The Influence Of Antiflammin-1 On Adhesion Between Neutrophils And Endothelial Cells Induced By LPS

Chapter1 Comparison of different neutrophil separation methods from human peripheral bloodObjective:To study neutrophil's biological characteristic and function,it is the key step to isolate neutrophils from the peripheral blood. Percoll density gradient centrifugation,Ficoll-Hypaque density gradient centrifugation,red blood cells cracking and the natural erythrocyte sedimentation method with Dextran are the four popular methods.The objective of this work was to investigate a simple and efficient method from the four methods.Methods:Respectively using the four methods to separate neutrophil from healthy human peripheral blood,then the purity,recovery rate,and cell viability were compared.Results:Among the four methods,the neutrophil purity from Percoll density gradient centrifugation and Ficoll-Hypaque density gradient centrifugation were more than 90%,but there was no significant difference between them(P＞0.05).The neutrophil recovery rate from Percoll density gradient centrifugation,Ficoll-Hypaque density gradient centrifugation and red blood cells cracking were significantly higher than that from the natural erythrocyte sedimentation method with Dextran(P＜0.01 or P＜0.05).Cell viability from Percoll density gradient centrifugation was significantly higher than those from others(P＜0.01 or P＜0.05)Conclusion:Percoll density gradient centrifugation provides the most simple and efficient approach for isolation of human blood neutrophil with the high purity and viability. Chapter2 The influence of Antiflammin-1 on adhension between neutrophils and endothelial cells induced by LPSObjective:The accumulation of neutrophil is the initial factor in the acute lung injury(ALI).The adhension between neutrophils and pulmonary vascular endothelial cells is the primary step in the inflammatory response,which is the basis for neutrophils migration into the lung.Uteroglobin(UG)is a steroid-inducible,homodimerie, multifunctional secreted protein with potent anti-inflammatory, immunomodulatory,anti-tumorigenic and embryonic growth-stimulatory properties.Antiflammin-1(MQMKKVLDS,AF-1)is equivalent to the 9 C-terminal amino acids ofα-helix 3 of uteroglobin and have been showed have potent anti-inflammatory effects.But the mechanism of AF-1 which inhibites the inflammation is still unclear.The present study was determined to investigate the effects of AF-1 on adhension between neutrophils and human umbilical vein endothelial cells(HUVECs)and on the expression of adhesion molecule on human neutrophil and HUVEC induced by LPS.Furthermore,we detected the expression of uteroglobin-binding protein(UGBP)on neutrophils and endothelial cells, and the adhesion inhibition of anti-UGBP antibody and the adhesion molecules expression induced by AF-1.We also detected the effect of AF-1 and anti-UGBP antibody on signal pathway of P38/MAPK to provide a new target of ALI treatment.Methods:We analyzed in vitro the adherence of neutrophils to HUVEC-12 cell line after pretreatment with AF-1 of different time and different concentration,and observed the expression of adhesion molecules on human neutrophils and HUVECs using the flow cytometry (FCM).Furthermore,we investigated the expression of UGBP on neutrophils and endothelial cells by Immunofluorescence,FCM and RT-PCR.And we observed the adhesion inhibition of anti-UGBP antibody and the adhesion molecules expression induced by AF-1 using the flow cytometry(FCM).Moreover,the effect of AF-1 and anti-UGBP antibody on the signal pathway of P38/MAPK was detected using Western-Blot.Results:1.Neutrophils adhension to endothelial cells were significantly inhibited compared with the LPS-stimulated group(P＜0.01),while neutrophils or endothelial cells were pretreated with AF-1(100μmol/L) for 15min～2h,and the inhibitory effect reached the maximum at 30min, with prolonging the pretreatment time its inhibitory effect decreased,for example,its inhibitory effect does not have statistics difference compared with LPS group at 4h.When nentrophils or endothelial cells were treated with different concentration of AF-1 from 1μmol/L to 100μmol/L for 30min,we found that AF-1(1μmol/L)could inhibited the adhesion between nentrophils and endothelial cells(P＜0.05),with a concentration-dependent relationship.FCM assay found that AF-1 could inhibit the expression of adhesion molecule CD11b on neutrophils and CD54 on endothelial cells induced by LPS,but the AF-1 itself does not affect the expression of adhesion molecule on the resting neutrophils and endothelial cells.2.Immunofluorescence,FCM assay and RT-PCR indicated that there is UGBP expression in both neutrophils and endothelial cells,and UGBP expression in nentrophils was higher than in endothelial cells.3.Anti-UGBP antibody pretreatment could attenuate the inhibition of adhesion rate and the expression of adhesion molecules induced by AF-1.4.Western-Blot assay indicated that AF-1 could reduce LPS-induced the phosphorylation level of P38/MAPK in endothelial cells significantly, and anti-UGBP antibody could inhibit the down-regulated effect of AF-1.Conclusions:1.AF-1 can inhibit the adhesion between neutrophils and endothelial cells and the expression of adhesion molecules induced by LPS,which suggests that the anti-inflammatory effect of AF-1 may be related to modulate adhesion molecules expression.2.UGBP is expressed in both neutrophils and endothelial cells,but UGBP expression in neutrophils was higher than in endothelial cells. UGBP receptor expression in membrane was higher than in cytoplasm. 3.The pretreatment by anti-UGBP antibody can attenuate the inhibitory effect of the adhesion between neutrophils and endothelial cells and the expression of adhesion molecules induced by AF-1,which suggest the inhibitory effect of AF-1 on adhesion might be related to the UGBP.4.The AF-1 can decrease the intracellular phosphorylation level of P38/MAPK by binding to UGBP.These results provided evidences that AF-1 can inhibit the adhesion between neutrophils and endothelial cells and the adhesion molecules expression on them to produce anti-inflammatory effect,which mechanism is related to decreasing the phosphorylation level of P38/MAPK through combination with the UGBP.