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

Abstract

Background and aims: Barrier dysfunction is an important feature contributing to inflammation and diarrhoea in Crohn's disease (CD). Recently, tumour necrosis factor alpha (TNF-alpha) 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-alpha 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-alpha 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) Omegaxcm(2)) and improved to 34 (3) Omegaxcm(2) after therapy. Occludin, claudin 1, and claudin 4 were not affected by TNF-alpha 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-alpha 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.

Figure 1

Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) (A, D), 4′’,6-diamidino-2-phenylindole (B, E), and haematoxylin-eosin (C, F) stained thin sections of sigmoid colon from one representative Crohn’s disease patient before (A, B, C) and two weeks after (D, E, F) tumour necrosis factor α antibody therapy. Arrows indicate apoptotic enterocytes (magnification 200×).

Figure 2

Apoptotic ratio of controls (n = 8) and Crohn’s disease patients (n = 11) before and two weeks after tumour necrosis factor α (TNF-α) antibody treatment, as determined in terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) stained sections (A) and 4′,6-diamidino-2-phenylindole stained sections (B), respectively. Individual data are given as means (SEM). Upper limit of the normal range (NR), as obtained from the mean of control±2 SD, is indicated by the broken line.

Figure 3

Original impedance locus plots of human sigmoid colon. (A) Control. (B) Crohn’s disease (CD) before tumour necrosis factor α (TNF-α) antibody treatment. (C) CD after TNF-α antibody treatment. Z_real gives the ohmic component and Z_imaginary the reactive component of the complex impedance. Intersections between the semi circle and X axis at low and high frequencies represent R^t and R^sub, respectively (R^t−R^sub = R^e). R^e, epithelial electrical wall resistance; R^sub, subepithelial electrical wall resistance; R^t, transmural wall resistance.

Figure 4

Epithelial electrical wall resistance (R^e) in controls (n = 8) and Crohn’s disease patients (n = 9) before and two weeks after tumour necrosis factor α (TNF-α) antibody therapy. Data are given as means (SEM). Lower limit of the normal range (NR), as obtained from the mean of control ±2SD, is indicated by the broken line.

Figure 5

Expression of tight junction proteins occludin, claudin 1, and claudin 4, as obtained from crude membrane fractions in immunoblots. An original western blot is shown for three patients before and two weeks after tumour necrosis factor α (TNF-α) antibody therapy. Statistical evaluation was performed after densitometry and is presented at the bottom. Values represent means (SEM) of protein expression analysed in comparison with pretreatment values on the same blot, which were set to 100%.

Figure 6

Correlation of apoptotic ratio and transmural resistance (R^t) in human HT-29/B6 cell lines before (Control) and after induction of apoptosis with 0.2 μg/ml and 2.0 μg/ml camptothecin for 48 hours. Late stages of apoptosis were analysed using the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay, as indicated. Data are given as means (SEM) and are representative of at least six independent experiments.