Modulation of uPAR on the surface of macrophages regulates the phagocytosis of viable neutrophils in vitro and in vivo, and is dependent on macrophage-associated integrins. (A) Blockade of uPAR enhances the ability of WT macrophages to phagocytose viable WT neutrophils. WT macrophages were preincubated without (con) or with 1 μg/mL anti-uPAR antibodies or rabbit IgG for 30 minutes. The medium was then changed to RPMI plus 5% FBS, and viable WT neutrophils were added for 60 minutes and the phagocytic index determined. ***P < .001 compared with the control group. (B) suPAR dose-dependently decreases the ability of uPAR^−/− macrophages to phagocytose viable WT neutrophils. uPAR^−/− macrophages were preincubated without (con) or with suPAR at the indicated concentrations for 30 minutes. The medium was then changed to RPMI plus 5% FBS, and viable WT neutrophils were added for 60 minutes and the phagocytic index determined. **P < .01, ***P < .001 compared with the control group. (C) suPAR binds to the surface of uPAR^−/− and WT macrophages. The cells were incubated with Chromeo 488–labeled mouse suPAR (1 μg/mL) or mouse albumin for 30 minutes and then washed with PBS 5 times. Fluorescent intensity was determined by flow cytometry. (D) uPAR^−/− alveolar macrophages demonstrate enhanced ability to phagocytose viable WT neutrophils in vivo. A total of 10 × 10⁶ viable WT neutrophils were injected intratracheally into WT or uPAR^−/− mice (n = 5 in each group). After 90 minutes, the mice were killed and bronchoalveolar lavage performed with 3 mL PBS. Cytospin slides were prepared using 250 μL bronchoalveolar lavage fluids. Phagocytic indices were determined as described in “In vivo efferocytosis assay.” (E) Exposure to suPAR in vivo abrogates the enhanced ability of uPAR^−/− alveolar macrophages to phagocytose viable WT neutrophils in vivo. A total of 10 μg suPAR or mouse albumin was intratracheally instilled 1 hour before intratracheal administration of 10 × 10⁶ viable WT neutrophils into uPAR^−/− mice (n = 5 in each group). Bronchoalveolar lavage was performed 90 minutes after injection of neutrophils and phagocytic indices determined. n = 5 in each group. **P < .01. ***P < .001. (F) RGD-, but not RAD-containing, peptides abrogate the enhanced activity of uPAR^−/− macrophages to phagocytose viable WT neutrophils. uPAR^−/− macrophages were preincubated with 0.1 or 1 μg/mL RGD or RAD peptides for 30 minutes. The medium was then changed to RPMI plus 5% FBS, and viable WT neutrophils were added for 60 minutes, after which the phagocytic index was determined. **P < .01. ***P < .001. (G) Integrins participate in the enhanced ability of uPAR^−/− macrophages to phagocytose viable WT neutrophils. uPAR^−/− macrophages were preincubated with 1 μg/mL mouse IgG, or antibodies to the integrins α_M, α_V, β₁, β₂, β₃, or β₄ for 30 minutes. The medium was then changed to RPMI plus 5% FBS. and viable WT neutrophils were added for 60 minutes, after which the phagocytic index was determined. *P < .05, **P < .01, compared with the control group treated with IgG.