Dynamics of Neutrophil Aggregation in Couette Flow Revealed by Videomicroscopy: Effect of Shear Rate on Two-Body Collision Efficiency and Doublet Lifetime

Abstract

During inflammation, neutrophil capture by vascular endothelial cells is dependent on L-selectin and beta(2)-integrin adhesion receptors. One of us (S.I.S.) previously demonstrated that homotypic neutrophil aggregation is analogous to this process in that it is also mediated by these receptors, thus providing a model for studying the dynamics of neutrophil adhesion. In the present work, we set out to confirm the hypothesis that cell-cell adhesion via selectins serves to increase the lifetimes of neutrophil doublets formed through shear-induced two-body collisions. In turn, this would facilitate 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 unstimulated neutrophils were subjected to a uniform shear field in a transparent counter-rotating cone and plate rheoscope, and the formation of doublets and growth of aggregates recorded using high-speed videomicroscopy. The dependence of neutrophil doublet lifetime and two-body collision-capture efficiency on shear rate, G, from 14 to 220 s(-1) was investigated. Bond formation during a two-body collision was indicated by doublets rotating well past the orientation predicted for break-up of doublets of inert spheres. A striking 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 > or = 66 s(-1), doublet lifetimes increased, with increasing G reaching values twice those for the L-selectin-blocked controls. This correlated with capture efficiencies in excess of 20%, and, at G > or = 110 s(-1), led to the rapid formation of large aggregates, and this in the absence of exogenous chemotactic stimuli. Moreover, the aggregates almost completely broke up when the shear rate was reduced below 66 s(-1). Partial inhibition of aggregate formation was achieved by blocking beta(2)-integrin receptors with antibody. By direct observation of the shear-induced interactions between neutrophils, these data reveal that steady application of a threshold level of shear rate is sufficient to support homotypic neutrophil aggregation.

Hydrodynamic Shear Rate Regulates Melanoma-Leukocyte Aggregation, Melanoma Adhesion to the Endothelium, and Subsequent Extravasation

“…Previous studies have reported similar shear-rate dependent bond formation. For example, Goldsmith et al 13 showed shear-rate dependent PMN homotypic aggregations in a Couette flow; Chen and Springer 6 reported individual PMNs expressing P-selectin glycoprotein ligand 1 (PSGL-1) rolled along and tethered to the P-selectin-immobilized substrate was shear rate dependent. Our results here show PMN-WM9 aggregation facilitates melanoma cell adherence to the EC, which is predominantly regulated by the shear rate.…”

Similarities between heterophilic and homophilic cadherin adhesion

“…At 1-to 3-rpm shaking, E-CAD and P-CAD cells formed mixed aggregates, whereas only uniform aggregates formed at the typical higher mixing speeds of 75-100 rpm (19). These cell aggregation assays use agitated (sheared) cell suspensions, and it is well known that particle (e.g., cell) coagulation rates under shear depend on the bond kinetics and the shear rate (30)(31)(32)(33).…”

Shear modulation of intercellular contact area between two deformable cells colliding under flow

“…Prior work has demonstrated that steady application of a threshold level of shear rate is necessary to support PMN homotypic aggregation in bulk suspensions (Goldsmith, et al, 2001). The presence of the shear threshold phenomenon, by which a reduction of shear rate below a threshold value diminishes the probability of cell adhesion, was also detected during the interaction of free-flowing and surface-adherent PMNs (Kadash, et al, 2004).…”

“…Given the evidence suggesting that cellular deformation during shear-induced collisions affects the intercellular contact area, and thus, the probability of receptor-ligand bond formation between the interacting cells (Goldsmith, et al, 2001;Kadash, et al, 2004), our attention focuses on the development of mathematical models that incorporate cellular deformation.…”