1. Volume 129, Issue 2, Pages 550-564 (August 2005)
Interleukin-13 Is the Key Effector Th2 Cytokine in Ulcerative Colitis That Affects Epithelial Tight Junctions, Apoptosis, and Cell Restitution 
Frank Heller, Peter Florian, Christian Bojarski, Jan Richter, Melanie Christ, Bernd Hillenbrand, Joachim Mankertz, Alfred H. Gitter, Nataly Bürgel, Michael Fromm, Martin Zeitz, Ivan Fuss, Warren Strober, Jörg D. Schulzke 
Gastroenterology 
Volume 129, Issue 2, Pages (August 2005)
DOI: /j.gastro
Copyright © 2005 American Gastroenterological Association Terms and Conditions

2. Figure 1
(A) IL-13 and (B) IFN-γ production of LPMCs. LPMCs were isolated from colectomy specimens of noninflammatory controls (NIC), patients with active CD, and patients with UC. T cells were stimulated with soluble antibodies for CD2 and CD28. Cytokines were measured by enzyme-linked immunosorbent assay in culture supernatants collected after 48 hours of culture. Cells did not produce any cytokines without stimulation. (C) IL-13 receptors are expressed on HT-29/B6 cells and epithelial cells in the colon. RNA was isolated from untreated (lanes 1–6) and IL-13-treated (lanes 7–12) HT-29/B6 cells. After reverse transcription, DNA of IL-13Rα1 (lanes 1, 4, 7, and 10), IL-13Rα2 (lanes 2, 5, 8, and 11), and IL-4Rα (lanes 3, 6, 9, and 12) was amplified. (D) HT-29/B6 monolayers and colonic biopsy specimens from noninflammatory controls and patients with UC were stained with antibodies for IL-4R (green) and IL-13Rα1 (red).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

3. Figure 2
Effect of IL-13 on Rt. (A) Time course of Rt after basolateral addition of 10 ng/mL IL-13 and without IL-13. Values are means ± SEM of 6–9 filters. (B) Dose-response curve 48 hours after basolateral addition of IL-13 (n = 6–9; **P < .01, ***P < .001). (C) Rt after addition of IL-13, IL-4, TNF-α, or blocking antibodies to IL-4Rα. (D) IL-13 leads to STAT-6 phosphorylation but not nuclear translocation of NF-κB. Western blot of untreated (lanes 1 and 3) and IL-13-treated (lanes 2 and 4) HT-29/B6 cells for phosphorylated STAT-6 at indicated times. Immunofluorescence localization of the p65 subunit of NF-κB (red) and nuclei (4′,6-diamidino-2-phenylindole, blue). Only HT-29/B6 cells treated with TNF-α but not control or IL-13-treated cells show nuclear translocation of NF-κB.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

4. Figure 3
Epithelial cell apoptosis is induced by IL-13. (A) Apoptosis of HT-29/B6 monolayers stained with TUNEL (original magnification 20×). Condensed chromatin fragments in nuclei and segmentation of the nuclei indicate epithelial cell apoptosis. Apoptotic rate is the percentage of TUNEL-positive cells per total counted cells. (B) HT-29/B6 monolayers were treated with the indicated cytokines and/or Z-VAD-FMK as apoptosis inhibitor. After 48 hours, cells were brought into single-cell suspension and stained with TUNEL. Cell suspensions were then analyzed by FACS. ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

5. Figure 4
IL-13 causes an increase in conductance of apoptotic leaks. (A) Representative image of a single-cell apoptosis (arrows) in colonic epithelial cells after staining of the tight junction proteins occludin (red) and ZO-1 (green) and merging those 2 images (overlay in yellow). The blue lines in the z-stacks indicate the level of focus for the top image. (B) After identification of the single-cell apoptosis, conductance scanning revealed an increase in conductance of single apoptosis after IL-13 incubation. In control specimens, median conductance was 100 nS (white columns) and after IL-13 incubation was 630 nS (gray columns). (C) Z-VAD-FMK or Z-DEVD-FMK can inhibit the effect of IL-13 only partially. HT29/B6 cells were incubated with or without IL-13 and the apoptosis inhibitors Z-VAD-FMK or Z-DEVD-FMK. The decrease of Rt after IL-13 incubation is significantly ameliorated (**P < .001) but not completely prevented.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

6. Figure 5
Immunofluorescence localization of the tight junction proteins ZO-1, occludin, and claudin-2 in HT-29/B6 epithelial cells. Under control conditions and after incubation with IL-13 (10 ng/mL) for 48 hours (original magnification 6300×), some regions of the monolayer slightly leave the focus level of single pictures. Refocusing of these respective regions clearly revealed an intact monolayer at all sites.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

7. Figure 6
Expression of the pore-forming tight junction protein claudin-2 is increased by IL-13 in HT-29/B6 cells. (A) Expression of tight junction proteins in crude membrane fractions by Western blotting. Lanes 1–3 represent samples from controls, and lanes 4–6 represent samples from IL-13-treated cells. Protein expression was quantified by densitometry. The Table shows protein expression after IL-13 incubation in percent of the control. (B) RNA was isolated from control or IL-13-treated cells. After adjusting RNA concentration and reverse transcription, claudin-2 cDNA was quantified by real-time PCR (n = 5 each, ***P < .001).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

8. Figure 7
The velocity of epithelial restitution is impaired by IL-13. HT29/B6 cells were treated with 10 ng/mL IL-13 (gray column) and compared with untreated control samples (white column). After scraping of standardized 200-μm-wide gaps, the velocity of epithelial restitution was estimated from the time until the gap is completely closed.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions

9. Figure 8
Expression of claudin-2 is increased in UC. Expression of tight junction proteins in crude membrane fractions of biopsy specimens by immunoblotting. Lanes 1–3 represent samples from noninflammatory controls, and lanes 4–6 represent samples from patients with active UC. Statistical data of densitometric quantification represent protein expression of the samples from 6 to 8 controls and from 5 to 7 patients with UC (in percent of the controls).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2005 American Gastroenterological Association Terms and Conditions