No abstract

Background and purpose: The pro-inflammatory cytokine, interleukin-1b (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-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-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 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.

Signal transduction pathways activated by Toll-like Receptors and the IL-1 family of cytokines are fundamental to mounting an innate immune response and thus to clearing pathogens and promoting wound healing. Whilst mechanistic understanding of the regulation of innate signalling pathways has advanced considerably in recent years, there are still a number of critical controllers to be discovered. In order to characterise novel regulators of macrophage inflammation, we have carried out an extensive, cDNA-based forward genetic screen and identified 34 novel activators, based on their ability to induce the expression of cxcl2. Many are physiologically expressed in macrophages, although the majority of genes uncovered in our screen have not previously been linked to innate immunity. We show that expression of particular activators has profound but distinct impacts on LPS-induced inflammatory gene expression, including switch-type, amplifier and sensitiser behaviours. Furthermore, the novel genes identified here interact with the canonical inflammatory signalling network via specific mechanisms, as demonstrated by the use of dominant negative forms of IL1/TLR signalling mediators.