Interleukin-13 Is the Key Effector Th2 Cytokine in Ulcerative Colitis That Affects Epithelial Tight Junctions, Apoptosis, and Cell Restitution

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-13Rα1 and IL-4Rα 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 μm/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. 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-13Rα1 and IL-4Rα 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 μm/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. Ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory bowel diseases (IBDs) characterized by an activated mucosal immune system leading to impaired epithelial barrier function and tissue destruction. Current experimental data suggest that the intestinal flora is an important antigenic stimulus. This has been shown in animal models of IBD, where the gut flora is essential for disease induction, and in humans, because antibiotic treatment or diversion of the fecal stream can ameliorate disease activity.1Sartor R.B. Current concepts of the etiology and pathogenesis of ulcerative colitis and Crohn’s disease.Gastroenterol Clin North Am. 1995; 24: 475-507PubMed Google Scholar Whereas an inflammatory response to these antigens from the lumen is suppressed in healthy individuals, a destructive immune response is initiated in patients with IBD. This immune response is mediated by lymphocytes that can either be of a Th1 or a Th2 phenotype. In CD, the inflammation is clearly a Th1 response because it is associated with high levels of interleukin (IL)-12 and interferon (IFN)-γ secretion.2Fuss I.J. Neurath M. Boirivant M. Klein J.S. de la Motte C. Strong S.A. Fiocchi C. Strober W. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5.J Immunol. 1996; 157: 1261-1270PubMed Google Scholar These cytokines lead to macrophage and granulocyte activation and thus the release of multiple downstream inflammatory cytokines such as tumor necrosis factor (TNF)-α and IL-6. The result is a transmural (sometimes granulomatous) inflammation that is not primarily centered on epithelial cells, although collateral damage to the epithelium may occur. Until recently, the inflammation in UC has been difficult to classify using the Th1/Th2 paradigm. Whereas in patients with UC the secretion of IFN-γ or IL-12 is not increased and thus the inflammation is clearly not a Th1 response, IL-4 (messenger RNA [mRNA] or protein) is also reduced.2Fuss I.J. Neurath M. Boirivant M. Klein J.S. de la Motte C. Strong S.A. Fiocchi C. Strober W. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5.J Immunol. 1996; 157: 1261-1270PubMed Google Scholar, 3West G.A. Matsuura T. Levine A.D. Klein J.S. Fiocchi C. Interleukin 4 in inflammatory bowel disease and mucosal immune reactivity.Gastroenterology. 1996; 110: 1683-1695Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar A breakthrough in our understanding of the disease came initially from the study of oxazolone colitis, a murine model of mucosal inflammation that resembles UC and is caused by IL-13-producing natural killer T cells (NKT cells). Based on these observations, we have recently shown that UC is also associated with increased IL-13 production by NKT cells and that the latter cells manifest reactivity to antigens presented by epithelial cells.4Fuss I.J. Heller F. Boirivant M. Leon F. Yoshida M. Fichtner-Feigl S. Yang Z. Exley M. Kitani A. Blumberg R.S. Mannon P. Strober W. Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis.J Clin Invest. 2004; 113: 1490-1497Crossref PubMed Scopus (653) Google Scholar These immunologic changes may explain the fact that the inflammation in UC is different from that in CD in that it is a relatively superficial process marked by abnormalities of the epithelium. Ulcers ranging in size from microerosions to large defects disrupt the line of epithelial cells. Abscesses can be found in the base of the crypts. Already early in the disease process, the barrier function of the mucosa is severely impaired.5Schmitz H. Barmeyer C. Fromm M. Runkel N. Foss H.D. Bentzel C.J. Riecken E.O. Schulzke J.D. Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis.Gastroenterology. 