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. 2007 May;151(1):115-27.
doi: 10.1038/sj.bjp.0707213. Epub 2007 Mar 12.

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

H L Wilson  1 , R W Varcoe, L Stokes, K L Holland, S E Francis, S K Dower, A Surprenant, D C Crossman
Affiliations

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

H L Wilson et al. Br J Pharmacol. 2007 May.

Abstract

Background and purpose: 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.

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-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 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.

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Figures

Figure 1
Figure 1
RT-PCR assay of P2X receptor subtypes in HUVEC. (a) Quantitative RT-PCR was optimized for ‘housekeeping genes' to detect human 18S rRNA, β2-microglobulin (β2M) and human hydroxymethylbilane synthase (HMBS), under various stimulatory conditions. HUVECs were cultured in the presence of pro-inflammatory mediators for 24 h before total RNA was extracted and cDNA synthesized. h18S rRNA, hβ2M and hHMBS qRT-PCR primers were then tested against these cDNA samples to observe changes in expression levels. (b) RT-PCR on total RNA from unstimulated HUVECs using standard P2X receptor and β-actin primers, separated by agarose gel electrophoresis. RT-control is for thermal cycling in the absence of template.
Figure 2
Figure 2
Effect of pro-inflammatory cytokines on expression of P2X4 and P2X7 receptors in HUVEC. (a) qRT-PCR for P2X4 receptor message relative to untreated control under various stimulatory conditions for 24 and 48 h. (b) q-RT-PCR for P2X7 receptor message relative to untreated control under various stimulatory conditions for 24 and 48 h. (c) Relative expression of P2X4 and P2X7 receptor message compared to untreated control, for IFNγ (100 ng ml−1)+TNFα (10 ng ml−1) treatment over 72 h. Values shown are (in a and b) means±s.d., n=2; (in c) means±s.e.m, n=6.
Figure 3
Figure 3
Expression of P2X7 receptor protein and functional responses in HUVEC. (a) Flow cytometry using an antibody to the extracellular domain of the human P2X7 receptor to detect protein expression at 24 h for IFNγ (100 ng ml−1) and TNFα (10 ng ml−1) stimulation. (b) Functional whole cell electrophysiology responses in HUVECs. Typical membrane currents for 5 s application of Bz-ATP (300 μM) in low divalent cation solution (LDV), recorded from untreated HUVEC (left), HUVEC treated for 48 h with IFNγ (100 ng ml−1) and TNFα (10 ng ml−1) (centre) and HUVEC over-expressing hP2X7 receptor by adenoviral transfer (right). (c) Whole cell electrophysiology responses to Bz-ATP (300 μM) in LDV, in the absence and presence of KN-62 (1 μM). (d) Mean (±s.d.) current density values from untreated HUVEC (nine cells), HUVEC treated for 48 h with IFNγ (100 ng ml−1) + TNFα (10 ng ml−1) (seven cells) and HUVEC overexpressing hP2X7 receptor by adenoviral transfer (eight cells).
Figure 4
Figure 4
Expression of P2X4 receptor protein and functional responses in HUVEC. (a) Immunoblotting to detect P2X4 receptor protein, for IFNγ (100 ng ml−1)+TNFα (10 ng ml−1) treatment over time. 20 μg protein was loaded in each lane. (b) Cell surface-labelled P2X4 receptor protein blotting using biotinylation. (c) Histogram showing mean current densities from HUVEC, or HEK293 cells overexpressing hP2X7 or hP2X4 receptors for stimulation with 30 μM ATP in the absence (black) or presence (grey) of ivermectin (3 μM), which acts to enhance hP2X4 responses. Values shown are means±s.d. from n=4, HEK/P2X7; n=6, HEK/P2X4; HUVEC, n=12.
Figure 5
Figure 5
