Chlamydia Pneumoniae Infection Induces The Transendothelial Migration Of Monocytes By Enhancing Endothelial Permeability Via Promoting VE-cadherin Phosphorylation

Objective: Using the model of human umbilical vein endothelium cell line Ea.hy926 cells infected with Chlamydia pneumoniae(C.pn) in vitro, to investigate the effects of C.pn infection on the transendothelial migration of monocytes, and to explore whether C.pn infection promotes the transendothelial migration of monocytes by increasing the permeability of vascular endothelial cells(VECs). We demonstrated that C.pn infection induces the transendothelial migration of monocytes by enhancing endothelial permeability induced by the increased VE-cadherin phosphorylation at Y658 via Src. Methods: 1. Immunofluorescence was performed to identify human umbilical vein endothelial cell line Ea.hy926 cel s. 2. Human peripheral monocytes were isolated from the blood samplings from health donors by discontinuous density gradient centrifugation in Perco ll; Transendothelial migration assay was used to investigate the effects of C.pn infection on the transendothelial migration of monocytes. 3. Transendothelial electrical resistance(TEER) assay was used to detect the permeability of C.pn-infected VECs. 4. Confocal laser scanning microscopy was performed to observe the distribution of VE-cadherin after C.pn infection. 5. Western blot was employed to determine the expression levels of total VE-cadherin and phosphorylated VE-cadherin(Y658) in VECs at different time potins after C.pn infection. 6. Co-immunoprecipitation was performed to collect protein substrate of Src kinase; Src activity was measured by incorporation of [γ-32P]ATP into exogenous substrate after stimulated by phosphatidylserine and diolein using a liquid scintillation counter. 7. Western blot was performed to detect the tyrosine phosphorylation of VE-cadherin(Y658) in the infected VECs pretreated with PP2. 8. Confocal laser scanning microscopy was performed to observe the distribution of VE-cadherin in VECs pretreated with PP2 before C.pn infection.9. TEER assay was used to detect the changes in the permeability of the infected VECs pretreated with PP2. 10. Transendothelial migration assay was used to determine the changes in the transendothelial migration of monocytes after the infected VECs were pretreated with PP2. Results: 1. The results from the immunofluorescence with the antibody of Factor Ⅷ showed that green fluorescence was observed in human umbilical vein endothelial cell line Ea.hy926 cells, indicating a successful identification of the Ea.hy926 cell as an endothelial cel. 2. The results from transendothelial migration assay showed that the number of monocytes migrated aross C.pn-infected VECs was significantly larger than that of the control group(P < 0.05).3. In TEER assay, the TEER values in C.pn- infected VECs decreased in a time-dependent manner. The TEER values decreased quickly between 12 h and 24 h after infection, and then gradually decreased till 48 h after infection. 4. Under a confocal laser scanning microscope, VE-cadherin was located at interendothelial junctions and the borders of interendothelial junctions were intact. After C.pn infection, VE-cadherin was partly translocated from membrane to cytoplasm, and was distributed diffusely in the cytoplasm. Several small gaps were observed at the interendothelial junctions. 5. Compared with the control group, there are no significant differences in the expressions of VE-cadherin in VECs at different time points after C.pn infection, whereas the tyrosine phosphorylation levels of VE-cadherin(Y658) at 24 h and 48 h postinfection were both much higher than that in the control group(P < 0.05). However, there is no significant difference in the tyrosine phosphorylation levels of VE-cadherin(Y658) between 24 h and 48 h after infection. 6. Src activity in C.pn- infected VECs detected by liquid scintillation co unter began to elevate at 12 h postinfection, and reached a peak at 24 h postinfection, and then decreased till 48 h after infection. Src activities in C.pn- infected VECs at different time points were all higher than that in the control group(P < 0.05). 7. Western blot analysis showed that the tyrosine phosphorylation levels ofVE-cadherin(Y658) at 24 h postinfection were much higher than that in the control group(P < 0.05), whereas the tyrosine phosphorylation level of VE-cadherin(Y658) in VECs pretreated with PP2 at 24 h postinfection was significantly lower than that in C.pn-infected VECs(P < 0.05). 8. Under a confocal laser scanning microscopy, the translocation of VE-cadherin from membrane to cytoplasm in C.pn-infected VECs was inhibited by the pretreatment with PP2. There were fewer and smaller gaps at the interendothelial junctions than that in C.pn-infected VECs. 9. The TEER results showed that the time-dependent rapid decreases in TEER values in C.pn-infected VECs can be partly reversed, and displayed a slow decrease after infection(P < 0.05). 10. In the transendothelial migration assay, C.pn infection-induced increases in the number of monocytes migrated across the infected VECs were significantly inhibited by the pretreatment of VECs with PP2(P < 0.05). Conclusions: C.pn infection promotes the transendothelial migration of monocytes possibly by enhancing endothelial permeability induced by the increased VE-cadherin tyrosine phosphorylation at Y658 via Src.