Local barrier dysfunction identified by confocal laser endomicroscopy predicts relapse in inflammatory bowel disease

Abstract

Objectives: Loss of intestinal barrier function plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Shedding of intestinal epithelial cells is a potential cause of barrier loss during inflammation. The objectives of the study were (1) to determine whether cell shedding and barrier loss in humans can be detected by confocal endomicroscopy and (2) whether these parameters predict relapse of IBD.

Methods: Confocal endomicroscopy was performed in IBD and control patients using intravenous fluorescein to determine the relationship between cell shedding and local barrier dysfunction. A grading system based on appearances at confocal endomicroscopy in humans was devised and used to predict relapse in a prospective pilot study of 47 patients with ulcerative colitis and 11 patients with Crohn's disease.

Results: Confocal endomicroscopy in humans detected shedding epithelial cells and local barrier defects as plumes of fluorescein effluxing through the epithelium. Mouse experiments demonstrated inward flow through some leakage-associated shedding events, which was increased when luminal osmolarity was decreased. In IBD patients in clinical remission, increased cell shedding with fluorescein leakage was associated with subsequent relapse within 12 months after endomicroscopic examination (p<0.001). The sensitivity, specificity and accuracy for the grading system to predict a flare were 62.5% (95% CI 40.8% to 80.4%), 91.2% (95% CI 75.2 to 97.7) and 79% (95% CI 57.7 to 95.5), respectively.

Conclusions: Cell shedding and barrier loss detected by confocal endomicroscopy predicts relapse of IBD and has potential as a diagnostic tool for the management of the disease.

Figure 1

Confocal endomicroscopic imaging of epithelial cell shedding in the terminal ileum. (A) Fluorescein images capillaries beneath epithelial cells (block arrow) and the lateral intracellular space between epithelial cells (line arrow). (B) Epithelial cells become permeable to fluorescein prior to shedding (line arrow). (C–E) Fluorescein fluorescence signal is intense as shedding cells move out of the epithelial monolayer. (F) Intensely fluorescent shedding epithelial cells seen en face.

Figure 2

Loss of barrier function visualised by confocal endomicroscopy. (A) Intact barrier function with no escape of fluorescein into the gut lumen (arrow). (B) Fluorescein in the gap in the epithelium left by a shedding cell (arrow). Cellular debris from the shedding cell can be seen in the lumen. (C) efflux of fluorescein out of blood vessels (block arrow) into the lateral intercellular space. Efflux into the lumen is constrained at the apical border (block arrows). A plume of fluorescein effluxing through the gap left behind a shedding cell (line arrow). (D) Multiple sites of efflux of fluorescein through the epithelium into gut lumen (arrows). Note the increased fluorescence in the gut lumen. (E) Microerosion (arrow) where more than one epithelial cell has been lost at one site exposing a capillary to the lumen. Note the functional relevance of this lesion as there is efflux of fluorescein into the lumen.

Figure 3

(A) Localised fluorescein leak is preceded by cell shedding. Intravenous Alexa-dextran 647 (MW 10 000) shown in red and Hoechst 33342 labelled nuclei in blue. Images at 0, 5, 15 and 28 min relative to the start of cell shedding. (B–E) Dextran movement across small intestinal epithelia. Luminal FITC-dextran (MW 4000) shown in green. Intravenous Alexa-dextran 647 (MW 10 000) shown in red and combined images shown with Hoechst 33342 labelled nuclei. Dextran leakage from circulation into lumen in an outward direction (B–D), from lumen in an inward direction (E–G), in both inward and outward directions (H–J) and no movement of inward or outward dextran (K–M).

Figure 4

Analysis of loss of local barrier function and prediction of relapse of inflammatory bowel disease (IBD). Dextran movement (Luminal FITC-dextran (MW 4000) and intravenous Alexa-dextran 647 (MW 10 000)) through small intestinal epithelium at the site of epithelial cell shedding from luminal perfusion solutions of either 300 mOsm/l (black) and 246 mOsm/l (grey) solutions. (A) Percentage of events that show no dextran movement (sealed) and dextran leakage (leakage) in either the inward or the outward directions. (B) Percentage movement of dextran in an inward, outward or inward and outward direction from the leakage group. *p<0.05, **p<0.01, error bars show SEM (n=4 mice per group). (C) Kaplan–Meier plot of relapse of IBD patients over 12 months after confocal laser endomicroscopy stratified according to their Watson grade.