Fig 1.
Effects of diesel exhaust particulates on viability of nasal fibroblasts.
Cytotoxic effects of diesel exhaust particulates in nasal fibroblasts. Nasal fibroblasts were treated with various concentrations (0–800 μg/mL) of diesel exhaust particulates for 72 hours. Cytotoxicity tests were performed by MTT assay. DEP did not affect cell survival until the concentration reached 400 μg/mL. The graphic data represents the means ± SEM of three independent experiments. * p<0.05 compared to control.
Fig 2.
Cytokine and chemokine array in DEP-induced nasal fibroblasts.
After nasal fibroblasts were untreated or treated with DEP (50 μg/ml) for 72 hours, cytokine and chemokine array was performed. Profiles of mean spot pixel density were measured using Quantity One software (Bio-Rad). The relative levels of IL-6 and IL-8 were much higher in DEP-induced nasal fibroblasts than in untreated cells. The levels of MIF, pentraxin-3, and uPAR were also higher in DEP-induced nasal fibroblasts. The graphic data represents the means ± S.E.M. for four donors. * p<0.05; ** p<0.01 compared to untreated.
Fig 3.
Effect of DEP on expression of IL-6 and IL-8 in nasal fibroblasts.
(A) After treatment with DEP (0–50 μg/mL) for 12 hours, mRNA expression of IL-6 and IL-8 was increased in a dose-dependent manner. (B) After treatment with DEP for 48 hours, protein expression levels of IL-6 and IL-8 were also increased in a dose-dependent manner. The graphic data represents the means ± SEM of three independent experiments. * p<0.05; ** p<0.01 compared to control.
Fig 4.
Signaling pathway of DEP-induced IL-6 and IL-8 expression in nasal fibroblasts.
(A) The expression level of phosphorylated p38 (p-p38) and Akt (p-Akt) was determined by western blot in nasal fibroblast treated by DEP (50 μg/mL), in the in the presence or absence of SB203580 (p38 inhibitor, 10 μmol/L) or LY294002 (Akt inhibitor, 10 μmol/L). β-actin was used as an internal control. (B) Expression levels of IL-6 and IL-8 were measured by ELISA after treatment with DEP with or without SB203580 or LY294002. The graphic data represents the means ± SEM of three independent experiments. * p<0.05, ** p<0.01 compared to control; † p<0.05 compared to treatment with DEP alone.
Fig 5.
NF-κB activation in DEP-induced IL-6 and IL-8 expression in nasal fibroblasts.
(A) Nasal fibroblasts were treated DEP (50 μg/mL) in the presence or absence of BAY117082 (NF-κB inhibitor, 1 μM). Protein expression levels of NF-κB p50 were examined using western blotting. (B) The transcriptional activity of NF-κB promoter by DEP was measured using luciferase assay. Firefly luciferase activity was normalized to Renilla luciferase activity, and the level of induction was reported. (C) IL-6 and IL-8 expression levels by DEP via NF-κB signal pathway were assessed by ELISA. The graphic data represents the means ± SEM of three independent experiments. * p<0.05 compared to control; † p<0.05 compared to treatment with DEP alone.
Fig 6.
DEP-induced IL-6 and IL-8 expression and its suppression by signaling pathway inhibitors in ex vivo organ culture.
Nasal inferior turbinate tissues were cultured ex vivo and treated with DEP (50 μg/mL). Expression of IL-6 and IL-8 was increased after treatment, as determined by ELISA. The increased expression was blocked by pretreatment with SB203580 (10 μmol/L), LY294002 (10 μmol/L), or BAY117082 (1uM). The graphic data represents the means ± SEM of three independent experiments. * p<0.05 compared to control; † p<0.05, †† p<0.01 compared to treatment with DEP alone.
Fig 7.
Signaling pathway for DEP-induced expression of IL-6 and IL-8 in nasal fibroblasts and organ culture of nasal inferior turbinate.
DEP-induced expression of IL-6 and IL-8 is mediated by the dual signaling pathways of p38 and Akt, which converge on the NF-κB pathway.