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.
Insights
Human corneal cells, including keratocytes and epithelial cells, produce IL-8 when exposed to inflammatory cytokines. Keratocytes release significantly more IL-8 than epithelial cells after IL-1 alpha stimulation.
Area of Science:
- Ophthalmology
- Immunology
- Cell Biology
Background:
- Interleukin-8 (IL-8) is a key chemokine in inflammatory responses.
- Corneal cells play a role in ocular inflammation.
- Understanding cytokine-induced IL-8 production in corneal cells is crucial for managing eye inflammation.
Purpose of the Study:
- To investigate if human corneal keratocytes and epithelial cells synthesize and release IL-8.
- To determine the effect of pro-inflammatory cytokines, specifically IL-1 alpha and TNF-alpha, on IL-8 production in these corneal cells.
Main Methods:
- Isolation and in vitro culture of human corneal keratocytes and epithelial cells.
- Stimulation of cell cultures with varying concentrations of IL-1 alpha or TNF-alpha.
- Quantification of IL-8 in culture supernatants using ELISA and monitoring mRNA levels via Northern blot.
Main Results:
- Both corneal keratocytes and epithelial cells demonstrated dose-dependent IL-8 mRNA synthesis and IL-8 production upon stimulation with IL-1 alpha or TNF-alpha.
- TNF-alpha induced comparable IL-8 synthesis in both cell types.
- Corneal keratocytes produced 33 times more IL-8 than epithelial cells when stimulated with IL-1 alpha.
Conclusions:
- Pro-inflammatory cytokines effectively induce IL-8 synthesis in both human corneal epithelial cells and keratocytes.
- IL-1 alpha appears to have a more significant role in amplifying inflammatory responses within the corneal stroma compared to the epithelial layer.
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.
