J. D. Schulzke

Downregulation of epithelial apoptosis and barrier repair in active Crohn’s disease by tumour necrosis factor α antibody treatment

Background and aims: Barrier dysfunction is an important feature contributing to inflammation and diarrhoea in Crohn’s disease (CD). Recently, tumour necrosis factor α (TNF-α) antibodies were recognised as effective in steroid refractory CD. The aim of this study was to characterise the effects of this therapy on the epithelial barrier. Patients and methods: Forceps biopsies were obtained from the sigmoid colon before and 14 days after TNF-α antibody therapy in 11 patients treated for chronic active CD (Crohn’s disease activity index >150). Epithelial apoptoses were measured after terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) and 4′,6-diamidino-2-phenylindole staining. Epithelial resistance was determined by alternating current impedance analysis in miniaturised Ussing chambers. Occludin, claudin 1, and claudin 4 expression was quantified in immunoblots. Results: The epithelial apoptotic ratio was 2.1 (0.2)% in controls and increased to 5.3 (1.0)% in CD. TNF-α antibody therapy decreased the apoptotic ratio to 2.9 (1.0)% (normalised in 10 of 11 patients). In parallel, epithelial resistance was lower in CD than in controls (24 (3) v 42 (3) Ω×cm2) and improved to 34 (3) Ω×cm2 after therapy. Occludin, claudin 1, and claudin 4 were not affected by TNF-α antibody therapy. In support of a functional role of epithelial apoptoses in CD, a similar decrease in resistance of −40% was observed when the apoptotic rate was selectively upregulated from 2.6% to 5.4% with camptothecin in HT-29/B6 cells. Conclusions: Epithelial apoptoses were upregulated in the colon in CD and restored to normal in 10 of 11 patients by TNF-α antibody therapy. This is the structural correlate of epithelial barrier dysfunction measured as epithelial resistance while expression of tight junction proteins did not contribute to this therapeutic effect.

Effect of chronic Giardia lamblia infection on epithelial transport and barrier function in human duodenum

Background:Giardia lamblia causes infection of the small intestine, which leads to malabsorption and chronic diarrhoea. Aim: To characterise the inherent pathomechanisms of G lamblia infection. Methods: Duodenal biopsy specimens from 13 patients with chronic giardiasis and from controls were obtained endoscopically. Short-circuit current (ISC) and mannitol fluxes were measured in miniaturised Ussing chambers. Epithelial and subepithelial resistances were determined by impedance spectroscopy. Mucosal morphometry was performed and tight junction proteins were characterised by immunoblotting. Apoptotic ratio was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling staining. Results: In giardiasis, mucosal surface area per unit serosa area was decreased to 75% (3%) of control, as a result of which epithelial resistance should increase. Instead, epithelial resistance of giardiasis biopsy specimens was decreased (19 (2) vs 25 (2) Ω cm2; p<0.05) whereas mannitol flux was not significantly altered (140 (27) vs 105 (16) nmol/h/cm2). As structural correlate, reduced claudin 1 expression and increased epithelial apoptosis were detected. Furthermore, basal ISC increased from 191 (20) in control to 261 (12) µA/h/cm2 in giardiasis. The bumetanide-sensitive portion of ISC in giardiasis was also increased (51 (5) vs 20 (9) µA/h/cm2 in control; p<0.05). Finally, phlorizin-sensitive Na+–glucose symport was reduced in patients with giardiasis (121 (9) vs 83 (14) µA/h/cm2). Conclusions:G lamblia infection causes epithelial barrier dysfunction owing to down regulation of the tight junction protein claudin 1 and increased epithelial apoptoses. Na+-dependent d-glucose absorption is impaired and active electrogenic anion secretion is activated. Thus, the mechanisms of diarrhoea in human chronic giardiasis comprise leak flux, malabsorptive and secretory components.

