Abstract 134: Extracellular ATP Inhibits Natural Killer Cell Recruitment in Inflamed Tissues and Protects Endothelial Cells From NK-Mediated Vascular Injury

Abstract

Background: Natural killer cells (NK) represent the main source of IFN-gamma that contributes to atherosclerotic plaque progression and instability. Fractalkine (CX3CL1) expressed by endothelial cells (ECs) mediate NK recruitment and elicit NK-mediated ECs killing. Moreover high CX3CL1 mRNA expression has been found in advanced atherosclerotic lesions of human arteries as well as in the deep intima of diabetic patients. Nucleotides can be massively released in the extracellular space by ECs under shear stress, activated platelets and injured cells and stimulate purinergic receptors expressed in different cell types including NK. Hypothesis: that extracellular nucleotides modify NK responses to CX3CL1 or SDF-1 (CXCL12) and their ability to mediate vascular injury. Methods: NK were isolated by negative immunomagnetic selection from peripheral blood of healthy blood donors and P2 receptor genes expression pattern studied by RT-PCR. NK chemotaxis was measured by transwell cell culture system followed by flow cytometric analysis of migrated cells. NK were co-cultured with HUVEC cells to analyze NK-mediated ECs killing. Results: we show for the first time that NK express P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, P2Y14 and P2X1, P2X4, P2X7 receptors. Neither ATP or UTP alone induced NK cell migration. However ATP but not UTP increased non-directional cell movement (chemokinesis). This effect was mimicked by the cell permeable cyclic AMP analogue 8-Br-cAMP and was dependent on Protein kinase A activity since it was blocked by the specific inhibitor H89. In addition, ATP but not UTP, inhibited NK cell chemotaxis to CX3CL1 whereas expression for CXCR4 and migration following CXCL12 stimulation were increased. Moreover the NK-mediated killing of HUVEC cells was downregulated following exposure to extracellular ATP. Conclusion: CX3CL1 has been shown to participate to leukocyte adhesion to ECs and extravasation. In addition membrane bounds form of CX3CL1 expressed by ECs triggers NK cytotoxicity. ATP release from endothelial cells upon shear stress and in inflammatory states might represent a protective mechanism from NK cell-mediated cytotoxicity and point out P2 purinergic receptors as novel potential therapeutic targets.