Involvement of ERK1/2 MAPK in IL-6 and IL-8 release.

<p>(A, B) Cells were treated with UDP alone or co-incubated with ERK1/2 MAPK inhibitor, PD 98059 (50 µM), for 6 h. IL-6 (A) and IL-8 (B) levels were measured using an ELISA. Levels of IL-6 and IL-8 were corrected with vehicle control alone and normalized to UDP-stimulated cytokine release. Each column represents the mean ± S.E. (<i>n</i> = 4–6). Statistically significant inhibitory effects compared with UDP-treated controls are marked with an asterisk (*<i>p</i><0.05, Student’s <i>t</i>-test). (C) Effect of PD 98059 (50 µM) alone on basal IL-6 (16HBE14o- cells) or IL-8 (16HBE14o- cells and primary HBE cells) release. Each column represents the mean ± S.E. (<i>n</i> = 4–6). (D) The 16HBE14o- cells were stimulated with 100 µM UDP for the indicated times. Protein matched lysates were analyzed by western blotting with antibodies against phospho-ERK1/2 and ERK1/2 MAPK. The graph represents fold change in phosphorylation signal over total MAPK, normalized to GAPDH. The phosphoMAPK∶total MAPK ratio of the vehicle control was arbitrarily set to 1. Data shown are representative of six independent experiments (*<i>p</i><0.05 compared with vehicle control, Student’s <i>t</i>-test). (E) Similar western blot experiments were performed to show the inhibitory effect of PD 98059 on UDP-stimulated ERK1/2 MAPK activation in the absence or presence of inhibitor (50 µM) for 30 min. Data shown are representative of five independent experiments (*<i>p</i><0.05 compared with the UDP control, Student’s <i>t</i>-test). (F) Similar to (A) and (B), treating primary HBE cells with PD 98059 inhibited UDP, but not MRS 2693-stimulated IL-8 release (<i>n</i> = 5–6). The cells were stimulated with the P2Y₆ agonists for 6 h.</p

Involvement of P2Y₆ receptors in the poly-L-arginine-induced cytokine response.

<p>Human bronchial epithelial cells (16HBE14o-) were treated with a P2Y₆ receptor antagonist, MRS 2578, alone or with poly-L-arginine for 3 h in the absence or presence of different concentrations of the blocker. Cell-free supernatants were assayed by ELISA for IL-6 (A) and IL-8 (B). Levels of IL-6 and IL-8 were corrected with vehicle control alone and normalized to 3 µM poly-L-arginine. Each column represents the mean ± S.E. (<i>n</i> = 5–7). Statistically significant inhibitory effects compared with poly-L-arginine-treated controls are marked with an asterisk (*<i>p</i><0.05, Student’s <i>t</i>-test). (C-F) Similar experiments were performed by stimulating 16HBE14o- cells with the natural ligand for the P2Y₆ receptor, UDP (10 µM and 100 µM), or a specific P2Y₆ agonist, MRS 2693. Cell supernatants were analyzed for IL-6 (C, E) and IL-8 (D, F). Each column represents the mean ± S.E. (<i>n</i> = 4–8). Statistically significant stimulatory effects compared with vehicle control (normalized to 1) are marked with an asterisk (*<i>p</i><0.05, Student’s <i>t</i>-test). (G, H) UDP or MRS 2693 also stimulated IL-8 release in primary HBE cells (<i>n</i> = 5–7).</p

Involvement of p38 MAPK in IL-6 and IL-8 release.