1999; 116: 301-309Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar This arises from widespread apoptosis of epithelial cells and a decreased complexity of the tight junctions between epithelial cells causing increased paracellular permeability.6Gitter A.H. Wullstein F. Fromm M. Schulzke J.D. Epithelial barrier defects in ulcerative colitis characterization and quantification by electrophysiological imaging.Gastroenterology. 2001; 121: 1320-1328Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar Here we report findings indicating that most of the functional defects of the mucosa can in fact be traced to direct effects of IL-13 on epithelial cells. This includes epithelial tight junction alterations and stimulation of epithelial apoptosis together with epithelial restitution arrest. Thus, IL-13 emerges as a key effector cytokine in UC acting adversely on various aspects of epithelial cell function that ultimately lead to the severe destructive inflammation seen in patients with UC. Lamina propria mononuclear cells (LPMCs) were isolated from surgical specimens of patients undergoing colectomy as described previously.2Fuss I.J. Neurath M. Boirivant M. Klein J.S. de la Motte C. Strong S.A. Fiocchi C. Strober W. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5.J Immunol. 1996; 157: 1261-1270PubMed Google Scholar Six patients had chronic active UC, and 5 patients had CD; 4 patients without intestinal inflammation with colonic adenocarcinoma served as noninflammatory controls. The institutional review boards approved the collection of surgical specimens. Because repetitive culture of cells from the same surgical specimen showed almost identical cytokine production, we used each specimen only once for LPMC stimulation experiments and each measurement was obtained from another specimen. Freshly isolated LPMCs were cultured and stimulated in vitro with soluble antibodies to CD2 and CD28. IL-13, IL-4, and IFN-γ were measured by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN; Pierce Chemical Co, Rockford, IL; and BD PharMingen, New York, NY) in culture supernatants collected 48 hours after stimulation as described previously.4Fuss I.J. Heller F. Boirivant M. Leon F. Yoshida M. Fichtner-Feigl S. Yang Z. Exley M. Kitani A. Blumberg R.S. Mannon P. Strober W. Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis.J Clin Invest. 2004; 113: 1490-1497Crossref PubMed Scopus (653) Google Scholar IL-13Rα1, IL-13Rα2, and IL-4Rα were detected by reverse-transcription polymerase chain reaction (PCR) and immunofluorescence. Claudin-2 mRNA was quantified by real-time PCR. RNA was isolated from IL-13-treated (48 hours; 10 ng/mL) or control cultures of HT-29/B6 cells with RNAzol B (Wak-Chemie Medical GmbH, Steinbach, Germany) according to the manufacturer’s protocol. After reverse transcription of 1.5 μg total RNA for real-time PCR or 2 μg total RNA for receptor PCR (Omniscript RT Kit; Qiagen, Hilden, Germany) (60 minutes at 37°C, 5 minutes at 93°C), IL-13 or IL-4 receptor complementary DNA (cDNA) was amplified (35 cycles of 45 seconds at 95°C, 60 seconds at 60°C, and 60 seconds at 68°C) with the following primer pairs: hIL13Rα1FOR ggagccagctcaatttgtag, hIL13Rα1REV cacacgggaagttaaaggca, hIL13Rα2FOR ggagagaggcaatatcaagg, hIL13Rα2REV ggccatgactggaaactgt, hIL4RFOR gacctggagcaacccgtatc, and hIL4RREV catagcacaacaggcagacg. The amplified products were verified by agarose gel electrophoresis and showed single bands of predicted sizes for each sample and no products in negative controls. In biopsy samples from patients with UC or noninflammatory controls, IL13Rα1 and IL4Rα (both with antibodies from R&D Systems, Minneapolis, MN) were detected by immunofluorescence according to the protocol listed in the following text. cDNA prepared from untreated or IL-13-treated HT-29/B6 cells as described previously was amplified with claudin-2 specific primers: CLDN2F gaatcccgagccaaagacagagtg and CLDN2B tcagggagaacagggaagaaataa. Quantitative LightCycler-PCR was performed using the FastStart DNA Master SYBR Green I Kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). The final MgCl2 concentration was 3 mmol/L. Each sample contained 3 μL cDNA preparation. The reaction mixture was denatured for 10 