Allergy Asthma Immunol Res.  2014 Sep;6(5):449-457. 10.4168/aair.2014.6.5.449.

Prostaglandin E2 Induces IL-6 and IL-8 Production by the EP Receptors/Akt/NF-kappaB Pathways in Nasal Polyp-Derived Fibroblasts

• Cho JS
• Han IH
• Lee HR
• Lee HM

• KMID: 2181082
• DOI: http://doi.org/10.4168/aair.2014.6.5.449

Abstract

PURPOSE
Interleukin 6 (IL-6) and IL-8 participate in the pathogenesis of chronic rhinosinusitis with nasal polyps, and their levels are increased by prostaglandin E2 (PGE2) in different cell types. The purposes of this study were to determine whether PGE2 has any effect on the increase in the levels of IL-6 and IL-8 in nasal polyp-derived fibroblasts (NPDFs) and subsequently investigate the possible mechanism of this effect.
METHODS
Different concentrations of PGE2 were used to stimulate NPDFs at different time intervals. NPDFs were treated with agonists and antagonists of E prostanoid (EP) receptors. To determine the signaling pathway for the expression of PGE2-induced IL-6 and IL-8, PGE2 was treated with Akt and NF-kappaB inhibitors in NPDFs. Reverse transcription-polymerase chain reaction for IL-6 and IL-8 mRNAs was performed. IL-6 and IL-8 levels were measured byenzyme-linked immunosorbent assay (ELISA). The activation of Akt and NF-kappaB was evaluated by western blot analysis.
RESULTS
PGE2 significantly increased the mRNA and protein expression levels of IL-6 and IL-8 in NPDFs. The EP2 and EP4 agonists and antagonists induced and inhibited IL-6 expression. However, the EP4 agonist and antagonist were only observed to induce and inhibit IL-8 expression level. The Akt and NF-kappaB inhibitors significantly blocked PGE2-induced expression of IL-6 and IL-8.
CONCLUSIONS
PGE2 increases IL-6 expression via EP2 and EP4 receptors, and IL-8 expression via the EP4 receptor in NPDFs. It also activates the Akt and NF-kappaB signal pathways for the production of IL-6 and IL-8 in NPDFs. These results suggest that signaling pathway for IL-6 and IL-8 expression induced by PGE2 might be a useful therapeutic target for the treatment of nasal polyposis.

Keyword

Nasal polyps; prostaglandins E; interleukin-6; interleukin-8; E prostanoid receptor; Akt

MeSH Terms

Blotting, Western
Dinoprostone*
Fibroblasts*
Interleukin-6*
Interleukin-8*
Nasal Polyps
NF-kappa B
Prostaglandins E
RNA, Messenger
Signal Transduction
Dinoprostone
Interleukin-6
Interleukin-8
NF-kappa B
Prostaglandins E
RNA, Messenger

Figure

• 

  Fig. 1 Effect of PGE2 on IL-6 expression in NPDFs. (A) NPDFs were stimulated with PGE2 (20 µM) in a time-dependent manner. The expression level of IL-6 mRNA was examined using RT-PCR and quantified. (B, C) NPDFs were stimulated with PGE2 in a dose-dependent manner. The mRNA and protein expression levels of IL-6 were examined using RT-PCR for 12 hours (B) and ELISA for 48 hours (C). Values are the mean ± SEM of three independent samples. *P<0.05, †P<0.01 as compared to the mean IL-6 values of control cells.

• 

  Fig. 2 Effect of PGE2 on IL-8 expression in NPDFs. (A) NPDFs were stimulated with PGE2 (20 µM) in a time-dependent manner. The expression level of IL-8 mRNA was examined using RT-PCR and quantified. (B, C) NPDFs were stimulated with PGE2 in a dose-dependent manner. The mRNA and protein expression levels of IL-8 were examined using RT-PCR for 12 hours (B) and ELISA for 48 hours (C). *P<0.05 as compared to the mean IL-8 values of control cells.

• 

  Fig. 3 Effect of EP receptor agonists and antagonists on IL-6 expression in NPDFs. NPDFs were stimulated with the EP receptor agonists, and PGE2 (20 µM), with or without the EP receptor antagonists. IL-6 mRNA expression was determined by RT-PCR for 12 hours to study the effects of EP agonists (A) and antagonists (B). The production of IL-6 was analyzed by ELISA for 48 hours (C) and (D). *P<0.05 as compared to the mean IL-6 values of control cells; †P<0.05 as compared to the mean IL-6 values of PGE2-stimulated cells. EP receptor agonists; Butaprost (10 µM), CAY10580 (10 µM), Sulprostone (10 nM). EP receptor antagonists; AH23848 (10 µM), AH6809 (10 µM).

• 

  Fig. 4 Effect of EP receptor agonists and antagonists on IL-8 expression in NPDFs. NPDFs were stimulated with the EP receptor agonist, and PGE2 (20 µM), with or without the EP receptor antagonist. IL-8 mRNA expression was determined by RT-PCR for 12 hours to study the effects of EP agonists (A) and antagonists (B). Production of IL-8 was analyzed by ELISA for 48 hours (C) and (D). *P<0.05 as compared to the mean IL-8 values of control cells; †P<0.05 as compared to the mean IL-8 values of PGE2-stimulated cells. EP receptor agonists; Butaprost (10 µM), CAY10580 (10 µM), Sulprostone (10 nM). EP receptor antagonists; AH23848 (10 µM), AH6809 (10 µM).

• 

  Fig. 5 The role of Akt in the expression of IL-6 and IL-8 in NPDFs. NPDFs were stimulated with PGE2 (20 µM), with or without the Akt inhibitor (LY294002, 10 µM). The activation of Akt was determined by western blot analysis (A). Akt inhibition of IL-6 expression was analyzed by RT-PCR for 12 hours and ELISA for 48 hours (B and C). Akt inhibition of IL-8 expression was analyzed by RT-PCR for 12 hours and ELISA for 48 hours (D and E). *P<0.05 as compared to the mean IL-6 or IL-8 values of control cells; †P<0.05 as compared to the mean IL-6 or IL-8 values of PGE2-stimulated cells.

• 

  Fig. 6 Role of NF-κB on the expression of IL-6 and IL-8 in NPDFs. NPDFs were stimulated with PGE2 (20 µM), with or without the NF-κB inhibitor (BAY-11, 1 µM). Western blot analysis showed increased expression of NF-κB in case of when PGE2-treated NPDFs and decreased expression of NF-κB in case of BAY-11-treated NPDFs (A). Inhibition of IL-6 or IL-8 expression by BAY-11 treatment was analyzed by RT-PCR for 12 hours (B and D) and ELISA for 48 hours (C and E). *P<0.05 as compared to the mean IL-6 or IL-8 values of control cells; †P<0.05 as compared to the mean IL-6 or IL-8 values of PGE2-stimulated cells.

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