Figure 8

Entry of mammary and urinary epithelial cells by neutrophils. Isolated fresh bovine blood neutrophils were placed on a monolayer of murine mammary EPH-4 cells (A, B) or human urinary epithelial cell line 5637 (C, D) grown on glass cover slides in 24 wells culture plate. After 12 h cells were fixed with PFA and stained with DAPI (blue in A, B) and phalloidin-TRITC (red in A, B) and or prepared for TEM (C, D). Representative images of confocal laser microscopy (A, B) and TEM (C, D). The xy image is on the plan indicated by the horizontal and vertical dashed lines shown in the xz and yz images, respectively (A, B). Scale bars 7 µm (A, B), 10 µm (C) and 5000 nm (D). Entry preceded by elongation and crawling of live neutrophils along the intercellular borders of the polar epithelial cell monolayer (white arrows in A, C, D). The entry process is associated with the formation of an actin-rich tunnel (white arrow in B) and required extensive elongation and rearrangement of the nucleus to accommodate this entrance route. Intraepithelial live (yellow arrows in B) and apoptotic (yellow arrows in C, D) neutrophils are also visible. All images are representative of > 3 similar experiments.