IL-1β and IL-1α production in HUVEC. (a) The expression of IL-1β in HUVECs under various inflammatory stimuli was detected by immunoblotting cell extracts. The 31 kDa band represents the unprocessed biologically inactive pro-IL-1β, and the 17 kDa band corresponds to processed biologically active IL-1β. rhIL-1β is recombinant human IL-1β run as control, UnTx are untreated HUVEC controls. Twenty micrograms of protein was loaded in each lane. (b) Stimulation of IL-1β and caspase-1 in HUVECs and THP-1 monocytes. The time-course of induction of IL-1β and caspase-1 was determined by immuno-blotting, for HUVECs treated with TNFα (10 ng ml−1) and IFNγ (100 ng ml−1). Differentiated THP-1 monocytes and HEK293 cells treated for 24 h with the respective inflammatory stimuli, were used as controls. The positive control (con) is recombinant human IL-1β (20 μg), or HL-60 cell lysates for caspase-1. For each set of conditions, initially samples were blotted using anti-human IL-1β, and were then stripped and re-probed with anti-caspase-1 antibodies. Twenty micrograms of protein was loaded into each lane. (c) HUVEC supernatants were compared for IL-1β (left) and IL-1α production (right) following IFNγ/TNFα treatment. Positive controls are recombinant human IL-1β and IL-1α (R&D) and THP-1 cells treated for 24 h with LPS. Twenty micrograms of protein was loaded in each lane.
Figure 6
Figure 6
Released IL-1β from stimulated HUVEC. Cell lysates (a) and supernatants (b), collected from HUVECs treated with TNFα (10 ng ml−1) and IFNγ (100 ng ml−1) for 48 h were tested by ELISA for the presence of IL-1β. (c) Release of IL-1β from HUVEC over-expressing P2X7 receptors. HUVECs were transformed with adenoviral human P2X7-GFP, adenoviral human P2X7-EE or adenovirus alone (Adeno) and treated for 48 h with TNFα (10 ng ml−1) and IFNγ (100 ng ml−1), then for 60 min with buffer (control) or 300 μM Bz-ATP (Bz), or 300 μM Bz-ATP in the presence of 100 nM AZ11645373 P2X7 receptor antagonist (Ant). (a) Supernatants were collected and tested by ELISA (c) or (d), by immunoblotting for the presence of IL-1β. The LDH activity of the HUVEC supernatants was tested as a measure of cell death (e), compared to control Triton X (1%, v/v)-treated cells. Data are mean±s.e.m. for six separate wells. **P<0.01 and *P<0.05 vs control, where data were analysed by one-way ANOVA with Tukey's post-test (b), or by two-way ANOVA with Bonferroni's post-test (c).
Figure 7
Figure 7
Biological activity of supernatants from stimulated HUVEC at the IL-1 receptor. (a) Effect of supernatants from stimulated HUVECs on IL-1 bioassay. Buffer only (Buffer) or supernatants from HUVEC treated for 48 h with TNFα (10 ng ml−1)+IFNγ (100 ng ml−1), then for 60 min with extracellular solution (Con SN), ATP (3 mM, ATP SN) or Bz-ATP (300 μM, Bz SN) were added to the IL-1-sensitive IL-8 luciferase reporter assay in HeLa cells in the absence (clear bars) or presence (filled bars) of 1 pg ml-1 IL-1β. The effect of the HUVEC samples on the IL-8 luciferase activity was compared to the effect of exogenously applied recombinant IL-1Ra (Recomb RA) at 100 ng ml-1. HUVEC cell lysates (Cells) and supernatants (SN) from these same treatments in (a) were tested for the presence of IL-1Ra by ELISA (b) and Western blotting (c) Values in (a) and (b) are means±s.e.m., n=4. (d) HUVEC were transformed with adenoviral human P2X7-GFP, adenoviral human P2X7-EE or adenovirus alone (Adeno) and treated for 48 h with TNFα (10 ng ml−1) and IFNγ (100 ng ml−1), then for 60 min with buffer (control) or 300 μM Bz-ATP (Bz), or 300 μM Bz-ATP in the presence of 100 nM AZ11645373 P2X7 receptor antagonist (Ant). Supernatants were collected and tested by ELISA for IL-1Ra levels. These samples correspond to those tested for IL-1β levels in Figure 6c. Values shown are means±s.e.m., n=3.
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