KLF2Regulate CXCR1、CXCR2、L-selectin、CD11a And Other To Affecting Adhesion And Migration Of Neutrophil-like Cells

Background and Objectiveasthma is a chronic inflammatory disease of the airways; neutrophil is one of themajor effector cells, which is a common feature of severe-type asthma, but themechanism of the increase of neutrophils is still not clear. The study here mainly aims,with cellular level, to elaborate whether KLF2affects neutrophil’s adhesion andmigration by regulating the expression of CXCR1, CXCR2, L-selectin, CD11a,CD11b, CD18.Method(1) By using DMSO HL-60cells become neutrophil-like cells, Cell morphology canbe identified by Diff staining and the expression of mRNA of CD11b is tested byrealtime-PCR(2) Transfection the loaded and KLF2-RNAi lentiviral vectors to infect neutrophils,Randomly divide into the blank group, load group and siRNA intervention group.Transfection identified by fluorescence microscopy and western blot.(3) The expression of mRNA of CXCR1, CXCR2, L-selectin, CD11a, CD11b andCD18are tested by Realtime-PCR respectively. The expression of protein ofCXCR1, CXCR2and L-selectin are tested by western blot respectively. Meanfluorescence intensity of CD11a, CD11b, CD18is measured by Flow cytometry.(4) adhesion of neutrophil which is labeled with calcein-AM, are tested byFluorescence microscopy, migration of neutrophils which response to10ng/mlIL-8, is test by transwell.Results1. Identification of neutrophil-like cellsCD11b is one of signatures of maturation of neutrophil differentiation. TheHL-60cells were cultured with1.3%DMSO for5days，then cells were dyed bydiff-quik cell stain，we found the cells were smaller than the HL-60cell，and the nuclear was renal nuclear。The expression of mRNA of CD11b were tested byReatime-PCR，the result was neutrophil-like cell CD11b expression was significantlyincreased (P <0.01).2. Interference by KLF2Observation of infection efficiency of KLF2siRNA group and load group by thefluorescence microscope，both of the two groups’infection efficiency were over90%. Western blot：the expression of KLF2protein of KLF2knocded down groupwas lower than the other two groups significantly (P <0.01), KLF2protein expressionof load group and blank group was basically same (P>0.05), the rate of knockdownwas76%.3. mRNA and protein expression(1) Real-time PCR results show that, comparing the KLF2-siRNA group with theblank group and the load group, mRNA expression of CXCR1, CXCR2,L-selectin, CD11a, CD11b, CD18is significantly increased(P<0.01), while theexpression of L-selectin mRNA is significantly reduced(P<0.01), blank group andload group was basically same (P>0.05).(2) Western blot analysis shows that, comparing the KLF2-interfered group with theblank group and the load group, the CXCR1, CXCR2protein expression issignificantly increased(P<0.01), while the expression of L-selectin protein issignificantly reduced(P<0.01). blank group and load group was basically same(P>0.05).(3) Flow cytometry shows that mean fluorescence intensity of CD11a, CD11b, CD18of KLF2-siRNA group is significantly increased (P <0.01), blank group and loadgroup was basically same (P>0.05).4. Adhesion and migration experiments(1) Fluorescence microscopy shows that, comparing the KLF2-interfered group withthe blank group and the load group, number of adhesion neutrophils issignificantly increased(P<0.01), blank group and load group was basically same(P>0.05).(2) transwell show that, comparing the KLF2-interfered group with the blank groupand the load group, neutrophil migration rate is significantly increased (P<0.01),blank group and load group was basically same (P>0.05). Conclusion(1) KLF2affect neutrophil adhesion by regulating the expression of CD11a, CD11b,CD18, However, KLF2affecting neutrophil adhesion by L-selectin, May not be soimportant.(2) KLF2affect neutrophil migration by CXCR1, CXCR2in the chemotactic role ofIL-8.