Neutrophil (PMN) spreading on endothelium, mediated by the interactions between surface-bound β2 integrin and intercellular adhesion molecule-1 (ICAM-1) in the inflammatory cascade, is crucial for PMN post-adhesion and trans-migration in blood flow. The underlying mechanisms by which shear flow regulates PMN spreading dynamics are not well understood. Here, a parallel-plate flow chamber assay was applied to quantify the time course of PMN adhesion and spreading on an ICAM-1-immobilized substrate. Two types of shear flow, steady flows at shear stresses of 0.2, 0.5, and 1 dyne/cm2 and stepwise flows at 0, 1, and 10 dyne/cm2, were used to elucidate the impact of shear flow on cell adhesion and spreading. The number of adhered PMNs, the fraction of spreading PMNs and the projected area of spread PMNs were determined and were found to correlate with the distribution of surface-bound β2 integrin subunit (CD11a, CD11b, or CD18). The results indicate that PMN spreading on an ICAM-1 substrate is bi-directionally regulated under shear flow. CD11a, CD11b and CD18 subunits of β2 integrin contribute distinctly to PMN spreading on ICAM-1 substrates. This work provides new insights into understanding PMN spreading on the endothelium, mediated by β2 integrin and ICAM-1 under shear flow.
Receptor-ligand bond dissociation under applied force is crucial to elucidate its biological functionality when the molecular bond is usually connected to a mechanical probe. While the impact of probe stiffness, k, on bond rupture force has recently attracted more and more attention, the mechanism of how it affects the bond lifetime, however, remains unclear. Here we quantified the dissociation lifetime of selectin-ligand bond using an optical trap assay with low stiffness ranging from 3.5×10-3 to 4.7×10-2 pN/nm. Our results indicated that bond lifetime yielded distinct distributions with different probe stiffness, implying the stochastic feature of bond dissociation. It was also found that the mean lifetime varied with probe stiffness and that the catch bond nature was visualized at k■3.0×10-2 pN/nm. This work furthered the understanding of the forced dissociation of selectin-ligand bond at varied probe stiffness, which is physiologically relevant to the tethered rolling of leukocytes under blood flow.
