Inflammation and thrombosis usually occur together in many diseases,such as cardiovascular disease(CVD)and stroke,which remain to be the mostdetrimental human health killers.The crucial relevant cellular and molecular...Inflammation and thrombosis usually occur together in many diseases,such as cardiovascular disease(CVD)and stroke,which remain to be the mostdetrimental human health killers.The crucial relevant cellular and molecular events include platelet-leukocyte interaction,platelet P-selectin secretion of activated platelet and activation of leukocyte integrin Mac-1(also known asαMβ2 or CD11b/CD18),which has binding site of platelet receptor glycoprotein lbα(GPlboα). Circulating leukocytes tethered to,rolled on and firmly adhered at the activated platelets on vascular wall,through interaction of platelet P-selectin with leukocyte P-selectin glycoprotein ligand-1(PSGL-1)and Mac-1 with GPlbα.We assume that there is a rapid signaling pathway in PSGL-1 ligation-induced activation of Mac-1,for forming a stable gap junction intracellular communication between platelet and leukocyte.To test this assumption,we observed the tethering events and calcium bursting of neutrophils on immobilized P-selectin only or plus GPlbαwith use of the parallel plate flow chamber(PPFC)technique and intracellular calcium ion detector Fluo-4 AM at various wall shear stresses,and examined the dynamic force spectrum for interaction of Mac-1 plus Mn2+and GPlbαby single-molecule atomic force microscopy(AFM).In the PPFC experiments,the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin only or plus GPlbαwas observed in real timeby fluorescence microscopy,and the tether events of neutrophils was recorded by an inverted microscope and a high speed CMOS acquisition system in 1280 pixels×1024 pixels at 100 frames per second(fps). Captured images were analyzed by Image Pro Plus.Our results indicated that force triggered,enhanced and quickened the cytoplasmic calcium bursting of neutrophils.Calcium bursting may be induced first by interaction of the activated neutrophil Integrin Mac-1 and GPlbα,but not by P-selectin ligation to its ligand PSGL-1.Being triggered and speeded up by wall shear stress,the P-selectin-induced activation of Mac-1 in neutrophils was a previous events of calcium bursting,and occurred within one second.The tether lifetime of neutrophils on immobilized P-selectin only or plus GPlbαincreased first and then decreased with wall shear stress,and the wall shear stress threshold is 0.3 dyn/cm2 for P-selectin only but 0.25 dyn/cm2 for P-selectin plus GPlbα.It suggested a mechanical regulation mechanism of'Catch-slip bond'transition for Mac-1/GPlbαcomplex,like P-selectin/PSGL-1 complex.It was also demonstrated through the single-molecule AFM data,which showed that force regulated binding of activated Mac-1 to GPlbαthrough the Catch bond mechanism for tensile force small than 31.25 pN,being matched with the PPFC experimental data.The tether lifetime of neutrophils on immobilized P-selectin plus GPlbαwas more long than that on P-selectin only,showing the P-selectin-induced activation of Mac-1 on neutrophils was a rapid but local events under flows.In summary,force rapidly triggers and promotes the PSGL-1 ligation-induced activation of Mac-1 on neutrophils,leading to formation of stable intercellular communication between leukocytes and platelets.This finding should be useful in understanding of platelet-leukocytes interaction-mediated biological process in cellular inflammation responses and thrombus formation.展开更多
基金supported by the National Natural Science Foundation of China ( 116272109,11432006)
文摘Inflammation and thrombosis usually occur together in many diseases,such as cardiovascular disease(CVD)and stroke,which remain to be the mostdetrimental human health killers.The crucial relevant cellular and molecular events include platelet-leukocyte interaction,platelet P-selectin secretion of activated platelet and activation of leukocyte integrin Mac-1(also known asαMβ2 or CD11b/CD18),which has binding site of platelet receptor glycoprotein lbα(GPlboα). Circulating leukocytes tethered to,rolled on and firmly adhered at the activated platelets on vascular wall,through interaction of platelet P-selectin with leukocyte P-selectin glycoprotein ligand-1(PSGL-1)and Mac-1 with GPlbα.We assume that there is a rapid signaling pathway in PSGL-1 ligation-induced activation of Mac-1,for forming a stable gap junction intracellular communication between platelet and leukocyte.To test this assumption,we observed the tethering events and calcium bursting of neutrophils on immobilized P-selectin only or plus GPlbαwith use of the parallel plate flow chamber(PPFC)technique and intracellular calcium ion detector Fluo-4 AM at various wall shear stresses,and examined the dynamic force spectrum for interaction of Mac-1 plus Mn2+and GPlbαby single-molecule atomic force microscopy(AFM).In the PPFC experiments,the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin only or plus GPlbαwas observed in real timeby fluorescence microscopy,and the tether events of neutrophils was recorded by an inverted microscope and a high speed CMOS acquisition system in 1280 pixels×1024 pixels at 100 frames per second(fps). Captured images were analyzed by Image Pro Plus.Our results indicated that force triggered,enhanced and quickened the cytoplasmic calcium bursting of neutrophils.Calcium bursting may be induced first by interaction of the activated neutrophil Integrin Mac-1 and GPlbα,but not by P-selectin ligation to its ligand PSGL-1.Being triggered and speeded up by wall shear stress,the P-selectin-induced activation of Mac-1 in neutrophils was a previous events of calcium bursting,and occurred within one second.The tether lifetime of neutrophils on immobilized P-selectin only or plus GPlbαincreased first and then decreased with wall shear stress,and the wall shear stress threshold is 0.3 dyn/cm2 for P-selectin only but 0.25 dyn/cm2 for P-selectin plus GPlbα.It suggested a mechanical regulation mechanism of'Catch-slip bond'transition for Mac-1/GPlbαcomplex,like P-selectin/PSGL-1 complex.It was also demonstrated through the single-molecule AFM data,which showed that force regulated binding of activated Mac-1 to GPlbαthrough the Catch bond mechanism for tensile force small than 31.25 pN,being matched with the PPFC experimental data.The tether lifetime of neutrophils on immobilized P-selectin plus GPlbαwas more long than that on P-selectin only,showing the P-selectin-induced activation of Mac-1 on neutrophils was a rapid but local events under flows.In summary,force rapidly triggers and promotes the PSGL-1 ligation-induced activation of Mac-1 on neutrophils,leading to formation of stable intercellular communication between leukocytes and platelets.This finding should be useful in understanding of platelet-leukocytes interaction-mediated biological process in cellular inflammation responses and thrombus formation.
