In Vitro Models of Ocular Inflammation using Normal Human Conjunctival Cells

Abstract

Abstract: : Purpose. The purpose of this study was to establish an in vitro system to investigate the inflammatory response of human conjunctival epithelial cells upon various pathogenic stimulation. Human conjunctival epithelial cells have been widely used to study the toxicity of a large spectrum of biological agents on their apoptotic/cell proliferative properties. However, it is challenging to induce an inflammatory response in the normal epithelial cell population due to an absence of costimulatory elements, which are often present in vivo during inflammation. Additionally, even inflammatory/immune activation markers such as ICAM-1 and/or MHC II were induced after inflammatory stimulation, there is a lack of functional measurements reflecting the true inflammatory response in these cells. Methods. The response elements of transcriptional factor NF-ΚB was introduced into the normal human conjunctival cells (NHC) via transient transfection. Various inflammatory stimuli such as TNF-α/LPS/IL-1 were used to induce epithelial cell inflammation. The level of endogenous NF-ΚB activation in NHC was assessed by the reporter Luciferase gene activity as well as Western blot analysis. The PMA/Ca²⁺ ionophore-stimulated Jurkat cells were co-cultured with IFN-γ-treated NHC to enforce the inflammatory stimulation. NHC cells were stained for B7.1, B7.2 and VCAM-1. Results. In a time-dependent manner, TNFα, to a lesser extent LPS and IL-1ß significantly induced luciferase activities in NF-ΚB transfected NHC cells. The activation of endogenous NF-ΚB in NHC was confirmed by NF-ΚB nuclear translocation following pro-inflammatory stimulation. The induced inflammatory response can be inhibited by treating cells with prednisolone. Co-culturing NHC with activated T cells increased the expression level of the costimulatory molecules. Conclusion. We have established a cell-based NF-ΚB transfection system for epithelial cell inflammation. By monitoring NF-ΚB signaling pathway, we were able to evaluate/quantify the inflammatory response of NHC cells to pathogenic stimulation. The co-culture between NHC and activated T cells facilitated the inflammatory process in the normally less-responsive normal conjunctival epithelial cells in vitro .

Keywords: inflammation • conjunctiva • transcription factors