Treatment of neutrophils with cytochalasins converts rolling to stationary adhesion on P-selectin

Abstract

We investigated how disruption of the actin cytoskeleton with cytochalasins modified adhesion of neutrophils rolling on a platelet monolayer in vitro at 37 degrees C. When perfused at a wall shear stress of 0.1 Pa over rolling cells, cytochalasin B, cytochalasin D and dihydro-cytochalasin B each induced dose-dependent (approximately 1-10 microg/ml) conversion to stationary attachment over minutes. Stopping was associated with cell elongation to a teardrop shape. Increased deformability of cytochalasin-treated cells was independently evidenced by more rapid entry into a micropipette. Spherical shape and rolling were reestablished concurrently on washout of the cytochalasins, while increasing the shear stress in the range 0.2 to 1.0 Pa induced tear-drop-shaped cells to restart rolling even in the continued presence of cytochalasin. When cells were pretreated with cytochalasin B, they attached efficiently at 0.1 Pa, rolled initially and only stopped after approximately 30 seconds when elongation had been established. Adhesion was selectin-mediated in the presence or absence of cytochalasin B, as judged by inhibition of attachment by antibody against P-selectin and failure of antibody against beta2-integrin CD18 to influence adhesion. Cessation of rolling is unlikely to have arisen from an increase in adhesive contact area induced by deformation because stopped cells were found to be attached only at their pointed end. Failure of adhesive bonds to peel may have arisen because selectin ligands freed of cytoskeletal restraint were dragged into this tethered region and clustered there, and because force applied to bonds was influenced by the change in cell shape. These results suggest that cytoskeletal structure is an important modulator of dynamic adhesive responses of leukocytes, via effects on adhesion receptors and cellular mechanics.

Affinity, lateral mobility, and clustering contribute independently to β2-integrin-mediated adhesion

“…A more deformable cell will tend to elongate more, potentially increasing the area of contact and, thus, the number of adhesion molecules available to form bonds. A standard method of quantifying this deformability is by calculating a deformation index, which is the ratio of cell length to cell width (7,25,29).…”

“…Thus cytochalasin D-induced shape changes could have consequences for cell-substrate contact area. This effect is often quantified by calculating a deformation index, which is the ratio of cell length to cell width (7,25,29). Therefore, deformability indexes were calculated for rolling cells as a function of cytochalasin D concentration for both unfixed and fixed cells.…”

Clostridium difficileToxin A Alters In Vitro–Adherent Neutrophil Morphology and Function

“…Cytochalasin B counteracts F-actin polymerization, resulting in less F-actin, whereas toxin A, by disrupting Rho proteins, prevents the normal organization of polymerized F-actin in stimulated cells [39,41]. It has been shown that cytochalasin-treated neutrophils exhibit a teardrop shape, which is not mediated by b 2 integrins [42]. Unlike toxin A, TNF-a is able to activate Rho [43,44], an event that may lead not only to the organization of the actin cytoskeleton but also to the early expression of integrins.…”