C L Cubitt1, R N Lausch, J E Oakes. 1. Department of Microbiology and Immunology, College of Medicine, Mobile, Alabama.
Abstract
PURPOSE: To determine whether interleukin-6 (IL-6) can be synthesized by human corneal keratocytes and epithelial cells. METHODS: Epithelial cells and keratocytes isolated from the same donor corneas were grown in vitro. After 2 to 3 passages, the cultures were exposed to varying concentrations of recombinant human interleukin-1 (IL-1 alpha) or tumor necrosis factor (TNF-alpha). Culture supernatants subsequently underwent enzyme-linked immunosorbent assays for cytokine content. The levels of cytokine mRNA in cell lysates were monitored by the reverse transcription-polymerase chain reaction. RESULTS: Cultured human keratocytes stimulated with 100 U/ml IL-1 alpha for 18 hours produced more than 160 ng IL-6 per 10(6) cells. Under the same conditions 500 U/ml TNF-alpha induced approximately 5 ng IL-6. IL-6 mRNA, evident by 3 hours after exposure to either cytokine, accumulated and persisted through 18 hours. Exposure of epithelial cells to IL-1 alpha or TNF-alpha induced minimal and transient expression of IL-6 mRNA and < 0.5 ng protein product per 10(6) cells. The poor production of IL-6 did not reflect an inability of epithelial cells to respond to IL-1 alpha and TNF-alpha because both cytokines induced these cells to make copious amounts of IL-8. CONCLUSIONS: These results demonstrate that both IL-1 alpha and TNF-alpha could induce keratocytes to produce nanogram levels of IL-6 but IL-1 alpha was a 30-fold more effective inducer. In contrast, neither cytokine could stimulate epithelial cells to make more than picogram quantities of IL-6. The abundant IL-6 synthesized by keratocytes may promote various activities including specific immune responses in surrounding lymphoid tissues.
PURPOSE: To determine whether interleukin-6 (IL-6) can be synthesized by human corneal keratocytes and epithelial cells. METHODS: Epithelial cells and keratocytes isolated from the same donor corneas were grown in vitro. After 2 to 3 passages, the cultures were exposed to varying concentrations of recombinant humaninterleukin-1 (IL-1 alpha) or tumor necrosis factor (TNF-alpha). Culture supernatants subsequently underwent enzyme-linked immunosorbent assays for cytokine content. The levels of cytokine mRNA in cell lysates were monitored by the reverse transcription-polymerase chain reaction. RESULTS: Cultured human keratocytes stimulated with 100 U/ml IL-1 alpha for 18 hours produced more than 160 ng IL-6 per 10(6) cells. Under the same conditions 500 U/ml TNF-alpha induced approximately 5 ng IL-6. IL-6 mRNA, evident by 3 hours after exposure to either cytokine, accumulated and persisted through 18 hours. Exposure of epithelial cells to IL-1 alpha or TNF-alpha induced minimal and transient expression of IL-6 mRNA and < 0.5 ng protein product per 10(6) cells. The poor production of IL-6 did not reflect an inability of epithelial cells to respond to IL-1 alpha and TNF-alpha because both cytokines induced these cells to make copious amounts of IL-8. CONCLUSIONS: These results demonstrate that both IL-1 alpha and TNF-alpha could induce keratocytes to produce nanogram levels of IL-6 but IL-1 alpha was a 30-fold more effective inducer. In contrast, neither cytokine could stimulate epithelial cells to make more than picogram quantities of IL-6. The abundant IL-6 synthesized by keratocytes may promote various activities including specific immune responses in surrounding lymphoid tissues.