Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts

Background and Objective: The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleu...

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Main Authors: Montreekachon P., Chotjumlong P., Bolscher J.G.M., Nazmi K., Reutrakul V., Krisanaprakornkit S.
Format: Article
Language:English
Published: 2014
Online Access:http://www.scopus.com/inward/record.url?eid=2-s2.0-79954880573&partnerID=40&md5=f7d983f39f19b3901dd8eb263918312e
http://cmuir.cmu.ac.th/handle/6653943832/1127
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spelling th-cmuir.6653943832-11272014-08-29T09:17:47Z Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts Montreekachon P. Chotjumlong P. Bolscher J.G.M. Nazmi K. Reutrakul V. Krisanaprakornkit S. Background and Objective: The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleukin-8 (IL-8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL-8 induction. Material and Methods: Cultured fibroblasts were treated with different concentrations of LL-37 or interleukin-1β (IL-1β), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT-PCR and real-time PCR were conducted to analyze the expression of IL-8 mRNA, and the IL-8 levels in cell-free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL-37. Results: Nontoxic concentrations of LL-37 (up to 10μm) and IL-1β significantly up-regulated the expression of IL-8 mRNA in a dose-dependent manner (p<0.05). The IL-8 protein levels were consistently significantly elevated in conditioned media of LL-37-treated HGFs (p<0.05). IL-8 up-regulation by LL-37 was completely abrogated by 20μm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X7 receptor) and the neutralizing antibody against P2X7 blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X7 receptor in HGFs. Conclusion: These findings indicate that LL-37 induces IL-8 expression via the P2X7 receptor and the MEK1/2-dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues. © 2011 John Wiley & Sons A/S. 2014-08-29T09:17:47Z 2014-08-29T09:17:47Z 2011 Article 223484 10.1111/j.1600-0765.2011.01346.x 21338358 JPDRA http://www.scopus.com/inward/record.url?eid=2-s2.0-79954880573&partnerID=40&md5=f7d983f39f19b3901dd8eb263918312e http://cmuir.cmu.ac.th/handle/6653943832/1127 English
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
language English
description Background and Objective: The antimicrobial peptide LL-37, derived from human neutrophils, can directly chemoattract leukocytes and up-regulate the expression of several immune-related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL-37 on interleukin-8 (IL-8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL-8 induction. Material and Methods: Cultured fibroblasts were treated with different concentrations of LL-37 or interleukin-1β (IL-1β), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT-PCR and real-time PCR were conducted to analyze the expression of IL-8 mRNA, and the IL-8 levels in cell-free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL-37. Results: Nontoxic concentrations of LL-37 (up to 10μm) and IL-1β significantly up-regulated the expression of IL-8 mRNA in a dose-dependent manner (p<0.05). The IL-8 protein levels were consistently significantly elevated in conditioned media of LL-37-treated HGFs (p<0.05). IL-8 up-regulation by LL-37 was completely abrogated by 20μm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X7 receptor) and the neutralizing antibody against P2X7 blocked IL-8 up-regulation in a dose-dependent manner, consistent with expression of the P2X7 receptor in HGFs. Conclusion: These findings indicate that LL-37 induces IL-8 expression via the P2X7 receptor and the MEK1/2-dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL-37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues. © 2011 John Wiley & Sons A/S.
format Article
author Montreekachon P.
Chotjumlong P.
Bolscher J.G.M.
Nazmi K.
Reutrakul V.
Krisanaprakornkit S.
spellingShingle Montreekachon P.
Chotjumlong P.
Bolscher J.G.M.
Nazmi K.
Reutrakul V.
Krisanaprakornkit S.
Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts
author_facet Montreekachon P.
Chotjumlong P.
Bolscher J.G.M.
Nazmi K.
Reutrakul V.
Krisanaprakornkit S.
author_sort Montreekachon P.
title Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts
title_short Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts
title_full Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts
title_fullStr Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts
title_full_unstemmed Involvement of P2X7 purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts
title_sort involvement of p2x7 purinergic receptor and mek1/2 in interleukin-8 up-regulation by ll-37 in human gingival fibroblasts
publishDate 2014
url http://www.scopus.com/inward/record.url?eid=2-s2.0-79954880573&partnerID=40&md5=f7d983f39f19b3901dd8eb263918312e
http://cmuir.cmu.ac.th/handle/6653943832/1127
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