Disrupted Barrier Function through Epithelial Cell Apoptosis

Abstract:  Epithelial barrier function is determined by trans‐ and paracellular permeabilities, the latter of which is mainly influenced by tight junctions (TJs) and apoptotic leaks within the epithelium. The present article aims to present experimental evidence for a functional role of epithelial apoptoses by means of cell culture models as well as in tissues from patients with inflammatory bowel disease. It is shown that epithelial apoptoses are sites of elevated conductance within the intestinal epithelium and that proinflammatory cytokines like TNF‐α upregulate both the apoptotic rate and single apoptotic conductivity, making cytokine‐induced apoptosis functionally far more relevant than is spontaneous apoptosis. In ulcerative colitis and Crohns disease (CD), but not in collagenous colitis, apoptotic rates are increased to about 5%, in mild‐to‐moderately inflamed colon specimens, where as the control apoptotic rate is about 2%. Thus, epithelial apoptoses lead to a loss of ions and water into the intestinal lumen, causing leak flux diarrhea and enabling small antigens of <4,000 Da in the intestinal lumen to enter the intestinal mucosa, thereby perpetuating inflammatory responses. In addition to TNF‐α, interleukin (IL)‐13 is an important inductor of epithelial apoptosis in Th2 immune responses. Therapeutically,TNF‐α‐antibodies (infliximab) can restore barrier function in Crohns disease by downregulating epithelial apoptoses, while epithelial TJs are unaffected.

Mechanisms of epithelial translocation of the α2-gliadin-33mer in coeliac sprue

Background and aims: The α2-gliadin-33mer has been shown to be important in the pathogenesis of coeliac disease. We aimed to study mechanisms of its epithelial translocation and processing in respect to transcytotic and paracellular pathways. Methods: Transepithelial passage of a fluorescence-labelled α2-gliadin-33mer was studied in Caco-2 cells by using reverse-phase high-performance liquid chromatography, mass spectrometry, confocal laser scanning microscopy (LSM) and fluorescence activated cell sorting (FACS). Endocytosis mechanisms were characterised with rab-GFP constructs transiently transfected into Caco-2 cells and in human duodenal biopsy specimens. Results: The α2-gliadin-33mer dose-dependently crossed the epithelial barrier in the apical-to-basal direction. Degradation analysis revealed translocation of the 33mer polypeptide in the uncleaved as well as in the degraded form. Transcellular passage was identified by confocal LSM, inhibitor experiments and FACS. Rab5 but not rab4 or rab7 vesicles were shown to be part of the transcytotic pathway. After pre-incubation with interferon-γ, translocation of the 33mer was increased by 40%. In mucosal biopsies of the duodenum, epithelial 33mer uptake was significantly higher in untreated coeliac disease patients than in healthy controls or coeliac disease patients on a gluten-free diet. Conclusion: Epithelial translocation of the α2-gliadin-33mer occurs by transcytosis after partial degradation through a rab5 endocytosis compartment and is regulated by interferon-γ. Uptake of the 33mer is higher in untreated coeliac disease than in controls and coeliac disease patients on a gluten-free diet.

Regulation of mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and interferon gamma in vitro: A novel model for studying the pathomechanisms of inflammatory bowel disease cytokines

Objective. In Inflammatory bowel disease (IBD), elevated cytokines are responsible for disturbed intestinal transport and barrier function. The mechanisms of cytokine action have usually been studied in cell culture models only; therefore the aim of this study was to establish an in vitro model based on native intestine to analyze distinct cytokine effects on barrier function, mucosal structure, and inherent regulatory mechanisms. Material and methods. Rat colon was exposed to tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) in Ussing chambers. Transepithelial resistance (Rt) and 3H-mannitol fluxes were measured for characterization of the paracellular pathway. Transcellular transport was analyzed by horseradish peroxidase (HRP) flux measurements. Expression and distribution of tight junction proteins were characterized in immunoblots and by means of confocal laser-scanning microscopy (LSM). Results. Colonic viability could be preserved for 20 h in a specialized in vitro set-up. This was sufficient to alter mucosal architecture with crypt surface reduction. Rt was decreased (101±10 versus 189±10 Ω·cm2) with a parallel increase in mannitol permeability after cytokine exposure. Tight junction proteins claudin-1, -5, -7, and occludin decreased (45±10%, 16±7%, 42±8%, and 42±13% of controls, respectively), while claudin-2 increased to 208±32%. Occludin and claudin-1 translocated from the plasma membrane to the cytoplasm. HRP flux increased from 0.73±0.09 to 8.55±2.92 pmol·h−1·cm−2. Conclusions. A new experimental IBD model with native colon in vitro is presented. One-day exposure to TNFα and IFNγ alters mucosal morphology and impairs epithelial barrier function by up-regulation of the paracellular pore-former claudin-2 and down-regulation of the barrier-builders claudin-1, -5, and -7. These alterations resemble changes seen in IBD and thus underline their prominent role in IBD pathogenicity.

