Matthias Bruewer

Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms

It is well known that inflammatory conditions of the intestinal mucosa result in compromised barrier function. Inflammation is characterized by an influx into the mucosa of immune cells that influence epithelial function by releasing proinflammatory cytokines such as IFN-γ and TNF-α. Mucosal barrier function is regulated by the epithelial apical junctional complex (AJC) consisting of the tight junction and the adherens junction. Since the AJC regulates barrier function, we analyzed the influence of IFN-γ and TNF-α on its structure/function and determined the contribution of apoptosis to this process using a model intestinal epithelial cell line, T84, and IFN-γ and TNF-α. AJC structure/function was analyzed by confocal microscopy, biochemical analysis, and physiologic measurement of epithelial gate/fence function. Apoptosis was monitored by determining cytokeratin 18 cleavage and caspase-3 activation. IFN-γ induced time-dependent disruptions in epithelial gate function that were potentiated by coincubation with TNF-α. Tight junction fence function was somewhat disrupted. Cytokine treatment was associated with internalization of AJC transmembrane proteins, junction adhesion molecule 1, occludin, and claudin-1/4 with minimal effects on the cytoplasmic plaque protein zonula occludens 1. Detergent solubility profiles of junction adhesion molecule 1 and E-cadherin and their affiliation with “raft-like” membrane microdomains were modified by these cytokines. Inhibition of cytokine-induced apoptosis did not block induced permeability defects; further emphasizing their primary influence on the epithelial AJC structure and barrier function. Our findings for the first time clearly separate the proapoptotic effects of IFN-γ and TNF-α from their abilities to disrupt barrier function.

Probiotic mixture VSL#3 protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in a murine model of colitis

Changes in epithelial tight junction protein expression and apoptosis increase epithelial permeability in inflammatory bowel diseases. The effect of the probiotic mixture VSL#3 on the epithelial barrier was studied in dextran sodium sulfate (DSS)-induced colitis in mice. Acute colitis was induced in BALB/c mice (3.5% DSS for 7 days). Mice were treated with either 15 mg VSL#3 or placebo via gastric tube once daily during induction of colitis. Inflammation was assessed by clinical and histological scores. Colonic permeability to Evans blue was measured in vivo. Tight junction protein expression and epithelial apoptotic ratio were studied by immunofluorescence and Western blot. VSL#3 treatment reduced inflammation (histological colitis scores: healthy control 0.94 +/- 0.28, DSS + placebo 14.64 +/- 2.55, DSS + VSL#3 8.43 +/- 1.82; P = 0.011). A pronounced increase in epithelial permeability in acute colitis was completely prevented by VSL#3 therapy [healthy control 0.4 +/- 0.07 (extinction/g), DSS + placebo 5.75 +/- 1.67, DSS + VSL#3 0.26 +/- 0.08; P = 0.003]. In acute colitis, decreased expression and redistribution of the tight junction proteins occludin, zonula occludens-1, and claudin-1, -3, -4, and -5 were observed, whereas VSL#3 therapy prevented these changes. VSL#3 completely prevented the increase of epithelial apoptotic ratio in acute colitis [healthy control 1.58 +/- 0.01 (apoptotic cells/1,000 epithelial cells), DSS + placebo 13.33 +/- 1.29, DSS + VSL#3 1.72 +/- 0.1; P = 0.012]. Probiotic therapy protects the epithelial barrier in acute colitis by preventing 1) decreased tight junction protein expression and 2) increased apoptotic ratio.

Interferon-γ induces internalization of epithelial tight junction proteins via a macropinocytosis-like process

Increased epithelial permeability is observed in inflammatory states. However, the mechanism by which inflammatory mediators such as IFN‐γ increase epithelial permeability is unknown. We recently observed that IFN‐γ induces disassembly of tight junctions (TJ); in this study we asked whether such TJ disassembly is mediated by endocytosis of junctional proteins. The role of three major internalization pathways in disruption of TJ in IFN‐γ‐treated intestinal epithelial cells was analyzed using selective inhibitors and markers of the pathways. No role for the clathrin‐ and caveolar‐mediated endocytosis in the IFN‐γ‐induced internalization of TJ proteins was observed. However, inhibitors of macropinocytosis blocked internalization of TJ proteins and junctional proteins colocalized with macropinocytosis markers, dextran and phosphatidylinositol‐3,4,5‐trisphosphate. Internalized TJ proteins were identified in early and recycling endosomes but not in late endosomes/lysosomes. These results for the first time suggest that IFN‐γ produces a leaky epithelial barrier by inducing macropinoytosis of TJ proteins. Bruewer, M., Utechm, M., Ivanov, A. I., Hopkins, A. M., Parkos, C. A., Nusrat, A. Interferon‐γ induces internalization of epithelial tight junction proteins via a macropinocytosis‐like process. FASEB J. 19, 923–933 (2005)

