title: Involvement of P2X(7) purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts.
abstract: 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.
  
  
  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 μm) 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 μm 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Center of Excellence for Innovation in Chemistry, Department of Oral Biology and Diagnostic Sciences, Chiang Mai University, Thailand.
authors.forename: P
authors.initials: P
authors.lastname: Montreekachon
authors.forename: P
authors.initials: P
authors.lastname: Chotjumlong
authors.forename: J G M
authors.initials: JG
authors.lastname: Bolscher
authors.forename: K
authors.initials: K
authors.lastname: Nazmi
authors.forename: V
authors.initials: V
authors.lastname: Reutrakul
authors.forename: S
authors.initials: S
authors.lastname: Krisanaprakornkit
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides; D002070:Butadienes / Q000494:pharmacology; D054804:Cathelicidins / Q000494:pharmacology*; D002478:Cells, Cultured; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects*; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D016209:Interleukin-8 / Q000187:drug effects* / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3 / Q000502:physiology; D020556:Neutrophil Infiltration / Q000502:physiology; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D058486:Receptors, Purinergic P2X7 / Q000502:physiology*; D012394:Rosaniline Dyes / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D000900:Anti-Bacterial Agents; D057134:Antibodies, Neutralizing; D023181:Antimicrobial Cationic Peptides; D002070:Butadienes; D054804:Cathelicidins; D003470:Culture Media; D004791:Enzyme Inhibitors; D053583:Interleukin-1beta; D016209:Interleukin-8; D009570:Nitriles; D058920:Purinergic P2X Receptor Antagonists; D058486:Receptors, Purinergic P2X7; D012394:Rosaniline Dyes; C113580:U 0126; C482128:MAP2K2 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; C004692:coomassie Brilliant Blue