The Mechan-chemical Mechanism Of Calcium Response And LFA-1 Rapid Activation Of PMN Induced By P-selectin

During inflammation,leukocytes are induced by chemokines to be recruited to the site of inflammation blood vessels,and then roll,adhere and crawl on vascular endothelial cell surface,mediated mainly by selectins and PSGL-1,and ICAM-1 and ?2 integrin.P-selectin is one of the important molecules expressed on the surface of endothelial cells in the selectin family.It can rapidly perform the adhesion of leukocytes by binding to PSGL-1 on the surface of leukocytes to form initial rolling adhesion.?2 integrin and ICAM-1,expressed on the surface of endothelial cells,mediate the subsequent slow rolling,stable adhesion and creeping.However,the force-chemical signal transduction mechanism of selectin-activated ?2 integrin is not completely clear.Intracellular calcium signaling is thought to be an important indicator of leukocyte activation and is the most important second messenger molecule in the process of activating signal transduction.Selectin can mediate the elevation of intracellular calcium levels in leukocytes,requiring the presence of external forces,but this result is only demonstrated initially in rolling leukocytes.However,specific signaling processes and time courses of leukocyte calcium response remains unclear.Acute myeloid leukemia has the highest incidence of cancer under 35 years of age,mainly resulting from the abnormal proliferation of promyelocytic cells.HL-60 cells,belonging to be the precursor cells of neutrophils,are one of the acute myeloid leukemia cells,and are often considered to model cells of neutrophils.It has not been reported the selectin-mediated rolling adhesion and calcium response.ATRA is recognized as an important chemotherapeutic agent in acute myeloid leukemia,and can induce differentiation and apoptosis of leukemic cells.However,there is no idea about the force-chemical regulation mechanism of firm adhesion and calcium response of ATRA-treated or not treated leukemia cellsIn response to these scientific questions,we first loaded the indicator of the cytosolic calciu,Fluo-4 AM,into human PMNs,and perfused the cells in parallel plate flow chamber with P-selectin,P-selectin mixed ICAM-1 or ICAM-1 alon.The fluorescence of the adherent cells was recorded in real time by fluorescence microscopy.The activation ratio,peak intensity and delay time of the calcium signal were extracted by the software in image processing.To investigate the specificity of adhesion molecules activating calcium signaling in leukocytes,we performed experiments by altering the concentration of substrate adhesion molecules.To investigate the effect of force on the regulation to calcium reponse,we fabricated different fluid shear environments by varying the perfusion velocity.In order to study the signal transduction process,we added inhibitors of Syk,moesin,IP3,membrane ion channel,cytoskeleton in experiments,and in order to verify which has effect on ?2 integrin activation,we also used the LFA-1 and Mac-1 specific blocking antibodies to confirm the ?2 integrin types.The results show that we successfully exploited this system to extract parameters related to adherence to neutrophil adhesion and calcium response,and found that PSelectin mainly induced intracellular calcium release,while ICAM-1 induced Ca2+ influx through Ca2+ channel.In addition,we found an important P-selectin-activating integrin LFA-1 rapid pathway by comparing the delay time of calcium response in different adhesion conditions,which is mainly through Moesin-actin pathway.And with the Syk and lipid rafts in traditional signal molecules has little effect on this rapid pathway.Finally,we found that the calcium response of these PMNs and the rapid activation of LFA-1 were both regulated by the external forces.HL-60 cells were treated with ATRA for different times(0,24 h,48 h and 72 h).Fluo-4 AM was then loaded into the treated and untreated ATRA cells,and the cells were perfused into P-selectin covered parallel plate flow chamber with different shear stresses.We found that P-selectin can mediate HL-60 cells firm adhesion,which in turn produces a specific calcium response that is positively regulated by external forces.In addition,with the increase of ATRA treatment time,the level of calcium signal was gradually increased to the level of normal neutrophils direction.Moreover,the calcium response of ATRA-treated cells was also regulated by external forces.In summary,we firstly and systematically described the P-selectin-mediated leukocyte-calcium response and the force-chemical regulation of ?2 integrin,as well as the P-selectin-mediated calcium response of leukemic cells before and after chemotherapeutic treatment.The results will help us to deeply understand the role of calcium response in the process of P-selectin-activating integrin LFA-1 and its pathway.The change of signal transduction of leukemia cells before and after treatment ATRA will eventually help the medical and physiologic workers to understand the physiological activation of PMN from the mechanistic point of view,and provide them the reference information for the clinical treatment of inflammation.