TUMOR-NECROSIS-FACTOR-ALPHA INDUCES MORPHOLOGICAL AND FUNCTIONAL ALTERATIONS OF INTESTINAL HT29 CL.19A CELL MONOLAYERS

TNF-alpha is a widely distributed proinflammatory cytokine, involved in many disease states. Although it has widely distributed effects, a precise mechanism of action has never been described, in particular at the epithelial level. Morpho-functional changes of the intestinal epithelial monolayer HT29 cl.19A exposed to TNF-alpha were therefore assessed, using electron microscopy (including freeze-fracture replica analysis), as well as measurement of mannitol, Na+ and horseradish peroxidase fluxes across intestinal HT29 cl.19A cell monolayers using Ussing chambers. TNF-alpha receptors were induced on HT29 c1.19A cells by a small non-toxic dose of IFN-gamma (5 U/ml). After 4 h of the combined presence of TNF-alpha (10 ng/ml) and IFN-gamma (5U/ml), the tight junction structure was altered as shown by a significant decrease in the average strand number measured in the apico-basal direction (5.50 +/- 2.70 vs 3.73 +/- 1.39 in control and treated cells respectively, P < 0.0001) and by a significant decrease in junctional depth (0.27 +/- 0.14 and 0.17 +/- 0.10 mu m in control and treated cells respectively, P < 0.0001). These results are in agreement with a decrease in number of 'kiss' sites between contiguous membranes of TNF-alpha treated cells observed in ultrathin sections. In parallel, the paracellular permeability of intestinal monolayers increased in the presence of TNF-a compared to controls as shown by the increase in mannitol (32.4 +/- 4.7 vs 9.3 +/- 0.7 mu g/h.cm(2), P < 0.0003), Na (216 +/- 27 vs 83 +/- 4 mu g/h.cm(2), P < 0.0003) and HRP (274 +/- 67 vs 34 +/- 12 ng/h.cm(2), P < 0.004) fluxes and by the decrease in electrical resistance (43 +/- 7 vs 112 +/- 10 Omega.cm(2), P < 0.0001). These results indicate that, providing receptors are present, TNF-cr induces an alteration of epithelial tight junctions leading to a disruption of the epithelial barrier capacity. (C) 1995 Academic Press Limited.