Figure 1.
S1P induces IL-8 release in BEAS-2B cells.
BEAS-2B cells were stimulated with various concentrations of S1P for 4 hours. Culture supernatant was analyzed for concentration of IL-8 by ELISA (n = 5). Data are representative of means+SE. ANOVA with Tukey post hoc pairwise comparisons. *P<0.05.
Figure 2.
S1P induced IL-8 release is mediated by S1PR2 in BEAS-2B cells.
BEAS-2B cells were pretreated for 30 minutes with (A) W 123 (n = 5), (B) JTE 013 (n = 5) or (C) CAY 10444 (n = 8), specific inhibitors of S1PR1, S1PR2 and S1PR3 respectively and then stimulated with S1P or vehicle for 4 hours. Culture supernatant was analyzed for IL-8 concentration by ELISA. Human airway smooth muscle cells were loaded with 10 µM Fura 2-AM, then treated with CAY10444 or vehicle (n = 5) for 30 minutes and intracellular calcium was measured by ratiometric fluorescence microscopy (E). Cells were stimulated with S1P (n = 5) and increases in resting intracellular calcium were recorded (F). Data are representative of means+SE. ANOVA with Tukey post hoc pairwise comparisons. **P<0.01, ***P<0.001.
Figure 3.
S1P induced IL-8 release is mediated by NF-κB in BEAS-2B cells.
BEAS-2B cells were pretreated with (A) Helenalin (n = 3), (B) SR 11302 (n = 3), or c-JUN peptide (n = 3) inhibitors of NF-κB, AP-1 and AP-1 respectively, for 30 minutes and then stimulated with S1P for 4 hours. Culture supernatants were analyzed for IL-8 concentration by ELISA. Data are representative of means+SE. ANOVA with Tukey post hoc pairwise comparisons. *P<0.05, ***P<0.001.
Figure 4.
S1P induced Nf-κB activity is mediated by S1PR2 in BEAS-2B cells.
Nf-κB luciferase reporter BEAS-2B cells were pretreated with S1PR2 inhibitor JTE 013 for 30 minutes before stimulation with S1P for 4 hours (n = 3). Cell lysates were analyzed for luciferase activity by Tecan iControl plate reader. Data are representative of means+SE. ANOVA with Tukey post hoc pairwise comparisons. ***P<0.001.
Figure 5.
S1P induced IL-8 release is not dependent on the EGFR in BEAS-2B cells.
(A) BEAS-2B cells were pretreated for 30 minutes with the specific EGFR inhibitor AG 1478 and then stimulated with S1P for 4 hours (n = 7). Culture medium was assessed for IL-8 concentration by ELISA. (B) BEAS-2B cells transfected with control or EGFR specific siRNA were stimulated with S1P for 4 hours (n = 3). Culture medium was assessed for concentrations of IL-8 by ELISA (left). Knock-down efficacy is shown by western blot for total EGFR (170 kDa) with GAPDH loading control (37 kDa) (right). Quantification of total EGFR bands is shown (n = 4) (bottom left). (C) BEAS-2B cells were pretreated for 30 minutes with MMP inhibitors GM6001 or TAPI-1 and then stimulated with S1P for 4 hours (n = 3). Culture medium was assessed for IL-8 concentration by ELISA. Data are representative of means+SE. ANOVA with Tukey post hoc pairwise comparisons. *P<0.05, **P<0.01, ***P<0.001.
Figure 6.
S1P induced IL-8 release is not dependent on the production of reactive oxygen species in BEAS-2B cells.
(A) BEAS-2B cells were incubated for 30 minutes with 10 µM DCFH-DA. Free probe was washed with Hank’s buffer and baseline fluorescence was measured at 530 nm. Cells were then stimulated with S1P or vehicle and fluorescence intensity was measured every 5 minutes for one hour (n = 3). Analysis using repeated measures ANOVA revealed no difference between the two curves. (B) BEAS-2B cells were pretreated for 30 minutes with the general antioxidant N-acetyl cysteine (NAC) and then stimulated with S1P for 4 hours (n = 5). Culture medium was assessed for IL-8 concentration by ELISA. (C) BEAS-2B cells were pretreated for 30 minutes with the NADPH oxidase inhibitor DPI and then stimulated with S1P for 4 hours (n = 5). Culture medium was assessed for IL-8 concentration by ELISA. (n = 5). Data are representative of means+SE. ANOVA with Tukey post hoc pairwise comparisons *P<0.05, **P<0.01.