Fig 1: Corneal stromal cell-derived IL-1ß and CCL20 are involved in LPA-induced proliferation of corneal endothelial cells (CECs). A, Mitomycin C-pretreated human corneal stromal cells (HCSCs) were maintained in TC medium with or without 20 µmol/L LPA for 2 d before CM collection. ELISA showed that the concentrations of secreted IL-1ß and CCL20 were both significantly greater in the LPA-treated group. B, The dose-dependence of IL-1ß- and CCL20-induced B4G12 cell proliferation was determined by cell counts. The number of cells was significantly higher after treatment of recombinant IL-1ß alone for 2 d. In contrast, no difference was observed in the CCL20-treated group. C, The involvement of IL-1ß in LPA-induced proliferation of CECs was investigated via neutralization assay of the CM. Immortalized HCECs (B4G12) co-cultured with mitomycin C-pretreated HCSCs in TC medium for 5 d were greater in cell number in the LPA-treated group (20 µmol/L). Subsequent IL-1ß neutralization with 2 ng/mL IL-1ß-specific antibody (IL-1ß Ab, clone AS10) significantly attenuated the LPA-induced proliferation in co-cultures. D, The proliferation-promoting effect of IL-1ß on RCECs was also examined 2 d later. The fraction of RCECs in the co-culture increased significantly with increasing concentrations of IL-1ß (20, 200 or 1000 pg/mL). E, The morphology and ECD of the 200 pg/mL IL-1ß-treated tissue-cultured rabbit corneal endothelium was examined via immunostaining for ZO-1 on Day 2. The corneal ECD was significantly higher in the LPA-treated cells than that in controls and had the normal hexagonal phenotype typical of RCECs. (n = 3; *P < .5, **P < .05)