Dynamics of neutrophil aggregation in Couette flow revealed by videomicroscopy: Effect of shear rate on two-body collision efficiency and doublet lifetime

During inflammation, neutrophil capture by vascular endothelial cells is de pendent on L-selectin and beta (2)-integrin adhesion receptors. One of us ( S.I.S.) previously demonstrated that homotypic neutrophil aggregation is an alogous to this process in that it is also mediated by these receptors, thu s providing a model for studying the dynamics of neutrophil adhesion. In th e present work, we set out to confirm the hypothesis that cell-cell adhesio n via selectins serves to increase the lifetimes of neutrophil doublets for med through shear-induced two-body collisions. In turn, this would facilita te the engagement of more stable beta (2)-integrin bonds and thus increase the two-body collision efficiency (fraction of collisions resulting in the formation of nonseparating doublets). To this end, suspensions of unstimula ted neutrophils were subjected to a uniform shear field in a transparent co unter-rotating cone and plate rheoscope, and the formation of doublets and growth of aggregates recorded using high-speed videomicroscopy. The depende nce of neutrophil doublet lifetime and two-body collision-capture efficienc y on shear rate, G, from 14 to 220 s(-1) was investigated. Bond formation d uring a two-body collision was indicated by doublets rotating well past the orientation predicted for break-up of doublets of inert spheres. A strikin g dependence of doublet lifetime on shear rate was observed. At low shear ( G = 14 s(-1)), no collision capture occurred, and doublet lifetimes were no different from those of neutrophils pretreated with a blocking antibody to L-selectin, or in Ca++-depleted EDTA buffers. At G greater than or equal t o 66 s(-1), doublet lifetimes increased, with increasing G reaching values twice those for the L-selectin-blocked controls. This correlated with captu re efficiencies in excess of 20%, and, at G greater than or equal to 110 s( -1), led to the rapid formation of large aggregates, and this in the absenc e of exogenous chemotactic stimuli. Moreover, the aggregates almost complet ely broke up when the shear rate was reduced below 66 s(-1). Partial inhibi tion of aggregate formation was achieved by blocking beta (2)-integrin rece ptors with antibody. By direct observation of the shear-induced interaction s between neutrophils, these data reveal that steady application of a thres hold level of shear rate is sufficient to support homotypic neutrophil aggr egation.