Augmented increase in tight junction permeability by luminal stimuli in the non-inflamed ileum of Crohn's disease

Abstract

Background: Crohn's disease is associated with deranged intestinal permeability in vivo, suggesting dysfunction of tight junctions. The luminal contents are important for development of neoinflammation following resection. Regulation of tight junctions by luminal factors has not previously been studied in Crohn's disease.

Aims: The aim of the study was to investigate the effects of a luminal stimulus, known to affect tight junctions, on the distal ileum in patients with Crohn's disease.

Patients: Surgical specimens from the distal ileum of patients with Crohn's disease (n=12) were studied, and ileal specimens from colon cancer patients (n=13) served as controls.

Methods: Mucosal permeability to 51Cr-EDTA and electrical resistance were studied in Ussing chambers during luminal exposure to sodium caprate (a constituent of milk fat, affecting tight junctions) or to buffer only. The mechanisms involved were studied by mucosal ATP levels, and by electron and confocal microscopy.

Results: Baseline permeability was the same in non-inflamed ileum of Crohn's disease and controls. Sodium caprate induced a rapid increase in paracellular permeability--that is, increased permeation of 51Cr-EDTA and decreased electrical resistance--which was more pronounced in non-inflamed ileum of Crohn's disease, and electron microscopy showed dilatations within the tight junctions. Moreover, sodium caprate induced disassembly of perijunctional filamentous actin was more pronounced in Crohn's disease mucosa. Mucosal permeability changes were accompanied by mitochondrial swelling and a fall in epithelial ATP content, suggesting uncoupling of oxidative phosphorylation.

Conclusions: The tight junctions in the non-inflamed distal ileum of Crohn's disease were more reactive to luminal stimuli, possibly mediated via disturbed cytoskeletal contractility. This could contribute to the development of mucosal neoinflammation in Crohn's disease.

Figure 1

Electrical resistance in human ileal mucosa. (A) Electrical resistance (TER) during vehicle (control) experiments and during exposure to sodium caprate (C10) in mucosa from colon cancer patients (n=8; open symbols) and non-inflamed mucosa from Crohn's disease patients (n=5; filled symbols) mounted in Ussing chambers. *p<0.05 between groups, Mann-Whitney. (B) TER in inflamed mucosa from patients with Crohn's disease (n=4; filled symbols) mounted in Ussing chambers. Broken lines indicate the curves of non-inflamed mucosa from Crohn's disease patients and colon cancer patients in (A). *p<0.05 compared with non-inflamed mucosa, Mann-Whitney. The variables are expressed as per cent of initial values (median and 25–75th interquartile range). Washout of vehicle and C10 was performed at 10 minutes (arrows).

Figure 2

Transmission electron micrographs of the tight junction region in ileal enterocytes. Specimens were fixed after exposure to vehicle or sodium caprate (C10) in Ussing chambers. (A) Cell membranes of adjacent cells in close apposition in a tight junction exposed to vehicle (arrows). (B) Tight junction with dilatation (arrows) after exposure to C10. Bars indicate 0.2 μm. C10 induced an increased frequency of dilatations within tight junctions (37%) versus vehicle experiments (5%).

Figure 3

Perijunctional filamentous (F)-actin distribution in human ileal mucosa. Confocal en face sectioning at the apical level of enterocytes in the villus region in specimens stained with rhodamine-phalloidin. Graphs show specimens from a colon cancer patient (left) and non-inflamed mucosa from a Crohn's disease patient (right) after vehicle experiments (Vehicle; top) and after exposure to sodium caprate (C10; bottom). In the vehicle experiments, both groups showed F-actin arrayed in perijunctional rings. C10 exposed specimens demonstrated reorganisation of F-actin with marked differences in specimens from colon cancer patients and Crohn's disease patients, respectively. In cancer coli specimens (lower left), a more fragmented appearance of the perijunctional rings was seen, with occasional separation of the actin in adjacent cells (arrows). In non-inflamed ileum from Crohn's disease, F-actin staining was diminished at the junctional level (lower right), with staining seen only in patchy small areas. At the level of the microvilli, the F-actin from adjacent cells was separated after C10 exposure (arrows). Bars indicate 5 μm.

Figure 4

Representative transmission electron micrographs of the apical part of ileal enterocytes. Specimens were fixed after exposure to vehicle or sodium caprate (C10) in Ussing chambers. (A) Enterocyte exposed to vehicle with normal mitochondria (arrowheads) and unaffected tight junction region (arrows). (B) Swollen mitochondria with derangement of the cristae (arrowheads) and a dilated tight junction (arrows) after exposure to C10. Bars indicate 0.5 μm.