P2X receptor characterization and IL‐1/IL‐1Ra release from human endothelial cells

Abstract

Background and purpose:The pro‐inflammatory cytokine, interleukin‐1β (IL‐1β), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL‐1β in response to inflammatory stimuli, but the demonstration and mechanism of release of IL‐1 from ECs remains unclear. In activated monocytes, efficient release of bioactive IL‐1β occurred via activation of ATP‐gated P2X7 receptors (P2X7Rs). Activation of P2X7R in ECs from human umbilical vein (HUVECs) released IL‐1 receptor antagonist (IL‐1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL‐1β and IL‐1Ra synthesis, processing and release, in HUVECs under pro‐inflammatory conditions.Experimental approach:Quantitative RT‐PCR, immunoblotting, ELISA, flow cytometry, and whole‐cell patch clamp recordings were used to determine protein expression and receptor function. IL‐8‐luciferase‐reporter was used as an IL‐1 sensitive bioassay.Key results:HUVECs expressed P2X4R and P2X7R subtypes and both were significantly up‐regulated under inflammatory conditions. P2X7R currents were increased 3‐fold by inflammatory stimuli, whereas no P2X4R‐mediated currents were detected. Caspase‐1, but not IL‐1β, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro‐IL‐1β and increased caspase‐1 levels. Activation of P2X7Rs resulted in low‐level release of bioactive IL‐1β and simultaneous release of IL‐1Ra. The net biological effect of release was anti‐inflammatory.Conclusions and implications:Endothelial P2X7Rs induced secretion of both pro‐ and anti‐inflammatory IL‐1 receptor ligands, the balance of which may provide a means for altering the inflammatory state of the arterial vessel wall.British Journal of Pharmacology (2007) 151, 96–108. doi:10.1038/sj.bjp.0707213

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