Abstract
Purpose:
To test the hypothesis that IFN-γ inhibits conjunctival goblet cell growth and differentiation.
Methods:
Primary conjunctival epithelial cultures were established from fresh tissue explants of murine palpebral conjunctiva. Cultures were incubated with Keratinocyte SFM (KSFM) medium supplemented with 40 ng/mL murine EGF and 3% fetal bovine serum. Cultures were treated with 0.1 ng/mL of IFN-γ for seven or 14 days. IFN-γKO were also examined. Cell proliferation was measured by WST-1 assay. Keratin 7 (K7), MUC5AC, Annexin V were immunodetected by immunofluorescent staining. K7, MUC5AC, MHC class II mRNA expression were measured by quantitative real time PCR.
Results:
Positive K7 and MUC5AC immunofluorescent staining confirmed the growth of conjunctival epithelium and goblet cells in untreated control cultures. WST-1 cell viability assay showed cultures initiated from IFN-γKO mice had 2.24 fold and 2.01 fold greater number of viable cells at 7 and 14 days, respectively, compared to those from WT C57BL/6 mice. Conjunctival cultures treated with minuscule concentrations of IFN-γ (0.1 ng/ mL) do not express K7 or MUC5AC, manifest a spindle-like appearance, and are strongly Annexin V positive. PCR analysis indicated that IFN-γ-treated cultures had a two-fold decrease in MUC5AC expression and a 10-fold decrease in K7 expression compared to untreated cultures. In contrast, IFN-γ-treated cultures had 283-fold increase in expression of MHC class II compared to untreated cultures. A significant 2.58 fold elevation in MUC5AC mRNA transcripts was observed IFN-γKO cultures compared to WT C57BL/6 cultures, but K7 mRNA transcripts were not significantly different between the two strains.
Conclusions:
Our findings show that murine conjunctival cultures treated with very low concentrations of IFN-γ do not become goblet cells like untreated cultures. Conjunctival explants from IFN-γ deficient mice have increased growth and differentiation compared to wild-type mice and increased expression of MUC5AC. This study suggests that IFN-γ suppresses conjunctival epithelial growth and goblet cell differentiation.
Keywords: 474 conjunctiva •
555 immunomodulation/immunoregulation