Inflammatory Bowel Disease and the Apical Junctional Complex

Abstract:  A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the underlying interstitium. This intestinal barrier is primarily regulated by the apical junctional complex (AJC) consisting of tight junctions (TJs) and adherens junctions (AJs) and is compromised in a number of intestinal diseases, including inflammatory bowel disease (IBD). In vitro studies have demonstrated that proinflammatory cytokines, such as interferon‐gamma (IFN‐γ) and tumor necrosis factor‐alpha (TNF‐α), that are increased in the intestinal mucosa of patients with IBD can induce a leaky mucosal barrier. There is a growing evidence that the increased permeability and altered AJC structure observed in IBD are mediated by internalization of junctional proteins. This review summarizes barrier defects observed in IBD and addresses mechanisms by which proinflammatory cytokines, such as IFN‐γ and TNF‐α, modulate AJC structure and epithelial barrier function.

Regulation of the intestinal epithelial barrier by the apical junctional complex.

Purpose of review Epithelial intercellular junctions are important components of the epithelial barrier and are compromised in disorders such as Crohns disease. We will highlight recent progress in understanding the role of an intercellular junction referred to as the apical junctional complex in regulating small intestinal epithelial permeability in health and disease. Recent findings Recent studies have implicated aberrant regulation of the AJC as an underlying factor contributing to a leaky epithelial barrier in Crohns patients. Consequences of increased epithelial permeability include exposure of intestinal tissue to luminal antigens/pathogens which in turn influence disease activity. Furthermore, proinflammatory cytokines released into the milieu of the epithelium in patients with Crohns disease influence apical junctional complex and epithelial barrier function. Such cytokines induce disassembly of the apical junctional complex by promoting differential endocytosis of component proteins. Additionally, apical junctional complex proteins are targeted by pathogens that use the epithelium as a portal of entry to establish disease in the host. Summary The epithelial apical junctional complex is important in determining epithelial barrier properties. Recent studies have highlighted contribution of proinflammatory cytokines and endocytosis of apical junctional complex proteins to the epithelial barrier defect. Continued advances in understanding of this field will yield new therapeutic targets for intestinal disorders.

The Adenosine 2b Receptor Is Recruited to the Plasma Membrane and Associates with E3KARP and Ezrin upon Agonist Stimulation

We have previously shown that adenosine is formed in the intestinal lumen during active inflammation from neutrophil-derived 5′-AMP. Acting through the adenosine A2b receptor (A2bR), the luminally derived adenosine induces vectorial chloride secretion and a polarized secretion of interleukin-6 to the intestinal lumen. Although some G protein-coupled receptors interact with anchoring or signaling molecules, not much is known in this critical area for the A2bR. We used the model intestinal epithelial cell line, T84, and Caco2-BBE cells stably transfected with GFP-A2b receptor to study the intestinal A2bR. The A2bR is present in both the apical and basolateral membranes of intestinal epithelia. Apical or basolateral stimulation of the A2bR induces recruitment of the receptor to the plasma membrane and caveolar fractions. The A2bR co-immunoprecipitates with E3KARP and ezrin upon agonist stimulation. Ezrin interacts with E3KARP and PKA and the interaction between ezrin and E3KARP is enhanced by agonist stimulation. Our data suggest that the A2bR is recruited to the plasma membrane upon apical or basolateral agonist stimulation and interacts with E3KARP and ezrin. We speculate that such an interaction may not only anchor the A2bR to the plasma membrane but may also function to stabilize the receptor in a signaling complex in the plasma membrane.

Double Balloon Enteroscopy: A Useful Tool for Diagnostic and Therapeutic Procedures in the Pancreaticobiliary System