Cell polarity-determining proteins Par-3 and PP-1 are involved in epithelial tight junction defects in coeliac disease

Background Epithelial barrier defects are well known in coeliac disease, but the mechanisms are only poorly defined. It is unclear, whether barrier disturbance reflects upregulated epithelial transcytosis or paracellular leakage. Objective To characterise the molecular structure and function of the epithelial tight junction (TJ) and mechanisms of its dysregulation. Methods Molecular analysis of proteins involved in TJ assembly and their regulation was performed by western blotting and confocal microscopy correlated to electrophysiology. Results A complex alteration of the composition of epithelial TJ proteins (with more pore-forming claudins like claudin-2 and a reduction in tightening claudins like claudin-3, -5 and -7) was found for protein expression and subcellular localisation, responsible for an increase in paracellular biotin-NHS uptake. In contrast, epithelial apoptosis was only moderately elevated (accounting for a minor portion of barrier defects) and epithelial gross lesions—for example, at cell extrusion zones, were absent. This TJ alteration was linked to an altered localisation/expression of proteins regulating TJ assembly, the polarity complex protein Par-3 and the serine-/threonine phosphatase PP-1. Conclusions Changes in cell polarity proteins Par-3 and PP-1 are associated with altered expression and assembly of TJ proteins claudin-2, -3, -5 and -7 and ZO-1, causing paracellular leakage in active coeliac disease.

Epithelial barrier and ion transport in coeliac sprue: electrical measurements on intestinal aspiration biopsy specimens.

Epithelial barrier function and ion transport was studied in coeliac sprue using a miniaturised Ussing device for measurements on diagnostic aspiration biopsy specimens from the jejunum of untreated or gluten free nourished sprue patients, or from healthy controls. Pure epithelial resistance (Re) indicating epithelial barrier function was determined by transmural alternating current impedance analysis. It was reduced by 56% in acute sprue mean (SEM) (9 (1) omega.cm2) compared with controls (20(2) omega.cm2). In gluten free nourished sprue patients Re was only partly recovered (15 (1) omega.cm2). Subepithelial resistance (Rsub) was also changed from 28 (1) omega.cm2- in control to 17 (1) omega.cm2 in acute sprue because of the change in mucosal architecture, but was unchanged in gluten free nourished sprue patients (29 (4) omega.cm2). In acute sprue, unidirectional Na+ and Cl- fluxes were increased in both directions as a consequence of the decreased resistance. However, short circuit current (ISC) as well as Na+ and Cl- net fluxes were not significantly different from control. Subsequently, the electrogenic Cl- secretory system was investigated. After maximal stimulation with theophylline and prostaglandin E1, a Cl(-)-dependent increase in ISC was obtained in the sprue mucosa and control jejunum. It showed saturation characteristics and was blockable by serosal bumetanide. When compared with control, neither Km nor Vmax of this electrogenic Cl- secretion was significantly changed in coeliac sprue. In conclusion, a miniaturised Ussing device was used for transport measurements on intestinal biopsy specimens. In acute coeliac disease, the epithelial barrier of the jejunum was seriously disturbed. The active electrogenic Cl- secretory transport system was present in the sprue mucosa, but was not activated in the Ussing chamber in vitro when compared with control jejunum.

Glucocorticoids and Tumor Necrosis Factor-α Synergize to Induce Absorption by the Epithelial Sodium Channel in the Colon

BACKGROUND & AIMS The epithelial sodium channel (ENaC) mediates electrogenic sodium absorption in distal colon. In patients with inflammatory bowel disease (IBD), ENaC induction is impaired, mainly through transcriptional suppression by proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha. Glucocorticoid therapy rapidly increases sodium absorption; we investigated the molecular mechanisms underlying the interaction among TNF-alpha, glucocorticoids, and ENaC induction. METHODS ENaC-mediated sodium transport in glucocorticoid receptor (GR)-expressing HT-29/B6 cells and rat distal colon, under the influence of the synthetic glucocorticoid dexamethasone and TNF-alpha, was quantified in Ussing chambers. ENaC messenger RNA (mRNA) levels were monitored by real-time polymerase chain reaction. GR transactivation and expression were investigated by gene reporter, immunoblot, and confocal immunofluorescence microscopy analyses. The GR mRNA half-life was determined. Signaling pathways were characterized using mitogen-activated protein kinase inhibitors. RESULTS Dexamethasone not only prevented TNF-alpha-mediated ENaC suppression but caused synergistic induction of ENaC-dependent sodium absorption in HT-29/B6-GR cells and rat distal colon. This synergy resulted from TNF-alpha-mediated increases in GR protein levels because of GR mRNA stabilization and subsequent GR transactivation by dexamethasone. As a consequence, transcription of the ENaC beta- and gamma-subunits was up-regulated, increasing ENaC-dependent sodium absorption. p38 Mitogen-activated protein kinase is required for this synergistic effect: p38 inhibition blocked the increase in GR protein expression and ENaC-dependent sodium absorption. CONCLUSIONS TNF-alpha and dexamethasone induce ENaC, explaining the rapid and intense proabsorptive effect of glucocorticoid therapies.

