Interleukin–1 Receptor Antagonist (IL–1ra) Prevents Apoptosis in ex vivo Expanded Human Limbal Epithelial Cells Cultivated on Human Amniotic Membrane

Abstract

Abstract: : Purpose: To investigate the differential expression of interleukin–1 receptor antagonist (IL–1ra) in human limbal epithelial cells expanded on intact amniotic membrane (AM) or on plastic dishes, and to demonstrate the anti–apoptotic function of IL–1ra protein in this co–culture model. Methods: Corneoscleral buttons from human donor eyes were cut into 2 x 3 mm2 pieces and cultured on intact AM, or plastic dishes for 3 weeks. Cultures of either condition were subjected to phase contrast microscopic examination and annexin V staining to detect apoptotic cells. Differential expression of genes in between these two conditions was determined by cDNA microarray analysis, which was further confirmed by reverse transcription–polymerase chain reaction (RT–PCR) and enzyme–linked immunosorbent assay (ELISA). Cultures on plastic dishes at day 14 were added with media with or without 200 ng/ml of IL–1ra protein for one week, followed by apoptotic studies. Results: Limbal epithelial cells grown on intact AM demonstrated fewer apoptotic cells as compared with those on plastic dishes. IL–1ra over–expression in cultures on intact AM in both transcriptional and translational level was confirmed by cDNA microarray analysis, RT–PCR and ELISA. The prominent apoptosis detected in cultures on plastic dishes could be reversed by adding recombinant IL–1ra protein into the media. Conclusions: Human AM may prevent cultured human limbal epithelial cells from undergoing apoptosis. In addition to its well–recognized anti–inflammatory effect, IL–1ra may function as an anti–apoptotic molecule during the interaction between limbal epithelial cells and AM.

Keywords: cell death/apoptosis • cornea: epithelium • cytokines/chemokines