minutes at 95°C and subjected to 40 cycles in a 3-step PCR (95°C for 15 seconds, 60°C for 5 seconds, and 72°C for 10 seconds). Detection of fluorescence occurred at the end of the 72°C elongation step. Specificity of PCR products was verified by melting curve analysis subsequent to the amplification. Amplification, data acquisition, and analysis were performed by LightCycler (Roche). Standardization was performed with a standard dilution of a pCR2.1-TOPO vector construct (Invitrogen, Karlsruhe, Germany) containing the 199-base pair amplicon generated by the primer pair CLDN2F and CLDN2B. Claudin-2 mRNA copies were expressed per nanograms total RNA. HT-29/B6 cells represent a subclone of the human colorectal cancer cell line HT-29, which grow as highly differentiated polarized epithelial monolayers.7Kreusel K.M. Fromm M. Schulzke J.D. Hegel U. Cl-secretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6).Am J Physiol. 1991; 261: C574-C582PubMed Google Scholar The cells were routinely cultured in 25-cm2 culture flasks (Nunc, Wiesbaden, Germany). The culture medium contained RPMI 1640, 2% l-glutamine, and 10% fetal calf serum (all from Biochrom, Berlin, Germany), and the pH was 7.4 in all experiments. Cultures were incubated at 37°C in a 95% air/5% CO2 atmosphere. Cells were seeded on Millicell PCF filters (Millipore, Schwalbach, Germany; effective membrane area, 0.6 cm2) at an average concentration of 7 × 105 cells/cm2. Three inserts were placed together into one conventional culture dish (OD, 60 mm). Confluence of polarized monolayers was reached after 7 days. Experiments were performed on day 7 or 8, giving transepithelial resistances (Rt) of 400–600 Ω · cm2. The apical compartment was routinely filled with 600 μL culture medium, and the basolateral compartment contained 10 mL. Cytokines (TNF-α, TEBU, Offenbach, Germany; IL-4 and IL-13, R&D Systems, Wiesbaden, Germany) were added 24–48 hours before an experiment to cultures to the basolateral compartment at 10 ng/mL IL-13 unless stated otherwise. The IL-13 receptor type I was blocked with 100 ng/mL of anti-IL-4R antibodies (R&D Systems, Wiesbaden, Germany). Rt of the monolayers was measured by a modification of the method described by Kreusel et al.7Kreusel K.M. Fromm M. Schulzke J.D. Hegel U. Cl-secretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6).Am J Physiol. 1991; 261: C574-C582PubMed Google Scholar Electrical measurements were performed in the culture dishes by 2 fixed pairs of electrodes (World Precision Instruments, Sarasota, FL). Rt was calculated from the voltage deflections caused by an external ±10-μA, 21-Hz rectangular current. Depth of immersion and position of the filters were standardized mechanically. The temperature was maintained at 37°C during the measurements. Resistance values were corrected for the resistance of the empty filter and the bathing solution. For flux experiments, filters were incubated with 10 ng/mL IL-13 for 48 hours. The complete inserts were mounted into modified Ussing chambers, which were driven by a 6-channel computer-controlled voltage clamp device (CVC 6; Fiebig, Berlin, Germany). Measurements were performed under short-circuit conditions. The resistance of the bathing solution was determined before each experiment and subtracted from the raw data. Flux studies from the mucosal to the serosal side were performed with 3H-mannitol, 3H-lactulose, and 3H-polyethylene glycol (PEG) 4000 (Biotrend, Köln, Germany) as described previously.8Bojarski C. Gitter A.H. Bendfeldt K. Mankertz J. Schmitz H. Wagner S. Fromm M. Schulzke J.D. Permeability of human HT-29/B6 colonic epithelium as a function of apoptosis.J Physiol. 