Antisecretory Effect of Loperamide in Colon Epithelial Cells by Inhibition of Basolateral K+ Conductance

Background: The mechanism of the antisecretory effect of loperamide was investigated in cultured highly differentiated colon epithelial cells (HT-29/B6). Methods: Chloride secretion was stimulated via cAMP by forskolin (FSK, 10-5 M), via Ca2+ by the muscarinic agonist carbachol (CCh, 10-4 M), and via protein kinase C by the phorbol ester PMA (5·10-9 M). Stimulated Cl- secretion was quantified as short circuit current (ISC) of HT-29/B6 monolayers mounted in Ussing-type chambers. Results: Loperamide (5·10-5 M) inhibited ISC stimulated by FSK, CCh and PMA. The antisecretory action of loperamide was unaffected by preincubation with naloxone (10-5 M). Furthermore, loperamide strongly inhibited basolateral 86Rb efflux. Like loperamide, the calmodulin antagonist trifluoperazine (10-4 M) inhibited ISC induced by FSK, CCh or PMA. The Ca2+ channel blocker verapamil (5·10-5 M), on the other hand, inhibited only PMA-stimulated ISC, but had no effect on FSK or CCh-induced ISC. Conclusions: Loperamide exerts a direct antisecretory action on chloride secretion of colon epithelial cells independently of the respective stimulatory signal transduction pathway. This antisecretory effect is not mediated by opiate receptors and reflects inhibition of basolateral K+ conductance.BACKGROUND The mechanism of the antisecretory effect of loperamide was investigated in cultured highly differentiated colon epithelial cells (HT-29/B6). METHODS Chloride secretion was stimulated via cAMP by forskolin (FSK, 10(-5) M), via Ca2+ by the muscarinic agonist carbachol (CCh, 10(-4) M), and via protein kinase C by the phorbol ester PMA (5 x 10(-9) M). Stimulated Cl- secretion was quantified as short circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers. RESULTS Loperamide (5 x 10(-5) M) inhibited I(SC) stimulated by FSK, CCh and PMA. The antisecretory action of loperamide was unaffected by preincubation with naloxone (10(-5) M). Furthermore, loperamide strongly inhibited basolateral 86Rb efflux. Like loperamide, the calmodulin antagonist trifluoperazine (10(-4) M) inhibited I(SC) induced by FSK, CCh or PMA. The Ca2+ channel blocker verapamil (5 x 10(-5) M), on the other hand, inhibited only PMA-stimulated I(SC),but had no effect on FSK or CCh-induced I(SC) CONCLUSIONS: Loperamide exerts a direct antisecretory action on chloride secretion of colon epithelial cells independently of the respective stimulatory signal transduction pathway. This antisecretory effect is not mediated by opiate receptors and reflects inhibition of basolateral K+ conductance.

Aeromonas hydrophila Beta-Hemolysin Induces Active Chloride Secretion in Colon Epithelial Cells (HT-29/B6)

ABSTRACT The diarrheal mechanisms in Aeromonas enteritis are not completely understood. In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6). Ion secretion was determined as a short-circuit current (ISC) of HT-29/B6 monolayers mounted in Ussing-type chambers. Transepithelial resistance (Rt) served as a measure of permeability. A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant ISC (39 ± 3 μA/cm2) and decreased the Rt to ∼10% of the initial value. A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant ISC in totally stripped human colon. Tracer flux and ion replacement studies revealed the ISC to be mainly accounted for by electrogenic Cl− secretion. Supernatant applied serosally completely abolished basal ISC. The supernatant-induced ISC was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca2+ chelator (BAPTA-AM) had no effect. Physicochemical properties indicated that the supernatants active compound was an aerolysin-related Aeromonas beta-hemolysin. Accordingly, identical ISC and Rt responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin. Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp. strain Sb. In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl− secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C.