Abstract
The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-alpha) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell microporous filters and treated with TNF-alpha (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-alpha treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-alpha decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-alpha did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-alpha increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.
Highlights
The intestinal mucosal barrier plays an important role in the body’s protection against luminal pathogens and antigenic molecules
The effect of TNF-α on Caco-2 tight junctions (TJ) permeability was determined by measuring transepithelial electrical resistance (TER) and epithelial permeability to the paracellular marker Lucifer yellow
The intestinal barrier is primarily formed by the intestinal epithelial cells and the TJs between them
Summary
The intestinal mucosal barrier plays an important role in the body’s protection against luminal pathogens and antigenic molecules. The intestinal barrier includes secreted mucus and the epithelial cell itself, which serves as a selective barrier permitting the uptake of nutrients, ions and other desired solutes, helping to maintain homeostasis of the internal environment. Pathological events such as intestinal inflammation, sepsis, burn, end-stage liver diseases, and severe pancreatitis cause impairment of the intestinal epithelial barrier. Tumor necrosis factor alpha (TNF-α), which is increased in many disease states, is a proinflammatory cytokine. Epithelial cell apoptosis [3,4,8], protein kinase C, nuclear factor-kappaB, myosin light chain kinase, and mitogen-activated protein kinase are thought to be involved [9,10,11,12]
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