Polymorphonuclear Leukocyte Transmigration-Induced Endothelial Responses
| Autor: | Wen-Hong Su, 蘇文弘 |
|---|---|
| Rok vydání: | 2002 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 91 Vascular endothelium plays an important role in regulating the transendothelial migration of polymorphonuclear leukocytes (PMNs). In this study, we examined at single-cell level the intracellular calcium ion ([Ca2+]i) signaling of endothelial cells (ECs) during PMN transmigration. Human umbilical vein ECs were cultured on a thin layer of collagen gel. The ECs were labeled with fura-2 AM, immersed in formyl-Met-Leu-Phe, and subsequently perfused with fresh buffer in order to establish a gradient of chemoattractant across the EC monolayer. The entire process of PMN rolling on, adhering to, and transmigrating across the EC monolayer was recorded under both phase-contrast and fluorescence optics. We found that 1) At high concentration (~3 x106/ml), both PMN suspension and its supernatant stimulated frequent EC [Ca2+]i elevations across the monolayer; 2) when used at lower concentration (~5 x 105/ml) to avoid the interference of soluble factors, PMN transmigration, but not rolling nor adhesion, was accompanied by EC [Ca2+]i elevation; 3) the latter EC [Ca2+]i elevation occurred simultaneously in ECs adjacent to the transmigration site, but not in those which were not in direct contact with the transmigrating PMN; 4) this EC [Ca2+]i elevations was an initial and required event for PMN transmigration; 5) BAPTA-pretreated PMNs transmigrated with accompanying EC [Ca2+]i elevation but they became much elongated in the collagen gel. In conclusion, PMNs induce adjacent EC [Ca2+]i signaling that apparently mediates the ‘gating’ step for their subsequent transmigration. Most existing evidence regarding junction protein movements during transendothelial migration of leukocytes comes from taking post-fixation snap shots of the transendothelial migration process that happens on a cultured endothelial monolayer. In this study, we used junction protein-specific antibodies that did not interfere the transendothelial migration to examine the real-time movements of vascular endothelial-cadherin (VE-cadherin) and platelet/endothelial cell adhesion molecule-1 (PECAM-1) during transmigration of polymorphonuclear leukocytes (PMNs) either through a cultured endothelial monolayer or through the endothelium of a dissected human umbilical vein tissue. Both junction proteins showed relative movements, not transient disappearance, at the PMN transmigration sites under either experimental model system. VE-cadherin moved away from the transmigration site to different ends of it, whereas PECAM-1 opened to surround the periphery of a transmigrating PMN. Junction proteins usually moved back to their original positions when the PMN transmigration process was completed in less than 2 min. The relative positions of some junction proteins might rearrange to form a new inter-endothelial contour when PMNs preferentially transmigrated through multi-cellular corners. Although transmigrated PMNs maintained good mobility, they only moved laterally underneath the vascular endothelium instead of deeply into the vascular tissue. In conclusion, our results obtained from using either cultured cells or vascular tissues showed that VE-cadherin-containing adherent junctions were relocated aside, not opened or disrupted, while PECAM-1-containing junctions were opened, during PMN transendothelial migration. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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