OBJECTIVES:Diagnostic and therapeutic interventions in the biliary and pancreatic system in the previously operated patient by conventional endoscopic retrograde cholangiopancreaticography (ERCP) are difficult and, depending on the surgical procedure, in many cases unsuccessful. We describe our experience of ERCP performed with a double balloon enteroscope (DBE) as an alternative examination technique for these patients.METHODS:In a retrospective analysis of all DBE procedures at our department between November 2004 and June 2007, 11 patients were identified with various anatomic variations in whom ERCP was performed using a DBE.RESULTS:In 72% of the patients, previous conventional ERCP examinations failed (8/11). In these patients, DBE-ERCP was successful in 63%. The overall success rate of DBE-ERCP in all patients was 64% (7/11 patients). In those patients, interventions such as papillotomy, calculus extractions, as well as stent placement could be performed even though tools for DBE-ERCP are still very limited. Despite most of the DBE-ERCPs having included therapeutic interventions, no major complications occurred in our case series and minor side effects were restricted to meteorism and mild to moderate abdominal pain.CONCLUSIONS:DBE-ERCP is an alternative method for diagnostic as well as therapeutic interventions in the biliary as well pancreatic system in the operated patient. However, it should be limited to selected patients, e.g., with contraindications for PTC, as it is a time-consuming as well as a cost-intensive procedure.

Effect of Probiotics on Intestinal Barrier Function

Impairment of the intestinal barrier is a key event in various gastrointestinal diseases, including inflammatory bowel diseases, celiac disease, gastrointestinal infections, diarrhea, and critical illness. Recent studies demonstrated that probiotic bacteria have beneficial effects in these diseases by effectively improving intestinal barrier function. This article reviews available data on the effect of probiotics on intestinal barrier function in vitro, in animal models, and in clinical studies.

Effect of pyloric drainage procedures on gastric passage and bile reflux after esophagectomy with gastric conduit reconstruction

Background and aimsControversy still exists about the need for pyloric drainage procedures (pyloroplasty or pyloromyotomy) after esophagectomy with esophagogastrostomy and vagotomy. Although pyloric drainage may prevent postoperative delayed gastric emptying, it may also promote bile reflux into the oesophagus. We analysed pyloric drainage methods for their potential effect on gastric outlet obstruction and bile reflux in patients undergoing esophagectomy.Materials and methodsOne hundred and ninety-eight patients with esophageal carcinoma were treated by transthoracal esophagectomy with gastric conduit reconstruction either with pyloromyotomy (group II, n = 118), pyloroplasty (group III, n = 34) or without pyloric drainage (group I, n = 46) between January 2000 and December 2004. The postoperative gastrointestinal passage by radiological investigation, anastomotic leakage rate, mortality and incidence of gastroesophageal reflux by endoscopy within the first postoperative year were retrospectively analysed.ResultsPatient demographics and the types of surgical procedures did not differ between the three groups. There was no difference in hospital mortality, anastomotic leakage rate, gastrointestinal passage and postoperative hospital stay between the three groups. However, more patients with pyloric drainage showed bile reflux (I = 0% vs II+III=14.9%, p = 0.069) and reflux esophagitis (I = 10.3% vs II+III = 34.5%, p < 0.05) compared to patients without pyloric drainage. On the multivariate analysis, pyloric drainage and the anastomotic height were independent and were significant risk factors associated with postoperative reflux esophagitis.ConclusionPyloric drainage after esophagectomy with gastric conduit reconstruction should be omitted because it does not improve gastric emptying and may favour biliary reflux esophagitis.

Preoperative Steroid Administration: Effect on Morbidity among Patients Undergoing Intestinal Bowel Resection for Crohn’s Disease

Long-term steroid therapy may predispose to increased perioperative morbidity in patients undergoing surgery with bowel anastomoses. The aim of our study was to review our data to determine if the steroid dosage is associated with the incidence of early complications after bowel resection in patients with prolonged steroid therapy for Crohn’s disease (CD). Altogether, 397 patients underwent bowel resection with primary intestinal anastomoses for CD between 1982 and 2000 in our institution. The mortality and morbidity rates, anastomotic leakage, wound infections, intraabdominal abscesses, reoperation rate, and length of postoperative hospitalization in patients who were having high-dose (≥ 20 mg of prednisolone per day, n = 73) and low-dose (< 20 mg prednisolone per day, n = 146) steroid therapy for more than 1 month before surgery were compared with those of patients (n = 177) who were not receiving steroids. Statistical analysis was performed using Fisher’s exact test and Student’s t-test, with p < 0.05 considered significant. The three groups were similar in terms of gender, duration since first diagnosis, American Society of Anesthesiologists classification, and obesity. Mortality, morbidity, anastomotic leakage, wound infections, intraabdominal abscesses, reoperation rate, and average postoperative stay were not statistically different in patients with high-dose, low-dose, or no steroid therapy. The only factor associated with increased morbidity was a low preoperative hemoglobin level. Our results demonstrate that, in patients who are undergoing bowel resection for CD, even high-dose prolonged preoperative systemic steroid therapy is not associated with increased postoperative complications.