Abstract
The regulatory processes involved in eosinophil trafficking into tissues are poorly understood; therefore, it is crucial to elucidate these mechanisms to advance the quality of clinical care for patients with eosinophil-mediated diseases. The complex interactions between eosinophil integrin receptors and their corresponding ligands on the post-capillary venules of the bronchial endothelium result in distinct modifications to the cytoskeletal architecture that occur in coordinated, temporally regulated sequences. The current study utilizes real-time confocal microscopy and time-based immunofluorescence staining to further characterize the effects of physiologically relevant fluid shear stress on this novel phenomenon of perfusion-induced calcium response. We found that the mere perfusion of fluid over adhered human eosinophils induced a release of intracellular calcium observed in conjunction with changes in cell morphology (flattening onto the coverslip surface, an increase in surface area, and a loss of circularity), suggesting a previously unknown mechanosensing aspect of eosinophil migration out of the vasculature. Although changes in morphology and degree of calcium release remained consistent across varying perfusion rates, the latency of the response was highly dependent on the degree of shear stresses. Eosinophils were fixed post-perfusion at specific timepoints for immunofluorescence staining to track proteins of interest over time. The distribution of proteins was diffuse throughout the cell prior to perfusion; however, they quickly localized to the periphery of the cell within 5 min. The actin cytoskeleton became markedly built up at the cell edges rapidly after stimulation, forming punctate dots by 4 min, suggesting a pivotal role in directed cell motility.