uPAR^−/− neutrophils demonstrate enhanced adhesion to uPAR. (A) Adhesion of uPAR^−/− neutrophils to uPAR is increased. A total of 10⁶ viable WT and uPAR^−/− neutrophils were added into each well of a 96-well plate precoated with 1 μg/mL BSA or suPAR for 1 hour, and adhesion assays performed as described in “Adhesion assay.” *P < .05. (B) Adhesion of WT neutrophils to uPAR^−/− macrophages is increased. A total of 10⁶ viable WT neutrophils were added into each well of a 96-well plate plated with WT and uPAR^−/− macrophages, and adhesion assays performed as described in “Adhesion assay.” (C) RGD, but not RAD, peptides, abrogate the enhanced adhesion of uPAR^−/− neutrophils to suPAR. uPAR^−/− neutrophils were preincubated with 1 μg/mL BSA (con) or 1 μg/mL RGD or RAD peptides for 30 minutes and adhesion assays performed as in panel A. **P < .01. (D) Antibodies to integrins diminish the enhanced adhesion of viable uPAR^−/− neutrophils to suPAR. uPAR^−/− neutrophils 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 cells were then resuspended in RPMI 1640 medium and added to suPAR-coated plates for adhesion assays. **P < .01, ***P < .001, compared with the control group treated with control IgG. (E) suPAR and integins are colocalized on the surface of uPAR^−/− macrophages. uPAR^−/− macrophages were incubated with Chromeo 488–labeled mouse suPAR (1 μg/mL) for 30 minutes. The cells were fixed and then stained overnight without or with antibodies to the integrins α_V or β₃. Representative confocal images are shown. (F-K) Anti-integrin antibodies prevent the binding of suPAR to the surface of uPAR^−/− macrophages. Cells were preincubated without (F,G) or with antibodies (1 μg/mL) to the integrins α_V (H), β₃ (I), β₄ (J), or isotype IgG control (K) for 30 minutes. The cells were then incubated with Chromeo 488–labeled mouse suPAR (1 μg/mL) for 1 hour (G-K) and washed 5 times with PBS. Fluorescent intensity was determined by flow cytometry. (L) uPAR^−/− macrophages demonstrate greater Erk phosphorylation after exposure to viable WT neutrophils than do WT macrophages. A total of 4 × 10⁶ viable WT neutrophils were added for the indicated time into each well of a 12-well plate plated with WT or uPAR^−/− macrophages. The cells were then collected and the levels of phosphorylated Erk and total Erk determined by Western blot analysis.