Spatiotemporal Characteristics Of P-selectin-induced β₂ Integrin Activation Of Human Neutrophils Under Flow

As the most abundant circulating blood cells in the human body,human neutrophils constitute the first line of defense of human immunity,and participate in a series of physiological and pathological processes such as inflammation,thrombosis,and tumor metastasis.In the recruitment of flowing leukocytes to inflammatory or injured sites of vascular surface,there is a multistep cascade consisting of cell tethering,rolling,firm adhesion,crawling,and transmigration.As the dominant mediators for these events,P-selectin mediates cell tethering to and rolling on activated endothelium or platelet surfaces,and β2 integrin is responsible for slow rolling and firm adhesion.Current studies suggest that P-selectin mediates the tethering and rolling of human neutrophils,and initiates the activation of β2 integrin through the mitogen-activated protein kinases(MAPK)signaling pathway.Activated β2 integrins then regulate the subsequent slow rolling and stable adhesion.Therefore,activation of β2 integrin is a key but early event in leukocyte recruitment.Excessive or insufficient activation of β2 integrin on the surface of neutrophils will correspondingly cause an over-inflammatory response of neutrophils or result in a lack of immune function of neutrophils.The spatiotemporal characteristics of β2 integrin activation of flowing neutrophils should be assignable in β2 integrin-involved cell-cell and cell-extracellular matrix(ECM)interactions of inflammation,thrombosis,and cancer metastasis.Therefore,this research aims to answer the spatiotemporal characteristics of β2 integrin activation of flowing neutrophils.In this thesis,we used a fluorescence detection technology combined with a parallel plate flow chamber(PPFC)to detect stable adhesion,calcium response,cell tethering,and β2 integrin activation signaling of human neutrophils on functionalized substrates,showing that β2 integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase(MAPK)or non-MAPK signaling with a minute-level timescale in a force-dependent manner.In flow,MAPK signaling required intracellular Ca2+release to activate integrin within two minutes.Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1(PSGL-1)within sub-seconds,and then over the entire cell surface within one minute in an extracellular Ca2+influx-dependent manner.The transition from a local(but rapid)to global(but slow)activation mode was triggered by ligating the freshly activated integrin.Lipid rafts,moesin,actin,and talin were involved in non-MAPK signaling.Fluid loads had a slight effect on local integrin activation with a second-level timescale,but served as enhancers of global integrin activation.At the same time,in the micromechanical environment,using the β2 integrin specific structural recognition antibody and parallel-plate flow chamber,we found that after P-selectin mediated activation of β2 integrin,most of the β2 integrins transformed into the moderateaffinity extended form of β2 subunits,and a small number of β2 integrin with high-affinity transformations These findings help us to understand the force-regulated activation of β2 integrins.In addition,concerning the kinetic characteristics of β2 integrin(LFA-1,Mac-1)and its major ligand ICAM-1 on endothelial cells,and the kinetic characteristics of Mac-1 and its major ligand GPIbα on platelets,the kinetic characteristics of LFA-1/ICAM-1,and Mac-1/ICAM-1 have been reported,but the kinetic characteristics of Mac-1/GPIbα are lacking in relevant experimental studies.Therefore,we used atomic force microscopy(AFM)technology to describe the dynamic characteristics of Mac-1/GPIbα.We found that the kinetics of Mac1/GPIbα exhibited an inverse catch-slip bond transition in the range of 8.20-53.13 pN after Mac-1 was activated.This phenomenon provides a new dynamic perspective on the physiological process of neutrophil adhesion to platelets and also provides new clues for the treatment of thrombosis.In this thesis,we discovered and discussed the spatiotemporal characteristics of the coexistence of rapid and local β2 integrin activation,slow and global β2 integrin activation in human neutrophils,as well as the mechanical mechanism and mechanochemical signaling pathway of local activation of β2 integrin,explaining the human neutrophils in the circulation could timely and stably adhere to the site of vascular injury.It provides a new spatiotemporal and biomechanical perspective for the physiological and pathological processes in which neutrophils may participate in adhesion and immune function,such as inflammation,thrombosis,and cancer cell invasion.In the future,this research may provide new clues and molecular targets for anti-inflammatory,antithrombotic,anticancer,and other therapeutic schemes.