2001; 535: 541-552Crossref PubMed Scopus (97) Google Scholar After equilibration, samples for flux measurements were collected every 15 minutes. As a monitor of cell deterioration, lactate dehydrogenase (LDH) release from the cells was measured. The postexperimental LDH content in the supernatant of controls and of IL-13-treated cells was determined. After detergent extraction with 2% Triton X-100 for 30 minutes, the total LDH content of the residual cells was measured. Thereby, the percentage of LDH released into the supernatant could be calculated. Conductance scanning was performed as described previously.9Gitter A.H. Bertog M. Schulzke J. Fromm M. Measurement of paracellular epithelial conductivity by conductance scanning.Pflugers Arch. 1997; 434: 830-840Crossref PubMed Scopus (62) Google Scholar Confluent monolayers were mounted horizontally in a modified Ussing-type chamber, which gave access to a scanning probe to be positioned close to the apical side of the tissue. The probe consisted of a pair of microelectrodes with tips set apart vertically by 20–40 μm (Δz) that were connected to a differential amplifier and an AC bridge system with synchronous demodulation and kept at a distance of 25 μm from the epithelial surface. While a constant electric current (sinusoidal AC, 0.1 mA/cm2, 24 Hz) was passed through the monolayer, the resulting electric field was detected. The current in the probe induced a decrease in voltage across Δz and thereby the apparent conductivity GA of the probe could be calculated from the scanning signal. The conductivity of the cell culture filter support (22 mS/cm2) was subtracted, and the conductance of apoptotic leaks was computed by spatial integration of GA. Tight junction protein expression was determined by Western blot analysis of membrane extracts as described previously.10Bürgel N. Bojarski C. Mankertz J. Zeitz M. Fromm M. Schulzke J.D. Mechanisms of diarrhea in collagenous colitis.Gastroenterology. 2002; 123: 433-443Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar Briefly, cells or colonic biopsy specimens were homogenized with iced lysate buffer containing 20 mmol/L Tris, pH 7.4, 5 mmol/L MgCl2, 1 mmol/L EDTA, 0.3 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 μL/mL aprotinin, 16 μg/mL benzamidine HCl, 10 μg/mL phenanthroline, 10 μg/mL leupeptin, 10 μg/mL pepstatin, 1 mmol/L phenylmethylsulfonyl fluoride, 210 μg/mL sodium fluoride, 2.16 mg/mL β-glycerophosphate, 18.5 μg/mL NaVO4, and 1 μL/mL trypsin inhibitor (all from Sigma Chemical Co, St Louis, MO). Subsequently, the lysate was passaged through a 26-gauge needle and then centrifuged at 200g for 5 minutes at 4°C. The membrane fraction was then sedimented by centrifugation at 43,000g for 30 minutes at 4°C. Phosphorylated STAT-6 (P-STAT-6) was detected in total cell lysate. HT-29/B6 cells were homogenized with iced lysate buffer containing 10 mmol/L imidazole, pH 6.8, 0.1 mol/L KCl, 0.3 mol/L sucrose, 2 mmol/L MgCl2, 10 mmol/L EGTA, 1 mmol/L NaF, 1 mmol/L MbO42−, 1 mmol/L NaVO3, 0.2% Triton X-100 (vol/vol), and Complete Mini EDTA-Free Protease Inhibitor Cocktail Tablets (Roche, Mannheim, Germany) for 10 minutes at 4°C. After centrifugation (10 minutes, 4°C, 19000g), the protein content of the supernatant was determined. Protein concentrations were determined by bicinchoninic acid assay (Pierce Chemical Co). Aliquots of 2.5 μg (for occludin), 5 μg (for claudin-1, claudin-2, and claudin-4), or 20 μg protein (for P-STAT-6 or biopsy samples) were separated by polyacrylamide gel electrophoresis (8.5% for occludin and STAT-6, 12.5% for claudins) and transferred to a polyvinylidene difluoride transfer membrane (NEN, Boston, MA). Blots were first blocked for 2 hours in 5% milk powder (not for P-STAT-6) and then overnight or for 2 hours (for P-STAT-6) in 5% bovine serum albumin followed by incubation with a primary antibody (Zymed, San Francisco, CA, or New England Biolabs, Frankfurt am Main, Germany). POD-conjugated secondary antibodies and the chemiluminescence system Lumi-LightPLUS (Roche) were used to detect bound antibodies. Tight junctions were localized by immunofluorescence after 48 hours of incubation with 10 ng/mL IL-13. Nuclear factor κB (NF-κB) translocation was detected in confluent HT-29/B6 cells after 24 hours of serum-free culture. TNF-α (10,000 U/mL) or IL-13 (10 ng/mL) were added for 15 minutes. All samples were washed with phosphate-buffered saline (PBS) and fixed in ice-cold methanol for 10 minutes. Then, cells were washed with PBS and permeabilized with 0.5% Triton X-100 for 5 minutes. The samples were blocked with 0.5% goat serum for 30 minutes at room temperature before addition of the 1:50 diluted primary antibodies for tight junction proteins (Zymed; for monoclonal ZO-1, BD Transduction, Heidelberg, Germany) or 1:100 diluted antibody against the p65 subunit of NF-κB (Santa Cruz, Heidelberg, Germany). After 30 minutes of incubation at room temperature and 2 washing steps with PBS 0.5%, goat serum 1:500 diluted secondary antibodies (Alexa Fluor; MoBiTec, Göttingen, Germany) were added for 30 minutes at room temperature. In some samples, nuclei were counterstained with 4′,6-diamidino-2-phenylindole 1:1000. Finally, samples were mounted in ProTaqs MountFluor (Biocyc, Luckenwalde, Germany) and images were taken with a confocal microscope (Zeiss LSM 510 META, Jena, Germany). HT-29/B6 cells were cultured for 7–8 days in regular culture medium and then incubated with IL-13 (10 ng/mL, 48 hours), epidermal growth factor (20 nmol/L), or regular medium (control). A longitudinal defect with a width of 200 μm was scraped with a glass microelectrode driven by a computer-controlled micromanipulator. The monolayers were returned to regular cell culture conditions for 2 hours before the cells were fixed and stained for ZO-1. The exact width of the gap was measured in samples without restitution. As a marker of epithelial restitution, the velocity of defect closure was determined in micrometers per hour. Apoptotic cells were stained with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay (Roche) according to the manufacturer’s instructions. In one set of experiments, cells were grown on glass slides. After cytokine treatment, the cells were fixed with formaldehyde, stained for TUNEL, and analyzed by microscopy. Samples of IL-13-treated (10 ng/mL), IFN-γ-treated (100 U/mL), or untreated cells were compared. The apoptotic rate was calculated as percentage of TUNEL-positive cells within total cells. In other experiments, monolayers were brought into single-cell suspensions with trypsin and resuspended at 2 × 107 cells/mL in PBS. After fixation with paraformaldehyde, cells were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. Then, apoptotic cells were stained with a TUNEL assay according to the manufacturer’s instructions. Samples were analyzed by fluorescence-activator cell sorting (FACS) with a FACSCalibur (BD Biosciences, Heidelberg, Germany). The apoptotic rate was calculated as percentage of TUNEL-positive cells within total cells. Apoptosis was inhibited with 2 different caspase inhibitors: Z-VAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; Enzyme Systems Products, Livermore, CA) or Z-DEVD-FMK (Z-Asp-Glu-Val-Asp-fluoromethylketone; Alexis Deutschland, Grünberg, Germany). All values are given as mean ± SEM. The unpaired 2-tailed t test was used to determine the significance of differences. P < .05 was considered significant. To quantify cytokine production from inflammatory T cells, LPMCs were isolated from intestinal specimens of patients with UC and respective controls (obtained at surgery) and stimulated in culture with antibodies to CD2 and CD28. After 3 days of stimulation, cytokine release in the supernatant was measured by enzyme-linked immunosorbent assay. Cells from patients with UC produced significantly higher levels of IL-13 (985 ± 273 pg/mL) than cells from patients with CD or noninflammatory controls (216 ± 39 and 51 ± 24 pg/mL, respectively) (Figure 1A). In contrast, cells from patients with CD produced significantly higher levels of IFN-γ (26,496 ± 6365 pg/mL) than cells from patients with UC or controls (1548 ± 380 pg/mL and 1188 ± 406 pg/mL, respectively) (Figure 1B). As already shown,2Fuss I.J. Neurath M. Boirivant M. Klein J.S. de la Motte C. Strong S.A. Fiocchi C. Strober W. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5.J Immunol. 