Leukocyte adhesion and trafficking at the endothelium requires both cellular adhesion molecules and chemotactic factors. A newly identified CX3C chemokine, fractalkine, expressed on activated endothelial cells, plays an important role in leukocyte adhesion and migration. We examined the functional effects of fractalkine on β1 and β2 integrin-mediated adhesion using a macrophage-like cell line, THP-1 cells. In this study, we report that THP-1 cells express mRNA encoding a receptor for fractalkine, CX3CR1, determined by Northern blotting. Scatchard analysis using fractalkine-SEAP (secreted form of placental alkaline phosphatase) chimeric proteins revealed that THP-1 cells express a single class of CX3CR1 with a dissociation constant of 30 pM and a mean expression of 440 sites per cell. THP-1 cells efficiently adhered, in a fractalkine-dependent manner, to full-length of fractalkine immobilized onto plastic and to the membrane-bound form of fractalkine expressed on ECV304 cells or TNF-α-activated HUVECs. Moreover, soluble-fractalkine enhanced adhesion of THP-1 cells to fibronectin and ICAM-1 in a dose-dependent manner. Pertussis toxin, an inhibitor of Gi, inhibited the fractalkine-mediated enhancement of THP-1 cell adhesion to fibronectin and ICAM-1. Finally, we found that soluble-fractalkine also enhanced adhesion of freshly separated monocytes to fibronectin and ICAM-1. These results indicate that fractalkine may induce firm adhesion between monocytes and endothelial cells not only through an intrinsic adhesion function itself, but also through activation of integrin avidity for their ligands.

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Ceramide is now recognized as an intracellular lipid signal mediator, which induces various kinds of cell functions including apoptosis. Ceramide-induced apoptosis was reported to be blocked by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C (PKC) activator, but its mechanism remained unclear. Therefore, we investigated whether ceramide has any effects on PKC in the induction of apoptosis. We here report that N-acetylsphingosine (synthetic membrane-permeable ceramide) induced translocation of PKC-delta and -epsilon isozymes from the membrane to the cytosol within 5 min in human leukemia cell lines. Treatment with sphingomyelinase, tumor necrosis factor-alpha, or anti-Fas antibody, all of which can induce apoptosis by generating natural ceramide, similarly induced cytosolic translocation of PKC-delta and -epsilon. In Fas-resistant cells anti-Fas antibody did not induce cytosolic translocation of PKC-delta and -epsilon because of no generation of ceramide, whereas N-acetylsphingosine induced apoptosis with cytosolic translocation of PKC-delta and -epsilon. Furthermore, both 12-O-tetradecanoylphorbol 13-acetate and a nonspecific kinase inhibitor, staurosporine, prevented ceramide-induced apoptosis by inhibiting cytosolic translocation of PKC-delta and -epsilon. These data suggest that cytosolic translocation of PKC-delta and -epsilon plays an important role in ceramide-mediated apoptosis.

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Endothelial cells (ECs) are primary targets of immunological attack, and their injury can lead to vasculopathy and organ dysfunction in vascular leak syndrome and in rejection of allografts or xenografts. A newly identified CX3C-chemokine, fractalkine, expressed on activated ECs plays an important role in leukocyte adhesion and migration. In this study we examined the functional roles of fractalkine on NK cell activity and NK cell-mediated endothelial cell injury. Freshly separated NK cells expressed the fractalkine receptor (CX3CR1) determined by FACS analysis and efficiently adhered to immobilized full-length fractalkine, but not to the truncated forms of the chemokine domain or mucin domain, suggesting that fractalkine functions as an adhesion molecule on the interaction between NK cells and ECs. Soluble fractalkine enhanced NK cell cytolytic activity against K562 target cells in a dose- and time-dependent manner. This enhancement correlated well with increased granular exocytosis from NK cells, which was completely inhibited by the G protein inhibitor, pertussis toxin. Transfection of fractalkine cDNA into ECV304 cells or HUVECs resulted in increased adhesion of NK cells and susceptibility to NK cell-mediated cytolysis compared with control transfection. Moreover, both enhanced adhesion and susceptibility of fractalkine-transfected cells were markedly suppressed by soluble fractalkine or anti-CX3CR1 Ab. Our results suggest that fractalkine plays an important role not only in the binding of NK cells to endothelial cells, but also in NK cell-mediated endothelium damage, which may result in vascular injury.