Figure 4.

PMN recruitment by adherent platelets under flow requires P-selectin and αMβ2. (A) Human PMNs (5 × 10⁶/mL) were perfused across a platelet surface at a flow rate of 2 dynes/cm² for 2 minutes. Then, the chamber was perfused with fresh media for an additional 2 minutes at 5 dynes/cm², without interruption in the perfusion. The interaction of PMNs with adherent platelets was observed by contrast phase video microscopy with a 20×/0.40 NA objective. The total number of cells interacting at 2 minutes and the fractions of rolling and firmly adhered PMNs in the last 20 seconds of flow (4 minutes) were measured using an ad hoc software for image analysis as described in “Materials and methods.” (B) P-selectin and αIIbβ3 and αvβ3 were blocked by incubating the human platelet–coated slide with the mAbs WAPS12.2 or 7E3, respectively (20 μg/mL) for 15 minutes. β2 or α4/1 integrins on human PMNs were blocked by treating cells with the mAbs IB4 or 2B4 (20 μg/mL) for 15 minutes in ice. Experiments were performed as described for panel A and in “Materials and methods.” The results are presented as means ± SEM for 3 to 8 different experiments performed using cells from different donors. Black bars indicate the total number of PMNs recruited per field, and white bars indicate the number of PMNs that establish firm adhesion. *P < .05 for treatment versus control. (C) PMNs were isolated from wild-type or αM^−/− mice bone marrow. Platelets were isolated from wild-type or P-selectin–deficient (P-sel^−/−) mice. Experiments were performed as described for panel A and in “Materials and methods.” The results presented are means ± SEM from 4 different experiments each performed using cells collected from 5 wild-type or knock-out mice. *P < .05 for wild-type versus αM^−/− PMNs or P-selectin^−/− platelets.