<p>(A, B) Cells were treated with UDP alone or co-incubated with p38 MAPK inhibitor, SB 203580 (50 µM), for 6 h. IL-6 (A) and IL-8 (B) levels were measured with an ELISA. Levels of IL-6 and IL-8 were corrected with vehicle control alone and normalized to UDP-stimulated cytokine release. Each column represents the mean ± S.E. (<i>n</i> = 4–6). Statistically significant inhibitory effects compared with UDP-treated control are marked with an asterisk (*<i>p</i><0.05, Student’s <i>t</i>-test). (C) Effect of SB 203580 (50 µM) alone on basal IL-6 (16HBE14o- cells) or IL-8 (16HBE14o- and primary HBE cells) release. Each column represents the mean ± S.E. (<i>n</i> = 4–6). Statistically significant inhibitory effects compared with untreated control are marked with an asterisk (*<i>p</i><0.05, Student’s <i>t</i>-test). (D) The 16HBE14o- cells were stimulated with 100 µM UDP for the indicated times. Protein matched lysates were analyzed by western blotting with antibodies against phospho-p38 MAPK and p38 MAPK. The graph represents fold change in phosphorylation signal over total MAPK, normalized to GAPDH. The phosphoMAPK∶total MAPK ratio of the vehicle control was arbitrarily set to 1. Data shown are representative of 6–7 independent experiments (*<i>p</i><0.05 compared with vehicle control, Student’s <i>t</i>-test). (E) Similar western blot experiments were performed to show the inhibitory effect of SB 203580 on UDP-stimulated p38 MAPK activation in the absence or presence of the inhibitor (50 µM) for 60 min. Data shown are representative of five independent experiments (*<i>p</i><0.05 compared with the UDP control, Student’s <i>t</i>-test). (F) Similar to (A) and (B), primary HBE cells were stimulated with UDP or MRS 2693 for 6 h in the absence or presence of SB 203580 (<i>n</i> = 5–6).</p

Measurement of UDP release from 16HBE14o- cells and primary HBE cells damaged by poly-L-arginine.

<p>(A) Probe-1 fluorescence emission intensity as a function of UDP concentration. The line represents a linear regression applied to the data, with a <i>R²</i> of 0.9906. (B) 16HBE14o- cells were incubated in HEPES-buffered saline in the presence of poly-L-arginine (10 µM and 30 µM) for 1 h. Levels of secreted UDP were measured using Probe-1 as described. Each column represents the mean ± S.E. (<i>n</i> = 4). Statistically significant effects compared with vehicle control are marked with an asterisk (*<i>p</i><0.05, Student’s <i>t</i>-test). (C) Similar experiments were performed in primary HBE cells (<i>n</i> = 4).</p

UDP stimulated translocation of NF-κB.

<p>The 16HBE14o- cells were stimulated with vehicle control (A, B) and 100 µM UDP (C, D) for 60 min. The nuclei (A, C) were stained with Hoechst 33342, and NF-<i>κ</i>B (B, D) was stained with anti-NF-<i>κ</i>B and Alexa Fluor 488 goat anti-rabbit secondary antibody (magnification 20×). (E) Immunofluorescence of NF-<i>κ</i>B in the nuclear region and cytoplasmic region was quantified using NIH ImageJ software. Nucleus∶cytoplasm ratios of NF-κB staining were calculated. Each column represents the mean ± S.E. (<i>n</i> = 3) (*<i>p</i><0.01 compared with vehicle control, Student’s <i>t</i>-test).</p

UDP increased [Ca²⁺]_i, IL-6, and IL-8 mRNA expression.

<p>(A) Concentration-response of UDP upon changes in [Ca²⁺]_i in primary HBE cells. The cells were stimulated with different concentrations of UDP, and the Fura-2 ratio was quantified and plotted against the concentration of UDP (<i>n</i> = 3–7). Each data point represents the mean ± S.E. (B) The effect of 100 µM UDP on [Ca²⁺]_i in 16HBE14o- cells was significantly larger than that of primary HBE cells. (*<i>p</i><0.05, Student’s <i>t</i>-test, <i>n</i> = 8). (C–D) The qRT-PCR analysis of mRNA for IL-6 (C) and IL-8 (D). Epithelia were treated with UDP (100 µM) for 6 h. Relative expression of IL-6 and IL-8 was normalized to GAPDH and is shown as fold change relative to untreated controls. Each column represents the mean ± S.E. (<i>n</i> = 5).</p