S1782 Acetylcholine Protects Against Cytokine Induced Epithelial Barrier Dysfunction

Abstract

Background: Patients with Crohn's disease (CD) have a 'leaky gut' manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumor necrosis factor-α (TNFα) is a key pro-inflammatory cytokine that plays a central role in intestinal inflammation of CD. TNF-α has been shown to cause an increase in intestinal epithelial TJ permeability in cultured intestinal epithelial cell lines; and the TNF-α increase in TJ permeability has been postulated to be an important pathogenic mechanism leading to an increase in intestinal antigenic load and subsequent inflammatory response. However, the role of TNF-α on intestinal barrier function In-Vivo remains unclear. The major aim of this study was to examine the effect of TNF-α on intestinal permeability in an In-Vivo mouse model system and assess the mechanism involved. Methods: C57BL/6 mice (male, 9 weeks old) were administered TNF-α (5μg) intraperitoneally. Intestinal permeability was measured In-Vivo by recycling perfusion of isolated small intestine with perfusate solution containing paracellular probe (FITC-dextran-10Kd) for 2-h experimental period. Intestinal tissue resistance was measured by mounting in Ussing chamber. Results: TNF-α administration resulted in a significant increase in mouse intestinal permeability and decrease in transepithelial resistance in a time-dependent (0, 1, 3, 5, 7 days) manner. TNF-α increase in intestinal permeability was associated with an increase in intestinal tissue MLCK protein level and an increase in enterocyte MLCK mRNA level. (The mouse enterocytes were isolated from the mucosal surface by laser capture microdissection and the enterocyte mRNA level measured by realtime PCR.) ML-7 (MLCK inhibitor) administration (intraperitoneally) prior to TNF-α administration blocked the TNF-α increase in mouse intestinal permeability. Moreover, TNF-α treatment of MLCK knock-out mice (MLCK-/-) did not result in an increase in intestinal permeability or decrease in TER. Conclusions: TNF-α causes an increase in mouse intestinal permeability In-Vivo. The TNF-α increase in intestinal permeability In-Vivo requires an increase in MLCK protein expression in the enterocytes. These results suggest that MLCK plays a central role in the TNF-α induced increase in intestinal permeability in an In Vivo mice model.