CD99 Mediate Neutrophil Transmigration And Participate In Inflammation Mechanism After OGD In Vitro

Objective To investigate the role of CD99 in mediate neutrophils extravasation after OGD and providing new target for ischemic stroke treatment.Methods Immortalized mouse brain endothelial cells bend.3(bEnd.3)were used to do the experiment.Cells were cultured with Dulbecco's Modified Eagle Medium(DMEM)supplemented with 10%fetal bovine serum(FBS),1%Pen Strep(PS)and maintained in a humidified incubator(95%air/5%C02).Bone marrow neutrophils were extracted with the method of density gradient centrifugation.In brief,Euthanize mice and spray the animal surface with 70%ethanol.Dislocate the acetabulum from the hip joint,remove the remaining muscles from the femur and tibia and separate the femur from the tibia at the knee joint.exercising care to not break the bone ends.Place the bones in a Petri dish containing DMEM+10%FBS+1%PS.Flush the bone marrow cells from both ends of the bone shafts onto a 50 ml screw top Falcon tube fitted with a 100?m filter.Lyse the red blood cells by re-suspending the cell pellet in 10 ml of 0.2%NaCl for approximately 20 sec followed by addition of 10 ml of 1.6%NaCl.Then centrifuge for 7 min at 1,400 rpm at 4 °C.Collect and wash the cells.Then Separate neutrophils by Density Gradient Centrifugation.Add 3 ml of Histopaque-1119(density,1.119 g/ml)in a 15-ml conical tube.Overlay 3 ml of Histopaque-1077(density,1.077 g/ml)on the 3 ml of Histopaque-1119.Overlay the bone marrow cell suspension on top of the Histopaque-1077.Centrifuge for 30 min at 900g at 25 °C without brake.Wash the collected neutrophils between Histopaque-1119 and Histopaque-1077.At last wash the neutrophils twice with DMEM +10%FBS+1%PS.Neutrophils were confirmed by Stain with Wright-Giemsa.The expression of CD99 on bEnd.3 and neutrophils was confirmed with Immunofluorescence(bEnd.3 cultured on coverslips for 2 days,neutrophils were spread on glass slide and natural air drying).Then Consecutively cultured with primary and secondary antibody,and using microscope to observation.Western Blot were used to test the expression of CD99,Intercellular-adhesion molecule(ICAM-1),platelet-endothelial cell adhesion molecule(PECAM-1)protein in control,Oxygen Glucose Deprivation(OGD),Interleukin-l(IL-1?)and OGD+IL-1? group.The bEnd.3 were seeded in 6-well culture dishes,each group 3 wells.OGD were continued for 3.5h,the IL-1? concentration were 10ng/ml and stimulate cells for 16h,the OGD+IL-1? group were cultured with IL-1? for 16h and then managed with OGD.After that protein were extracted and tested by Western Blot.The test were done 3 times at least.The relationship between HIF-1? and CD99 were also tested with the method of Western Blot,bEnd.3 were cultured in 6-well culture dishes,and were divided into two groups,each group were given O?M,25?M,50?M concentration of HIF-1? inhibitor PX478 and each concentration had 3 wells,and then cells were placed in normoxia and OGD condition.After that extracting protein and tested with Western Blot.Of the neutrophil transmigration test,bEnd.3 cultured in 24-well,after grown up to 80 percent aggregation,the cells were processed with OGD?OGD+anti-CD99?anti-CD99,the concentration of anti-CD99 were 25?g/ml,and the culture time were 30min,after that neutrophils were added in.Consecutively taken photos every 20 seconds for 20minute,counting the number of neutrophils that crossed the monolayer and related to the number of phase bright neutrophils on top of endothelial cells using Image J 1.46r software.Each group repeat 3 times,The statistical analysis was performed with Graph Pad Prism 5.Data were presented as the means ± SD of at least three independent experiments and were analyzed using oneway analysis of variance(ANOVA)with Newman-Keuls's post hoc test.The level of significance was represented as*p<0.05,**p<0.01.Result both bEnd.3 and neutrophils express CD99 and the expression were all over the cell surface not only the cell contact;After OGD the expression of CD99,Intercellular-adhesion molecule(ICAM-1),platelet-endothelial cell adhesion molecule(PECAM-1)were up-regulated(control group vs.OGD group,p<0.05),after cultured with IL-1?,the expression of ICAM-1 were up-regulated(control group vs.IL-1? group,p<0.01)and CD99 were declined(OGD group vs.OGD+IL-1?group,p<0.01),and PECAM-1 has no change;After OGD,interfering the expression of hypoxia-inducible factor-1?(HIF-1?)does not influence the expression of CD99;Compared with control and OGD+anti-CD99 groups,the migrated neutrophils were significant high(OGD group vs.control group,p<0.01;OGD group vs.OGD+anti-CD99 group,p<0.01).Conclusion In conclusion,after OGD the expression of CD99 were up-regulated and the up-regulation were not dependent on HIF-1?,CD99 can mediate neutrophils cross endothelial cells and participate in inflammation mechanism after ischemic stroke.