Tumor necrosis factor-alpha (TNF alpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6

Cytokines are supposed to be mediators in diarrhoeal diseases. The aim of t his study is to characterize the effect of tumor necrosis factor-alpha (TNF alpha) on epithelial barrier function in the colonic epithelial cell line HT-29/B6. Active ion transport and barrier function were measured as short- circuit current and transepithelial electrical resistance (Rf), respectivel y. In parallel, freeze-fracture electron microscopy (EM) of tight junctions (TJ) and immunofluorescence microscopy of the zonula occludens protein-1 ( ZO-1) were performed. Serosal addition of TNF alpha (100 ng/ml) decreased R -t by 81%, This effect was dose-dependent and could be mimicked by antibodi es against the p55 form of the TNF receptor. Cytotoxic effects were exclude d by a negative lactate dehydrogenase (LDH) assay. Immunofluorescence local ization with anti-ZO-1 antibodies revealed no evidence for disruption of th e monolayer after TNF alpha treatment. In freeze-fracture EM, TJ complexity was decreased by TNF alpha, as indicated by a decrease in the number of st rands from 4.7 to 3.4, The tyrosine kinase blocker genistein and the protei n kinase A inhibitor H-8 reduced the effect of TNF alpha. A combination of TNF alpha with interferon-gamma acted synergistically on the epithelial bar rier. In conclusion, TNF alpha impairs epithelial barrier function by alter ing structure and function of the tight junction, which could be of pathoge nic relevance in intestinal inflammation.