Effects of Oxidant Stress on Equine Skin Epithelial Proinflammatory Cytokine and MMP Gene Expression
The epithelial cell is not only a target of inflammation, but has also been found to respond to inflammatory stimuli via expression of signaling molecules important in inflammatory injury including proinflammatory cytokines, chemokines and matrix metalloproteases (MMPs). We have recently found evidence of laminar oxidant stress in the early stages of laminitis. Cellular oxidant stress plays a central role in epithelial cell dysfunction in human sepsis-related inflammatory injury of organs; reactive oxygen and nitrogen species (ROS and RNS) responsible for cellular oxidant stress are produced by infiltrating leukocytes, and also reportedly by the host cells themselves during inflammatory signaling induced by Toll-like receptor-4 (TLR4) ligands. Due to these findings, we compared the inflammatory response of the equine epidermal epithelial cell (a critical cell type at the site of laminar failure) exposed directly to ROS to the epithelial response elicited by exposure to the TLR4 ligand lipopolysaccharide (LPS). Skin epithelial cells were obtained for primary culture from two horses. Epithelial cell cultures (passage 3-4) were exposed for 1H and 4H to ROS or LPS (5µg/ml). ROS included H2O2 (50uM, 200uM, 1mM) and xanthine/xanthine oxidase (X/XO, 100uM/3mU, 200uM/30mU; superoxide radical generation confirmed with nitro blue tetrazolium chloride). All experiments were performed in triplicate. Real time-quantitative PCR was used to assess mRNA concentrations of heme oxygenase-1 (HO-1, indicator of redox-stimulated gene expression), proinflammatory cytokines (IL-1β, IL-6 and IL-8) and MMPs (MMP-2 and MMP-9). Although variability in gene expression existed between cultures from the two horses, several patterns were evident. HO-1 gene expression was induced by oxidant stress (both H2O2 and X/XO) at 4H (P<0.05) but, in contrast to most reports, was not induced by exposure to LPS. IL-6, which was markedly induced by LPS at 1H and 4H (P<0.05), underwent minimal induction with oxidant stress. The potent neutrophil chemokine IL-8 was induced to a similar extent by both LPS (P<0.05) and oxidant stress (P<0.05). MMP-9 expression was significantly induced at 4H by oxidant stress but was unaffected by LPS. In conclusion, oxidant stress appears to produce a specific inflammatory response that differs markedly from the response elicited by LPS. Both the absence of HO-1 induction by LPS and the distinct differences in induction of cytokine expression between ROS and LPS exposure indicate that LPS may not cause significant changes in the redox state of the epidermal cells, and the TLR signaling is working through different signaling mechanisms than ROS. Coinciding HO-1 and MMP-9 upregulation indicates oxidant stress may be a contributing factor to MMP dysregulation reported to occur in laminitis. Future comparison of skin and laminar epithelium cell culture response will be helpful in understanding the heightened susceptibility of the laminar epithelium to injury during sepsis.