Neutrophil adhesion dynamics during flow

Neutrophils have been observed to adhere activated endothelium, platelets and neutrophils in vivo. We investigated the dynamics of neutrophil adhesion through assays that surveyed of interactions between neutrophils and adherent neutrophils or surface-presented ligands. We designed experiments that allowed combined the advantages of aggregometry (discrete events), rolling assays (physiological shear environment), micropipette aspiration (tether visualization) and atomic force microscopy (small contact zone). During observation of interactions between flowing and adherent neutrophils, we observed hydrodynamic thresholding in primary collision efficiency (ϵ) and neutrophil string formation, with a maximum in adhesion in both cases at a wall shear rate of γ w = 100 sec−1. High resolution imaging revealed deformation of the neutrophil at venous shear rates. Strings of neutrophils formed on ICAM-1, but not fibrinogen-coated surfaces, and analysis of collision lifetimes between neutrophils and the β2 integrin ligands revealed that poor capture to fibrinogen was the root cause of poor string formation on this substrate. We observed that both static adhesion and the subsequent onset of flow caused calcium mobilization in the neutrophil. When neutrophils were perfused over P-selectin presenting beads from γw = 25 to 200 sec−1, ϵ, was observed to decrease, while the membrane tethering fraction (f) and the average membrane tether length, Lmtether, increased. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters were: zero-stress off-rate, koff(0) = 0.56 sec −1 and reactive compliance, r = 0.10 nm, similar to published values for AFM experiments. For all adhesion events, with and without membrane tethers, the bond-lifetime distributions were more similar to those obtained in neutrophil rolling assays, and best simulated by Monte Carlo with the above Bell parameters and an average of 1.36 bonds. Pretreatment of neutrophils with actin depolymerizing agents had no effect on ϵ, but increased L mtether, and prolonged the average tether lifetime. Conversely, cholesterol-depletion with methyl-β-cyclodextrin or formaldehyde fixation had no effect on ϵ, but reduced Lmtether and average tether lifetime. These several results demonstrate a role for cellular processes in mediating neutrophil adhesion dynamics under flow, and provide useful assays for the development of diagnostic tools and therapeutics targeting cross-talk between inflammatory and coagulation pathways