Role of FUT7 during leukocyte adhesion and extravasation
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Leukocyte adhesion and transmigration from blood flow on the endothelial cells is an important cascade of processes during inflammation. The initial leukocyte capturing is mediated by binding of P-selectin expressed on the endothelium and PSGL-1 on the neutrophil which leads to decrease the leukocyte speed. E-selectin is another endothelial cell adhesion molecule that regulates leukocyte capture and this must be transcriptionally activated by thrombotic or inflammatory stimuli like IL-1β. In mouse, E-selectin binds to PSGL-1 and ESL-1 on the neutrophil aids the transition to firm cell binding. The E-selectin ligands on human leukocytes remain unknown. Once the cell is recruited, cell signaling events occur that lead to neutrophil deformation and migration through the endothelial cells. This phenomenon is known as leukocyte extravasation or diapedesis. Sialyl-Lewis x is a tetra-saccharide carbohydrate found in both PSGL-1 and ESL-1 structures which has an impact in cell-cell adhesion. The synthesis of sialyl-Lewis X is one of the responsibilities of the FUT7 gene. Selectins engagement is also involved in leukocyte activation and thus the disruption of selectin-ligands can impact not only the initial capture step but also steps in the cell adhesion cascade. To examine this aspect, in this project, the impact of FUT7 gene, on promyeloid HL-60, cell capture, firm adhesion and transmigration was examined using CRISPR-Cas9 to functionally ablate this specific gene. Results were compared with wild type HL-60 cells. Microfluidic flow cell was used to study cell adhesion to the selectins under shear flow. Transmigration assays were performed on activated HUVEC monolayers under static conditions. The results show a dominant role for FUT7in regulating P-selectin mediated cell adhesion with a much smaller role during E-selectin binding. FUT7 did not affect human HL-60 myeloid cell transmigration across HUVEC monolayers.