Abstract 1359: The Fractalkine Receptor is Expressed on Endothelial Cells, Mediates Upregulation of Intercellular Adhesion Molecule-1, and Promotes Neutrophil Adhesion
Background: Fractalkine (FKN) is a membrane-bound chemokine which can be released by proteolysis to produce soluble FKN (s-FKN). FKN and its receptor, CX3CR1, are believed to be important factors in atherosclerosis and may play an important role in the acute inflammatory response. Although FKN is expressed on endothelial cells (ECs), CX3CR1 is reported to reside only on certain leukocyte populations.
Methods and Results: To examine the role of FKN in post-hypoxic inflammation, cultured human umbilical vein ECs were exposed to hypoxia (H) and reoxygenation (R) for 2 hrs. FKN mRNA increased within 15 min R, and peaked at 60 min. Real-time R-T PCR and Western blotting confirmed that the ECs expressed CX3CR1 mRNA and protein. Confocal microscopic images demonstrated that CX3CR1 was located at the cell membrane and to a lesser extent in the cytoplasm. Addition of s-FKN to the cultured cells resulted in a dose-dependent increase in intercellular adhesion molecule-1 (ICAM-1) mRNA, which was much more marked following H/R. Transfection of CX3CR1 interfering RNA to knockdown the receptor resulted in decreased ICAM-1 expression after s-FKN stimulation. A cell-cell adhesion assay using human neutrophils labeled with the cytoplasmic fluorescent dye Calcein AM, demonstrated that significantly fewer neutrophils adhered to ECs in which CX3CR1 had been knocked down with the interfering RNA, compared to ECs transfected with control interfering RNA. Following exposure to s-FKN, ECs exhibited increased expression of phosphorylated ERK1/2, suggesting that s-FKN may act through the MAPK pathway.
Conclusion: We found for the first time that vascular endothelial cells not only express fractalkine, but also express its receptor CX3CR1. We demonstrate that s-FKN may act through its receptor in ECs to increase ICAM-1 expression and promote neutrophil adhesion.