Integrin αMβ2 interacts with a TSP-4 fragment composed of 3 type 2 (EGF-like) domains. (A) The 96-well plates were coated with the TSP-4 variant fragments or control GST (0-160 nM) for 16 hours at 4 °C, and resting or PMA-stimulated PMNs were allowed to adhere for 30 minutes at 37°C. (B) PMA-stimulated PMNs were pretreated with function-blocking mAbs to αMβ2, control mAb to MHC-1 (20 μg/mL), or NIF (10 nM) for 20 minutes at RT, and adhesion was assessed as described in “Materials and methods.” The data are means ± SEMs of quadruplets from 2 blood donors, and background adhesion to GST has been subtracted. (C) The 96-well plates were coated with recombinant-purified αMI-domain (50 μg/mL) for 20 hours at 4 °C and coated afterward with 3% BSA. 3EGF–TSP-4–GST fusion proteins (0-1.5 μM) were added and incubated for 4 hours at 37°C. After washings, the bound proteins were detected with anti-GST mAb by ELISA as described in “Materials and methods.” The results are means ± SEMs (n = 4). (D) Resting or PMA-stimulated (1 nM) PMNs were allowed to adhere to 3EGF–TSP-4–coated plates or control GST for 4 hours at 37°C. Hydrogen peroxide levels were measured as described in Figure 4. The data are means ± SEMs of quadruplet measurements from 3 blood donors. (Panels E and F) PMA-stimulated PMNs (1 nM) were allowed to adhere to the 3EGF–TSP-4 variants (80 nM) and control PVP for up to 30 minutes at 37°C. (E) Cells adherent for 30 minutes were stained with Ab to phosphorylated p38 MAPK and FITC-conjugated secondary Ab. Bar size, 10 μM. (F) Alternatively, equal amounts of total protein in lysates of adherent cells were analyzed by Western blot with Abs to phosphor-p38 MAPK and to p38 MAPK. Images were captured with a 40 ×/0.7 NA objective lens. Results are representative of experiments performed on PMNs isolated from 2 blood donors.
