E-selectin Mediated Calcium Response Of Neutrophils Under Flow Shear Stresses

During the inflammation response,leukocytes in the blood vessels are captured by endothelial cells.Then firmly adhered leukocyte will be activated,polarized deformation,and finally migrate to the inflammation site.Among them,the activation process of leukocytes will be accompanied by increased cytosolic calcium concentration,the study of this signal event has always been a hot topic.Therefore,we start to study leukocyte calcium signaling.In order to simulate the blood flow environment under physiological conditions,we used a parallel plate flow chamber combined with fluorescence microscopy to record real-time observation of E-selectin-mediated neutrophil stabilized calcium response in fluid environment with fluorescent protein Fluo 4-AM,so as to have a deep understanding of leukocytes activation in physiological conditions.The loaded Fluo 4-AM neutrophil fluid were perfused at various fluid shear stress(0 ~ 0.6 Pa)on 10 ?g/m L E-selectin substrates,and we observed the neutrophil adhesion in real time by the flow chamber system.We found that E-selectin could capture neutrophils from flow to the chamber substrate and induce further intracellular calcium flux of firmly adhered cells.The number of arrest cell and activation ratio increased gradually with the concentration increasing of E-selctin.Only immobilized E-selectin can conduct the external force-signal to effectively trigger the calcium response of neutrophils.By shear stress increasing,not only was the activation ratio raised from 23% to 70%,but also the calcium response peak intensity increased from 0.92 to 1.45,delay time between cell arrest and calcium response beginning greatly reduced from 70 s to 27 s.Shear force and E-selectin cooperated to modulate the calcium response of neutrophils,and the former positively regulated the activation rate and level of calcium response.Then We blocked the partial ligand of neutrophils with antibodies to discuss the effect of E-selectin with PSGL-1 alone on neutrophils calcium response in flow.By shear stress increasing from 0.02 Pa to 0.6 Pa,the activation ratio raised from 28% to 73%,the calcium response peak intensity increased from 0.69 to 1.43,delay time greatly reduced from 91 s to 28 s.The results of treated experiment showed that the activation ratio and calcium response peak intensity was similar to the untreated,but the delay time was different.In the low fluid shear stress(less than 0.06 Pa),blocking CD44 or blocking CD44 and L-selectin at the same time would affect the delay time.With the fluid shear stress increases,this effect gradually weakened.When the shear stress increased to 0.2 Pa or more,antibodies on E-selectin-mediated neutrophil calcium response had no effect.This research can deepen the understanding of leukocyte inflammation response in the blood environment.