1996; 157: 1261-1270PubMed Google Scholar LPMCs from all 3 groups did not produce significant levels of IL-4 (data not shown). Because the LPMCs were stimulated with antibodies to CD2 and CD28, unstimulated cells do not produce significant levels of cytokines, and CD2 is only expressed on T cells, this type of stimulation has to be assumed to induce cytokine production specifically by T cells. By reverse-transcription PCR, we found IL-13Rα1 and IL-4Rα but not IL-13Rα2 to be expressed on HT-29/B6 cells (Figure 1C). To identify receptors on epithelial cells, immunofluorescence was used for analysis of colonic biopsy specimens. Both receptors IL-13Rα1 and IL-4Rα were detected on colonic enterocytes of noninflammatory controls and patients with UC (Figure 1D). The expression of these receptors on human epithelial cells has also been shown previously by other groups.11Blanchard C. Durual S. Estienne M. Bouzakri K. Heim M.H. Blin N. Cuber J.C. IL-4 and IL-13 up-regulate intestinal trefoil factor expression requirement for STAT6 and de novo protein synthesis.J Immunol. 2004; 172: 3775-3783PubMed Google Scholar To study the effect of increased production of IL-13 on the function of the epithelial barrier, we determined the Rt of a monolayer of HT-29/B6 cells after the addition of IL-13 to the basolateral surface of the cells. IL-13 (10 ng/mL) caused a decrease in Rt from 512 ± 29 to 317 ± 36 Ω · cm2 after 48 hours, corresponding to a decrease to 60% ± 4% from the initial resistance (P < .001, n = 9; Figure 2A). In a subgroup of cultures, we extended Rt monitoring to 96 hours. At these late time points, Rt remained diminished to 51% ± 3% of the initial resistance (P < .001, n = 6). This effect of IL-13 on Rt was characterized by a sigmoidal curve of dose dependency with a point of inflection at 1 ng/mL (Figure 2B). A significant effect of IL-13 on the barrier function was only observed when IL-13 was added to the basolateral side, whereas apical addition, even at very high concentrations, did not affect Rt (80 ± 2% [n = 6] at 10 ng/mL IL-13 vs 91 ± 5% in controls [n = 6; not significant]). Within the initial 24 hours, 10 ng/mL IL-13 had only a modest effect on Rt (76% ± 2%). In parallel, at 500 U/mL, TNF-α induced a decrease in Rt to 73% ± 2% after 24 hours. However, this effect of small amounts of TNF-α on Rt was considerably intensified by IL-13. When combined with IL-13, TNF-α reduced Rt to 47% ± 1% of initial resistance (P < .0001) (Figure 2C). Finally, we studied the effect of IL-4 on epithelial barrier function. At concentrations of 10 ng/mL or 100 ng/mL, IL-4 did not affect Rt of HT-29/B6 monolayers (Figure 2C). Combined with TNF-α, IL-4 did not amplify the decrease of Rt induced by TNF-α but attenuated the effect of TNF-α (Figure 2C). Because both types of IL-13 receptors, the heterodimeric IL-4Rα/IL-13Rα1 and the IL-13Rα1 monomer, are expressed on colonic epithelial cells (see Discussion), we inhibited signaling with antibodies to the IL-4Rα chain to elucidate the functional role of the IL-13Rα1 monomer. In these experiments, Rt was decreased to 58% ± 1% after the addition of IL-13 compared with 53% ± 2% when both receptors remained active (Figure 2C). We performed Western blot analysis of P-STAT-6. As shown in Figure 2D, IL-13 induced the phosphorylation of STAT-6 in colonic HT-29/B6 epithelial cells. While untreated cells did not have detectable levels, P-STAT-6 became detectable after 30 minutes of incubation with IL-13. In contrast, translocation of the p65 subunit of NF-κB into the nucleus was only induced with TNF-α, not with IL-13 (Figure 2D). In the next set of studies, we determined if IL-13 alters the transepithelial transport of ions or large molecules (Table 1). Accordingly, the initial short-circuit current of HT-29/B6 cells incubated with or without 10 ng/mL IL-13 was measured. The initial short-circuit current of control and IL-13-treated cells was not different in both groups (9 ± 1 μA · cm2) and remained constant throughout the experiment. As shown previously, Rt was diminished after incubation with IL-13 for 48 hours (control cells, 562 ± 16 Ω · cm2; IL-13-treated cells, 274 ± 13 Ω · cm2; P < .001). Because short-circuit current was not affected by IL-13, the decrease in Rt is not due to activation of transporters involved in rheogenic ion transport.Table 1Effect of IL-13 on Mannitol, Lactulose, and PEG4000 Mucosal-to-Serosal Flux, Rt, and Short-Circuit Current of HT-29/B6 CellsJmannitolms (nmol·h−1·cm−2)Jlactulosems (nmol·h−1·cm−2)JPEG4000ms (nmol·h−1·cm−2)Rt (Ω·cm2)aControl, n = 15; IL-13, n = 16.Short-circuit current (μA·cm−2)aControl, n = 15; IL-13, n = 16.Control184 ± 860 ± 25.6 ± 0.1562 ± 169 ± 1IL-13572 ± 8881 ± 16.7 ± 0.2274 ± 139