Abstract
Purpose: :
RvE1 prevents proinflammatory mediator release from human corneal epithelial cells (HCEC), regulates innate and adaptive immune responses, as well as promotes corneal tissue repair through survival pathway stimulation. All of these effects contribute to hastening recovery of or prevent loss of corneal integrity during stress or infection. ChemR23 is a putative receptor for RvE1, which is expressed in corneal epithelial cells. We determined here if ChemR23 activation contributes to RvE1-induced suppression of IL-6 and IL-8 release.
Methods: :
Immunocytochemistry and Western blot analysis identified ChemR 23 expression in SV40-immortalized HCEC. ELISA monitored changes in IL-6 and IL-8 release. Dose dependent effects were compared of Resolvin E1 (RvE1) (0.01, 0.1, or 1 µM) or pertussis toxin (PTX) (10, 100 or 200 ng/ml) on their release in either isotonic (i.e. 300 mOsm) DMEM/F12 or NaCl supplemented hypertonic (i.e. 450 mOsm) medium. IL-6/8 release was normalized to protein. ChemR23 siRNA transfection knocked down gene and protein expression. Scrambled siRNA and ChemR 23 transfected knock down cells were preexposed to RvE1 and/or PTX for 2 h under isotonic conditions followed by an additional 22 h to the hypertonic stress.
Results: :
ChemR 23 expression was detected in HCEC cell membrane, and slightly increased during exposure to the hypertonic condition. In 450 mOsm, IL-6 and IL-8 release increased ~3/4-fold above the isotonic control levels. Such rises were dose-dependently reduced in the presence of RvE1. With 0.1 µM RvE1, their levels maximally declined by 41% and 52%, respectively. PTX at 10 ng/mL, an inhibitor of Gi-protein coupling, abolished the effects of RvE1. ChemR23 protein expression decreased by 75% in the knockdown cells, which eliminated suppression by RvE1 of hyperosmolar induced increases in IL-6 and IL-8 release.
Conclusions: :
PTX inhibition of RvE1 regulation of HCEC release of IL-6 and IL-8 during hyperosmolar stress is indicative of a Gi-protein coupled receptor engagement. ChemR23 is one potential receptor candidate as its gene silencing also abolished the inhibitory effects of RvE1 on IL-6 and IL-8 release to an extent similar to that seen with PTX.
Keywords: cornea: epithelium • receptors • cytokines/chemokines