title: Stimulation of specific cytokines in human conjunctival epithelial cells by defensins HNP1, HBD2, and HBD3.
abstract: OBJECTIVE
  To investigate the effect of human defensins HNP1, HBD2, and HBD3 on human conjunctival epithelial cell cytokine secretion.
  
  
  METHODS
  HNP1, HBD2, and HBD3 were used to test cytotoxicity (1-50 microg/mL) and to stimulate (1-20 microg/mL) primary cultured and immortalized human conjunctival epithelial (IOBA-NHC) cells. Cytokine concentrations in the culture medium were measured by cytokine array and a multiplexed microbead analysis. Protein kinase activation was determined by Western blot analysis after defensin stimulation and with specific inhibitors.
  
  
  RESULTS
  HBD3, but not HNP1 or HBD2, killed more than 50% of IOBA-NHC cells at concentrations greater than 12.5 microg/mL. Only IL-6, IL-8, and RANTES were detected in the culture medium in the absence of defensins. All three cytokines increased in the presence of HNP1, HBD2, and HBD3 at concentrations of 5 to 20 microg/mL and between 2 and 8 hours and further accumulated at 24 hours Stimulation with HBD2 and HBD3 increased the secretion of IL-2 and MIP-1beta in IOBA-NHC cells but only of MIP-1beta in primary cultured cells. Activation of p42/44 mitogen-activated protein (MAP) kinase, Akt, and STAT3 was observed in primary and IOBA-NHC cells after defensin stimulation. Cytokine secretion was significantly decreased by the inhibition of p42/44 MAPK in IOBA-NHC cells.
  
  
  CONCLUSIONS
  HNP and HBD selectively increase the secretion of specific proinflammatory cytokines in conjunctival epithelial cells in a time- and concentration-dependent manner, suggesting a supporting role to the innate immune system of the ocular surface.
authors.affiliation: Singapore Eye Research Institute, Singapore.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.forename: Hong Yuan
authors.initials: HY
authors.lastname: Zhu
authors.forename: Roger W
authors.initials: RW
authors.lastname: Beuerman
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D003228:Conjunctiva / Q000187:drug effects* / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D023084:alpha-Defensins / Q000494:pharmacology*; D023083:beta-Defensins / Q000494:pharmacology*
chemical_list: D000890:Anti-Infective Agents; D016207:Cytokines; C422950:DEFB103A protein, human; C106641:DEFB4A protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D023084:alpha-Defensins; D023083:beta-Defensins; C406413:human neutrophil peptide 1; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3