IL-8 gene expression in cultures of human corneal epithelial cells and keratocytes
C L Cubitt1, Q Tang, C A Monteiro
1Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile 36688.
Purpose:
To determine if human corneal keratocytes and epithelial cells synthesize and release IL-8 after stimulation with selected proinflammatory cytokines.
Methods:
Human corneal keratocytes and epithelial cells were isolated from human corneal buttons and grown independently in vitro. Epithelial cell cultures stained positive in immunofluorescent tests with antibody specific for keratin (AE1/AE3), whereas keratocyte cultures were unreactive. Both cell types reacted with anti-vimentin antibody. Cultures of the two cell types were treated with various concentrations of human recombinant interleukin-1 alpha or TNF-alpha. Culture supernatants were then assayed at timed intervals by enzyme-linked immunosorbent assay for IL-8 content. Cytokine mRNA levels in cell lysates were monitored by Northern blot analysis.
Results:
Exposure of corneal keratocytes and epithelial cells to either interleukin-1 alpha or TNF-alpha stimulated IL-8 mRNA synthesis and IL-8 production in a dose-response fashion. It was also found that TNF-alpha stimulated the synthesis of comparable amounts of IL-8 in both cell types. However, when IL-8 synthesis between the two cell types was compared after interleukin-1 alpha stimulation it was found that keratocytes synthesized 33 times more IL-8 than did epithelial cells.
Conclusions:
The results establish that pro-inflammatory cytokines can induce IL-8 synthesis in both human corneal epithelial cells and human corneal keratocytes. They also suggest that interleukin-1 alpha may play a more active role in amplifying inflammatory responses in the stroma than in the epithelial layer of the cornea.
