Cytochalasin D prevents PMNL adhesion to platelets, P-selectin–induced PMNL homologous aggregation, and the colocalization of Mac-1 clusters and F-actin patches in aggregated PMNLs.

(A) HE-loaded PMNLs were preincubated with increasing concentrations of cytochalasin D for 2 minutes at 37°C before the addition of PFA-fixed, BCECF-loaded activated platelets (♦). Mixed-cell suspensions were coincubated in standard conditions, the interaction was stopped at 2 minutes, and samples were processed for FACS analysis. Alternatively, cytochalasin D–pretreated PMNLs were challenged with P-selectin–IgG chimera (10 μg/mL) (■). The reaction was stopped at 2 minutes, and P-selectin–induced PMNL homologous aggregation was evaluated by counting nonaggregated PMNL by optical microscopy. The results are from a representative of 2 different experiments. (B) PMNLs were preincubated for 2 minutes at 37°C with ethanol (i-iii) or 20 μM cytochalasin D (iv-vi), before the addition of P-selectin–IgG chimera (10 μg/mL). Incubation in standard conditions was stopped at 2 minutes, and samples were processed for confocal laser scanning microscopy. Panels i, ii, and iii show an aggregate of 4 cells. Panels i and iv show F-actin staining, and ii and v show CD11b staining. In the overlay (iii, vi), yellow represents colocalization of the 2 stainings. The figure is representative of results obtained in 5 different experiments.