Role of the cytoskeleton in rapid activation of CD11b/CD18 function and its subsequent downregulation in neutrophils

When rolling adherent neutrophils are stimulated, they rapidly immobilise t hrough activation of integrin CD11b/CD18, and then modulate attachment thro ugh this integrin to allow migration. We investigated links between cytoske letal rearrangement and changes in function of integrin CD11b/CD18 in neutr ophils stimulated with formyl peptide (fMLP). Neutrophils treated with the actin-polymerising agent jasplakinolide became rolling adherent on monolaye rs of activated platelets, but could not use CD11b/CD18 to become immobilis ed when fMLP was perfused over them, If treated,vith jasplakinolide after f MLP, the cells stopped migrating but could not detach when fMLP was removed . Jasplakinolide did not inhibit changes in intracellular Ca2+ seen after f MLP treatment, or inhibit neutrophil immobilisation induced by externally a dded Mn2+. Thus cytoskeletal rearrangement was directly implicated in upreg ulation and, later, downregulation of CD11b/CD18 binding, Inhibition of Rho A with C3-transferase caused a dose-dependent reduction of initial rolling adhesion of neutrophils, and reduced the rate of migration after stimulatio n; however, neither the conversion of rolling to stationary adhesion, nor t he ability of neutrophils to detach on removal of the stimulus, were inhibi ted. Thus, Rho may regulate actin polymerisation and motility in neutrophil s, but did not appear to control integrin-mediated adhesion itself, Integri n binding may be promoted by disruption of links to the cytoskeleton, effec ted through depolymerisation of actin or cleavage of linking protein talin by calpain, Disruption of actin filaments with cytochalasin D did not, howe ver, cause integrin-mediated immobilisation of rolling neutrophils, Althoug h the calpain inhibitor calpeptin did inhibit the adhesion response to fMLP , this was only at doses where actin polymerisation was also ablated, We su ggest that the cytoskeleton actively regulates binding conformation of CD11 b/CD18 as well as its mobility in the membrane.