IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

Purpose. To determine time course of effect of lipopolysaccharide (LPS) on production of interleukin-8 (IL-8) and monocyte chemotactic protein (MCP) by cultured human corneal stromal cells. Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student's t-test. Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases ( ) after corneal stromal cell stimulation with LPS. Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS. 1. Introduction Leukocytic infiltration of the cornea is a common and important pathologic process observed in infection, autoimmune diseases, allograft rejection, and surgical and nonsurgical trauma. Soluble chemotaxins, such as interleukin-8 (IL-8) and monocyte chemotactic peptide (MCP), both appear to be essential to leukocyte recruitment, accumulation, and activation at sites of inflammation. IL-8 and MCP are distinct polypeptides that directly mediate leukocyte chemotaxis in vitro and in vivo and may be secreted by tissue-based cells exposed to inflammatory cytokines [1, 2]. Both IL-8 and MCP have been shown to be produced by human corneal tissue in response to inflammatory stimuli [3, 4]. Since the cornea is normally avascular, identification of chemotaxins that elicit corneal leukocyte infiltration may be pertinent to understanding pathogenetic mechanisms regulating corneal inflammation and immunity. Lipopolysaccharide (LPS) is a component of gram-negative bacteria cell membrane that is known to induce the innate immune response. The importance of corneal leukocytic infiltration in disease has prompted prior investigations of corneal-derived chemotactic factors, including lipopolysaccharide (LPS) [5–8], but the specific time- and dose-dependent properties of IL-8 and MCP in response to LPS have not previously been identified. In this study, we evaluated cultured human corneal stromal cells for the production of IL-8 and MCP using northern blot analysis to assess gene expression and