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

Background and purpose: The pro-inflammatory cytokine, interleukin-1 beta (IL-1b), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL-1b 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-1b occurred via activation of ATP-gated P2X(7) receptors (P2X(7)Rs). Activation of P2X(7)R in ECs from human umbilical vein (HUVECs) released IL-1 receptor antagonist (IL-anti-1Ra). The purpose of this study was to provide a quantitative investigation of P2XR expression and function, in parallel with IL-1b and IL-1Ra synthesis, processing and release, in HUVECs under pro-inflammatory conditions. Experimental approach: Quantitative RT-PCR, immunoblotting, ELISA, flow cytometry, and whole-ell 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 P2X(4)R and P2X(7)R subtypes and both were significantly up-regulated under inflammatory conditions. P2X(7)R currents were increased 3-fold by inflammatory stimuli, whereas no P2X(4)R-mediated currents were detected. Caspase-1, but not IL-1b, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro-IL-1b and increased caspase-1 levels. Activation of P2X(7)Rs resulted in low-level release of bioactive IL-1b and simultaneous release of IL-1Ra. The net biological effect of release was anti-inflammatory. Conclusions and implications: Endothelial P2X(7)Rs 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.