Figure 2.

Human macrophages sense pneumolysin (PLY)-bearing liposomes. (A) PLY does not bind to macrophages in the presence of cholesterol:sphingomyelin liposomes. Day 7 M-CSF differentiated macrophages, either pre-incubated with liposomes (Lipo) or left untreated, were stimulated with 4 µg/ml EGFP-PLY and serial confocal imaging was performed. Images from T = 2 min post PLY additions are shown. The toxin binds exclusively to untreated macrophages. (B) Uptake of sequestered PLY on liposomes by human macrophages in vitro (arrow). Human primary macrophages were stimulated with EGFP-PLY-liposomes and analyzed at T = 4 h by confocal microscopy for intracellular EGFP-PLY (bar 10 µm). (C) Stimulation of macrophages with PLY-Lipo induces NfkB translocation from the cytoplasm into the nucleus. Day 7 M-CSF differentiated macrophages were stimulated with PLY-Lipo for 4 h. Afterward, cells were fixed, permeabilized, and stained with a monoclonal antibody against NfkB (red) and Hoechst (chromatin, blue).