Abstract
Abstract: :
Purpose: Apoptosis plays significant roles in corneal development, wound healing and the response to inflammation and infection. Our aim was to determine the effects of proinflammatory cytokines on the survival of corneal stromal cells induced to undergo apoptosis by serum deprivation. Methods: Rabbit corneal stromal cells were grown to 2nd to 4th passage in DMEM containing 5% serum. Cells were then induced to undergo apoptosis by reducing the concentration of serum to 0.5%. Triplicate cultures were treated with a mixture of the proinflammatory cytokines IL 1–beta, TNF–alpha and rRaIFN–gamma or retained in medium containing 0.5% serum. The cellular response to the cytokines was monitored by nitrite and superoxide production. Cell death and apoptosis were measured by the Live/Dead Cell Assays and TUNEL staining respectively. Results: Cultures grown from the corneal stroma contained 2.3%±1.3% (n=8)(p<0.05) myofibroblasts and appeared primarily in a fibroblastic phenotype. Serum deprivation caused the number of dead cells in the attached cell population to rise from 2.8±2.2% to 38.4±6.7% (n=4) in 48hr. The addition of a mixture of the proinflammatory cytokines at the time of serum deprivation reduced the number of dead cells to 15.5±3.3% (n=4) (p<0.02). Flow cytometric analysis of TUNEL/propidium iodide stained serum–deprived cultures and serum–deprived cultures treated with IL1–beta, TNF–alpha and rRaIFN–gamma documented that cytokine treatment reduced the numbers of apoptotic cells from 2833±187 to 812±108 per 10^4 cells counted.(p<0.001). Increased nitrite production indicated that the serum–deprived cultures were responsive to the cytokines and suggested that the NFkß signal transduction system was activated. Inhibitors of inducible nitric oxide synthase 2, 1400W and L–NAME, had no affect on cytokine–induced cell survival, but PEG–SOD inhibited the anti–apoptotic affects. Conclusions: The mixture of proinflammatory cytokines promoted survival of rabbit corneal stromal cells by reversing or blocking apoptosis induced by serum deprivation via a superoxide dependent mechanism.
Keywords: cell death/apoptosis • cytokines/chemokines • cornea: stroma and keratocytes