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Involvement of P2X 7 purinergic receptor and MEK1/2 in interleukin‐8 up‐regulation by LL‐37 in human gingival fibroblasts
Author(s) -
Montreekachon P.,
Chotjumlong P.,
Bolscher J. G. M.,
Nazmi K.,
Reutrakul V.,
Krisanaprakornkit S.
Publication year - 2011
Publication title -
journal of periodontal research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.31
H-Index - 83
eISSN - 1600-0765
pISSN - 0022-3484
DOI - 10.1111/j.1600-0765.2011.01346.x
Subject(s) - purinergic receptor , receptor , interleukin , microbiology and biotechnology , messenger rna , intracellular , immune system , interleukin 8 , interleukin 2 , mtt assay , cytotoxicity , biology , medicine , endocrinology , immunology , chemistry , cell , cytokine , gene , biochemistry , in vitro
Montreekachon P, Chotjumlong P, Bolscher JGM, Nazmi K, Reutrakul V, Krisanaprakornkit S. Involvement of P2X7purinergic receptor and MEK1/2 in interleukin‐8 up‐regulation by LL‐37 in human gingival fibroblasts. J Periodont Res 2011; 46: 327–337.© 2011 John Wiley & Sons A/SBackground 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 10 μ m ) 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 20 μ m U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X 7 receptor) and the neutralizing antibody against P2X 7 blocked IL‐8 up‐regulation in a dose‐dependent manner, consistent with expression of the P2X 7 receptor in HGFs. Conclusion: These findings indicate that LL‐37 induces IL‐8 expression via the P2X 7 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.