uPAR^−/− macrophages demonstrate enhanced phagocytosis of viable neutrophils. (A) The phagocytosis of viable WT neutrophils by uPAR^−/− macrophages is significantly increased compared with phagocytic indices when WT neutrophils are incubated with WT macrophages, and is similar to that present when WT macrophages are exposed to apoptotic WT neutrophils. A total of 10⁶ viable or apoptotic WT neutrophils were added into each well of a 96-well plate containing adherent WT or uPAR^−/− macrophage monolayers, and efferocytosis assays performed as described in “In vitro efferocytosis assay.” Phagocytic indices are expressed as the percentage of macrophages containing at least 1 ingested neutrophil. *P < .05, **P < .01, compared with “WT macrophages + viable WT neutrophils.” (B) uPAR is expressed on the surface of WT, but not uPAR^−/− macrophages. WT and uPAR^−/− macrophages were incubated with anti-uPAR antibodies for 1 hour. The cells were then incubated with FITC-conjugated secondary antibodies and flow cytometry performed. (C) uPAR^−/− macrophages actively phagocytose viable WT neutrophils. Viable or apoptotic WT neutrophils were added to WT or uPAR^−/− macrophages. After 60 minutes, cytospin slides were prepared for Wright-Giemsa staining. Slides were viewed with a Labor LUX 12 microscope (Leitz) using a Phaco 2 lens at 40×/0.65. Images were acquired using a Sony cybershot camera model DSC-H2 and were processed with Adobe Photoshop version 7.0 software (Adobe Systems). As shown in the representative figures, the WT viable neutrophils ingested by uPAR^−/− macrophages showed normal nuclear and chromatin patterns, consistent with being viable and not apoptotic.