Figure 1.
Expression of inflammatory cytokines and their receptors in neutrophils.
Circulating neutrophils isolated from 6 different donors were treated with PBS, Ang1 (10−8 M) (A) or Ang2 (10−8 M) (B) for 90 minutes prior to RNA isolation. Data are expressed in a Volcano plot, as fold change in gene expression (x-axis) compared to PBS-treated cells; values outside the dotted vertical lines indicate significant fold increases (positive values) or fold decreases (negative values). Values below the dashed horizontal line (p<0.05) underline statistical significance (y-axis). Each circle corresponds to the fold-expression of a single gene.
Table 1.
Expression change of inflammatory cytokines and their receptors following Ang1 treatment.
Table 2.
Expression change of inflammatory cytokines and their receptors following Ang2 treatment.
Figure 2.
Kinetics of mRNA expression of IL-1β and IL-1RA.
Primers were designed to quantify changes in the mRNA levels of IL-1β (A) and IL-1RA (B), following treatment with PBS, angiopoietins (10−8 M) or LPS (1 µg/ml), for up to 6 hours. For each time point, basal (PBS) mRNA expression is set to unitary value, and the data are presented as fold change compared to its corresponding PBS. For angiopoietins, only values resulting from treatment with the highest concentration (10−8 M) are shown. Data are represented as the means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001 vs. PBS-control (Dunnett's test).
Table 3.
IL-1 mRNA and protein expression in freshly isolated and Ang1-stimulated neutrophils.
Figure 3.
Kinetics of IL-1β and IL-1RA protein expression.
Neutrophils were treated with PBS, angiopoietins (10−8 M) or LPS (1 µg/ml), for up to 24 hours. Intracellular IL-1β (A), IL-1RA (B) and extracellular IL-1RA (C) were quantified by ELISA. For angiopoietins, only values resulting from treatment with the highest concentration (10−8 M) are shown. No IL-1β was detected extracellularly. Data are represented as the means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, *** p<0.001 vs. PBS-control (Dunnett's test).
Figure 4.
Kinetics of pro-IL-1β protein expression.
Neutrophils were treated with PBS, angiopoietins (10−8 M) or LPS (1 µg/ml), for up to 24 hours. Only intracellular levels of pro-IL-1β were detectable, as no pro-IL-1β was detected in the supernatants at any time points and under any of the conditions tested. For angiopoietins, only values resulting from treatment with the highest concentration (10−8 M) are shown. Data are represented as the means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, *** p<0.001 vs. PBS-control (Dunnett's test).
Figure 5.
Effect of potassium depletion on IL-1β release.
Neutrophils were treated with PBS, angiopoietins (10−8 M) or LPS (1 µg/ml), for two hours, followed by an additional 45-minute treatment with potassium ionophore nigericin, CHX, or appropriate vehicles. Changes in intracellular (left panels) and extracellular (right panels) levels of pro-IL-1β (A) and IL-1β (B) before and after ionophore addition were quantified by ELISA. CHX: Cycloheximide. N: Nigericin. Vehicles: DMSO, ethanol. Data are represented as the means ± SEM of at least three independent experiments. *p<0.05, **p<0.01, *** p<0.001 vs. PBS-control (Dunnett's test).
Figure 6.
Effect of downstream signaling inhibitors on IL-1β and IL-1RA mRNA expression.
Neutrophils were pretreated with inhibitors of Akt (Triciribine; 5 µM), p38 MAPK (SB203580; 10 µM), and p42/44 MAPKK (U0126; 20 µM), vehicle-DMSO (0.2%) or PBS for 30 minutes prior to a 1-hour agonist challenge. Total mRNA was used in RT-qPCR for assessment of mRNA expression of IL-1β (A) and IL-1RA (B). Data are presented as mean ± SEM of at least three independent experiments. *p<0.05, **p<0.01, *** p<0.001 vs. PBS-control within each set (Dunnett's test); §p<0.05, §§p<0.01 vs corresponding agonist-DMSO (Tukey's test).
Figure 7.
Effect of downstream signaling inhibitors on IL-1β and IL-1RA protein synthesis and release.
Neutrophils were pretreated with inhibitors of Akt (Triciribine; 5 µM), p38 MAPK (SB203580; 10 µM), and p42/44 MAPKK (U0126; 20 µM), vehicle-DMSO (0.2%) or PBS for 30 minutes prior to a 2-hour agonist challenge. Concentrations of intracellular pro-IL-1β (A), IL-1β (B), IL-1RA (C) and released IL-1RA (D) were quantified by ELISA. Data are represented as mean ± SEM of at least three independent experiments. *p<0.05, **p<0.01, *** p<0.001 vs. PBS-control within each set (Dunnett's test); §p<0.05, §§p<0.01 and §§§p<0.001 vs corresponding agonist-DMSO (Tukey's test).