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W. J. O'Brien, T. Heimann; Proinflammatory Cytokines, Signal Transduction and Corneal Stromal Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2749.
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Proinflammatory cytokines interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) bind to receptors on corneal stromal cells and influence the expression of genes that determine the fait of the cells. The aim of these studies was to determine which signal transduction pathways activated by these cytokines mediate stroma fibroblast survival.
Rabbit corneal stromal cells were grown as the fibroblastic phenotype in media containing 5% FBS. Cultures of less than 4 passages were induced to undergo apoptosis by serum deprivation while being treated with various mixtures of IL-1ß, TNF-α and rRaIFN-γ. Apoptosis was assessed by caspase 3 activity and TUNEL staining. Steady state levels of proteins were evaluated by western blots. Cell viability was assessed by Live-Dead cell staining.
Serum deprivation induced apoptosis of rabbit corneal stromal fibroblasts by an extrinsic pathway as determined by TUNEL and annexin V staining, caspase 8, 9 and 3 activities, and DNA laddering. Individually and in combinations all 3 cytokines significantly promoted cell survival as measured by TUNEL staining and reduced caspase 3 activity (ANOVA p<0.05). TNF-α alone functioned as a "contextual modulator" in that it sometimes promoted apoptosis while other times promoted cell survival as shown by others. The mixture of all three cytokines, however, always expressed anti-apoptotic activity in stromal cells of the fibroblastic phenotype. Inhibitors of the PI 3 kinase-Akt pathway such as LY294002 but not triciribine inhibited the anti-apoptotic activity of the cytokine mixture. Rapamycin, the mTOR inhibitor, also significantly reduced the anti-apoptotic activity of the mixture of the three cytokines suggesting the p70 S6 kinase may be essential for anti-apoptotic activity. Cells treated with the cytokine mixture contained increased levels of phosphorylated Akt (ser-473), BAD (ser 112 and 155), and IAP-2.
Mixtures of the proinflammatory cytokines, IL-1ß, TNF-α and IFN-γ promoted corneal stromal cell survival. Nuclear localization of NF-ΚB did not appear essential. The survival pathway includes activation of the PI 3K, Akt, mTOR, and likely p70 S6 kinase. These studies suggest that stromal wound healing problems due to apoptosis of stromal fibroblasts should not be treated with agents that inhibit the anti-apoptotic activity of proinflammatory cytokines.
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