P2X receptor characterization and IL-1/IL-1Ra release from human endothelial cells.
OBJECTIVE
The pro-inflammatory cytokine, interleukin-1beta (IL-1beta), has been implicated in the pathogenesis of atherosclerosis, potentially via its release from vascular endothelium. Endothelial cells (EC) synthesize IL-1beta 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-1beta 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-1beta and IL-1Ra synthesis, processing and release, in HUVECs under pro-inflammatory conditions.


METHODS
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.


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-1beta, was present intracellularly under basal conditions; inflammatory stimuli activated the synthesis of intracellular pro-IL-1beta and increased caspase-1 levels. Activation of P2X(7)Rs resulted in low-level release of bioactive IL-1beta and simultaneous release of IL-1Ra. The net biological effect of release was anti-inflammatory.


CONCLUSIONS
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.
Wilson
H L
HL
School of Medicine and Biomedical Sciences, University of Sheffield, Royal Hallamshire Hospital, Sheffield, UK. h.l.wilson@sheffield.ac.uk
Varcoe
R W
RW
Stokes
L
L
Holland
K L
KL
Francis
S E
SE
Dower
S K
SK
Surprenant
A
A
Crossman
D C
DC
D042783:Endothelial Cells / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein / Q000378:metabolism*; D053583:Interleukin-1beta / Q000378:metabolism*; D008564:Membrane Potentials; D012333:RNA, Messenger / Q000032:analysis; D018048:Receptors, Purinergic P2 / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7
D053590:Interleukin 1 Receptor Antagonist Protein; D053583:Interleukin-1beta; C548226:P2RX4 protein, human; C546468:P2RX7 protein, human; D012333:RNA, Messenger; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7