arrow in panel F). MOMA-1⁺ metallophillic macrophages (G), ER-TR9⁺MZ macrophages (H), and F4/80⁺ macrophages of the red pulp also internalized apoptotic cells (in green; detail of cytospins in insets in panels G-I). Arrows in panels D to F show apoptotic cells in green. Bars in panels G to I indicate the approximate width of the MZ. Four animals were analyzed per time point. Yellow image denotes overlapping red and green images. Nuclei were counterstained with DAPI. Original magnification, × 200 for panels A to F; × 400 for panels G to I; all insets, × 1000.