Recognition of uPA by neutrophils is αMβ2 integrin- and uPAR-dependent.
(A) Migration to uPA. Human polymorphonuclear cells (PMNs) were resuspended in a serum-free DMEM/F-12 medium containing PMA (20 nM) and added (2 × 105 cells/well) to the upper chambers of transwells. HMW-tc-uPA was added to the lower chambers, and selected function-blocking mAbs (20 μg/mL) were added to both chambers. PMNs were allowed to migrate to the lower chamber for 6 hours in a humidified incubator at 37°C and 5% CO2. The number of migrated cells was determined as described in “Materials and methods.” The data are expressed as means ± SEM of duplicate wells from 3 independent experiments. (B) Adhesion to uPA. PMA-stimulated PMNs (1 × 105 cells/well) were seeded onto wells of microtiter plates coated with HMW-tc-uPA or BSA and allowed to adhere for 30 minutes at 37°C. Nonadherent PMNs were removed by 3 washings with PBS, and representative photomicrographs of the wells were taken (Original magnification, × 200). (C) Direct binding of uPA. Unstimulated or PMA-stimulated PMNs (20 nM, 20 minutes at 37°C) were incubated in DMEM/F-12 medium/0.1% BSA with 100 nM Alexa 488–HMW-tc-uPA in the absence or presence of the indicated blocking mAbs for 30 minutes at 37°C. Cells were centrifuged through a cushion of FCS twice and resuspended in 1% paraformaldehyde/PBS. Cell-bound HMW-tc-uPA was detected by FACS analysis.
