O-101: IL6 expression in human normal peritoneal and adhesion fibroblasts: Regulation by hypoxia

Abstract

We have previously identified phenotypic differences between fibroblasts isolated from normal peritoneum and from adhesions. Specifically, we have shown that adhesion fibroblasts manifest significantly higher levels of the inflammatory cytokines TGF-β1 and type I collagen as compared to normal peritoneal fibroblasts. IL6 is released in response to IL-1 and TNF-alpha and stimulates the acute-phase reaction which leads to a systemic reaction, including inflammation, fever and activation of the complement and clotting cascade. Therefore, we sought to determine the levels of IL6 mRNA in fibroblasts isolated from human normal peritoneum and adhesion tissues under normoxic and hypoxic conditions. Prospective experimental study. Primary cultures of fibroblasts were established from normal peritoneum and adhesion tissues of the same patients (n=3). Fibroblasts were cultured under normal (20% O2) and hypoxic (2% O2) conditions as follow. Hypoxic experiments were performed in an airtight Plexiglas chamber (Bellco Glass, Vineland, NJ). The chamber was deoxygenated by positive infusion of 2% Oxygen in Carbon Dioxide-Nitrogen gas mixture. Cultures were then placed in a standard humidified tissue incubator for 24 hours. Parallel cultures were placed in normoxia for the 24-hour time point. Cells were harvested and total RNA was isolated from each cell type. Complimentary DNA (cDNA) was generated by reverse transcriptase. Aliquots from cDNA were subjected to real-time RT-PCR to measure mRNA levels for IL6, using a QuantitTect SYBR Green kit (Qiagen) and Cepheid 1.2F Detection System. After PCR, a melting curve analysis was performed to demonstrate the specificity of the PCR product as a single peak. The amount of each mRNA was then normalized to the abundance of a housekeeping gene, β-actin. IL6 mRNA was present in both normal peritoneal and adhesion fibroblasts. Adhesion fibroblasts of all patients exhibited significantly higher levels of IL6 mRNA as compared to normal peritoneal fibroblasts (p<0.05). Hypoxia treatment significantly induced IL6 mRNA levels in both normal peritoneal and adhesion fibroblasts. The increase in IL6 mRNA levels of normal fibroblasts reached the levels observed for adhesion fibroblasts (20 fold increase with p<0.05). However, hypoxia had less effect on IL6 mRNA levels in adhesion fibroblasts. The differential expression of IL6 between normal peritoneal and adhesion fibroblasts and the regulation of IL6 by hypoxia suggest a possible inflammatory role in the development of adhesions. Modulation of IL6 may potentially provide the opportunity to reduce postoperative adhesion development.