Ulcerative Colitis: Florian P

Heller F, Florian P, Bojarski C, Richter J, Christ M, Hillenbrand B, Mankertz J, Gitter AH, Bürgel N, Fromm M, Zeitz M, Fuss I, Strober W, Schulzke JD. · Department of Gastroenterology, Charité, Campus Benjamin Franklin, Berlin, Germany. · Gastroenterology. · Pubmed #16083712 No free full text.

Abstract: BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by a Th2 immune response with inflammation and epithelial barrier dysfunction. So far, Th2 cytokines have not been shown to directly influence epithelial barrier function. METHODS: Lamina propria mononuclear cells (LPMCs) were stimulated and interleukin (IL)-13 was measured by enzyme-linked immunosorbent assay. Functional IL-13 and IL-4 effects were studied on HT-29/B6 colonic epithelial cells in Ussing chambers and by conductance scanning. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. IL-13/IL-4 receptors were analyzed by reverse-transcription polymerase chain reaction and immunofluorescence. Western blotting combined with immunofluorescence was used to detect tight junction proteins. Furthermore, restitution velocity was measured. Finally, mucosal biopsy specimens from patients with UC were compared with cultured cells for these features. RESULTS: LPMCs from patients with UC produced large amounts of IL-13 (985 +/- 73 pg/mL), much more than from controls or patients with Crohn's disease. IL-13Ralpha1 and IL-4Ralpha receptors were present in HT-29/B6 cells and colonic epithelial cells of control patients and patients with UC. IL-13 had a dose-dependent effect on transepithelial resistance of HT-29/B6 monolayers (reduction to 60% +/- 4%), whereas IL-4 had no effect. This was due to an increased number of apoptotic cells (5.6-fold +/- 0.9-fold) and an increased expression of the pore-forming tight junction protein claudin-2 to 295% +/- 37%, both of which contributed equally. Finally, epithelial restitution velocity decreased from 15.1 +/- 0.6 to 10.6 +/- 0.5 microm/h after treatment with IL-13. Parallel changes were observed in human samples, with an increase in claudin-2 expression to 956% +/- 252%. CONCLUSIONS: IL-13 was identified as an important effector cytokine in UC that impairs epithelial barrier function by affecting epithelial apoptosis, tight junctions, and restitution velocity.

Amasheh S, Barmeyer C, Koch CS, Tavalali S, Mankertz J, Epple HJ, Gehring MM, Florian P, Kroesen AJ, Zeitz M, Fromm M, Schulzke JD. · Department of Clinical Physiology, Campus Benjamin Franklin, Charité-University Medicine Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. · Gastroenterology. · Pubmed #15188166 No free full text.

Abstract: BACKGROUND & AIMS: The main limiting factor for sodium absorption in distal colon is the amiloride-sensitive epithelial sodium channel (ENaC). This study aimed to characterize mechanisms involved in the dysregulation of ENaC expression in ulcerative colitis (UC). METHODS: Epithelial preparations from surgically removed inflamed and control sigmoid colons were used. Active electrogenic Na(+) transport (J(Na)) was determined after 8-hour aldosterone stimulation in Ussing-chambers (corrected for the altered epithelial/subepithelial resistance ratio). Subsequently, ENaC alpha-, beta-, and gamma-subunits were analyzed immunohistochemically and in Western and Northern blots (corrected for the inflammatory increase in subepithelial protein content). To study gene regulation, the promoters of beta- and gamma-ENaC were analyzed in reporter gene assays. RESULTS: In controls, aldosterone stimulated J(Na) and induced ENaC beta- and gamma-subunit expression, whereas this response was virtually abolished in UC. Preservation of surface epithelium in UC was indicated by unchanged ENaC alpha-subunit expression, which points also against a mere immaturity or epithelial cell loss. Inhibition of electrogenic sodium transport as well as beta- and gamma-ENaC mRNA expression could be mimicked in control colon by in vitro preexposure for 8 hours to tumor necrosis factor alpha and interferon gamma. Promoter analysis revealed that down-regulation of beta- and gamma-ENaC gene expression was primarily induced by tumor necrosis factor alpha. CONCLUSIONS: We conclude that, in UC, elevated proinflammatory cytokines selectively impair beta- and gamma-ENaC expression, which contributes to diarrhea by reducing colonic sodium absorption.