title: Short-time exposure of hyperosmolarity triggers interleukin-6 expression in corneal epithelial cells.
abstract: OBJECTIVE
  Although tear hyperosmolarity is assumed to play a major role in dry eye disease, correlation between the level of hyperosmolarity and inflammation remains unclear. The purpose of this study was to examine the effect of short-time hyperosmolarity exposure in the production of inflammatory cytokines in corneal epithelial cells in vitro.
  
  
  METHODS
  Human corneal epithelial (HCE) cells were cultured under different osmotic conditions [310 (control), and 400-1000 mOsm]. Lactate dehydrogenase (LDH) release after short-term (10 minutes) or long-term (24 hours) hyperosmotic stress exposure was evaluated to determine HCE cell cytotoxicity. Production of inflammatory cytokines, including IL-6, IL-1β, IL-8, IL-23, and TGF-β1, due to hyperosmotic stress was also measured by enzyme-linked immunosorbent assay and semiquantitative real-time polymerase chain reaction.
  
  
  RESULTS
  After a 24-hour culture, exposures above 700 mOsm caused all HCE cells to die, 500 and 600 mOsm damaged the cells, whereas 400 mOsm caused no morphological changes. However, there was a significant increase in the release of LDH after 24-hour cultures, even in 400 mOsm. In contrast, LDH examination showed that there was no cytotoxicity for the 10-minute exposures, even at above 800 mOsm. The significant increases in IL-6 production and mRNA expression at 700 mOsm during the short-time exposures were both dependent on the osmolarity. Other cytokines such as IL-1β, IL-8, IL-23, and TGF-β1 were not detected.
  
  
  CONCLUSIONS
  Short-time hyperosmolarity exposure may activate IL-6 expression and production in HCE cells without cytotoxicity. These observations suggest that hyperosmolarity could cause inflammation on the ocular surface in dry eye disease.
authors.affiliation: *Department of Ophthalmology, Nippon Medical School, Tokyo, Japan; and †Department of Biochemistry and Molecular Biology, Division of Gene Therapy, Research Center for Advanced Medical Technology, Nippon Medical School, Tokyo, Japan.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Igarashi
authors.forename: Chiaki
authors.initials: C
authors.lastname: Fujimoto
authors.forename: Hisaharu
authors.initials: H
authors.lastname: Suzuki
authors.forename: Masafumi
authors.initials: M
authors.lastname: Ono
authors.forename: Osamu
authors.initials: O
authors.lastname: Iijima
authors.forename: Hisatomo
authors.initials: H
authors.lastname: Takahashi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takahashi
mesh_terms: D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism*; D007378:Interleukins / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009994:Osmolar Concentration; D009997:Osmotic Pressure / Q000187:drug effects; D060888:Real-Time Polymerase Chain Reaction; D012462:Saline Solution, Hypertonic / Q000633:toxicity*
chemical_list: C508600:IL6 protein, human; D053583:Interleukin-1beta; D015850:Interleukin-6; D007378:Interleukins; D012462:Saline Solution, Hypertonic; D007770:L-Lactate Dehydrogenase