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J. Huang, B. Stramer, M.E. Fini, J.V. Jester; IL-1 Induces Human Corneal Fibroblast but Not Cultured Corneal Keratocyte Apoptosis Through a Metalloproteinase and TGFß Regulated Cascade . Invest. Ophthalmol. Vis. Sci. 2003;44(13):872.
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Purpose: Previous work suggests that IL-1α induces keratocyte apoptosis during the initial stages of corneal repair. The purpose of this study was to more clearly identify the mechanism of IL-1α induced apoptosis. Methods: Cultured human corneal keratocytes and a telomerase immortalized human corneal fibroblast cell line (HTK) were treated with IL-1α (R&D;) alone or in combination with a MMP inhibitor (GM6001, Chemicon) and neutralizing antibody to TGFß (R&D;) under serum-starved conditions. Apoptosis was quantitatively evaluated by TUNEL and Caspase 3 assay. Cell function was evaluated by fluorescent microscopy to identify actin, fibronectin and vinculin organization and by zymogram to identify expression of MMPs. Results: Short-term treatment with IL-1α showed no effect (<24 hours), while treatment for 7 days increased TUNEL labeling of HTK cells from 0% to 5%, 14%, 22% and 63% with 0, 1, 5, 10 and 100 ng/ml respectively. TUNEL labeling was also associated with increasing Caspase 3 activity of 7%, 28%, 39% and 41%, and increasing MMP2 expression of 204%, 279% 318% and 287% compared to control at respectively day 2, 4, 6 and 8 as well as decreasing actin organization, focal adhesion assembly and fibronectin deposition. IL1-α induced TUNEL labeling was effectively blocked by both GM6001 (82%) and anti-TGFß (92%). No increase in TUNEL labeling was identified for cultured human or rabbit corneal keratocytes treated with the same concentrations of IL-1α. Conclusions: IL-1α induced apoptosis of human corneal fibroblasts through a metalloproteinase and TGF-beta-regulated cascade. The prolonged time course and selectivity for fibroblasts suggests that IL-1α may play a more important role during the later rather than the previously proposed initial stages of corneal wound repair.
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