Institut für Klinische Physiologie / Inst. of Clinical Physiology
Charité  > Departments  > Inst. Clin. Physiology  > Topics  > apoptosis

Apoptosis in colonic epithelia
Alfred H. Gitter

Necrosis and apoptosis

The death of a cell may be caused either by lack of oxygen or nutriment in a tissue region (necrosis) or by active self-destruction of an isolated single cell (apoptosis) (Fig. 1).

necrosis and apoptosis

Fig. 1 Necrosis and apoptosis. In necrosis the cellular metabolism breaks down, the cell swells and cellular membranes decompose. In apoptosis the cell rounds and shrinks; the DNA is systematically fragmented. In contrast to necrosis, the content of the apoptotic cell is packed into membrane vesicles (apoptotic bodies) and taken up via phagocytosis by adjacent cells.

Colonic epithelia

The intestinal wall is lined by a monolayered epithelium. In the colon the surface is enlarged by gland-like infoldings, the crypts of Lieberkühn, which have a predominantly secretory function (Fig. 2A).

human colon
A

 HT-29/B6 cells
B
Fig. 2 A Intestinal wall of the human colon, fixed, paraffin-embedded, and stained with hematoxylin and eosin. A monolayered epithelium forms the barrier between intestinal lumen and body fluids. B Cell culture of an epithelial HT-29/B6 monolayer, which shows properties of the native goblet cells in human colonic crypts. The dwindling cell, rich in contrast in the center of a rosette-like pattern is in apoptosis.

For experimental investigation of programmed cell death (apoptosis) in colonic epithelium a permanent cell line (HT-29/B6) was used (Fig. 2B). It showed reproducibly the functional characteristics of the cryptic goblet cells (chloride secretion, no sodium absorption, mucus secretion).

Demonstration of apoptosis

Viewed with light microscopy, apoptotic cells in living HT-29/B6 monolayers emerged as the reduced center of a "rosette". Histologically apoptosis was demonstrated in semi-thin sections using dyes or fluorescent markers. With highest resolution the morphological changes of apoptotic cells were shown by transmission electron microscopy (Fig. 3).

Apoptosis stainings 

Fig. 3 Demonstration of single HT-29/B6 apoptotic cells. A Living HT-29/B6 monolayer in bright field light microscopy (LM). B A fluorescent dye (Texas red) bound by means of antibodies to occludin, a component of the tight junctions, showed the cell borders in an apoptotic rosette. C With hematoxylin-eosin staining (H-E) the fragments of the decaying nucleus were well discerned. D The fluorescent dye 4,6-diamidino-2-phenylindole-2-HCl (DAPI) binds more strongly to nuclei, if they are apoptotic; hence these appeared brighter. E Blunt ends of double-stranded DNA exposed by strand breaks were visualized by means of the TUNEL assay (TdT-mediated X-dUTP nick end labeling); hence again the apoptotic nuclei appeared brighter. F With transmission electronen microscopy (TEM) apoptotic bodies of the fragmenbted cell were observed in the center of rosettes and engulfed by adjacent cells. Pictures taken by Kerstin Bendfeldt.

Apoptosis in ulcerative colitis

Ulcerative colitis is an inflammatory bowel disease, which impairs the epithelial barrier of the colon and causes severe inflammations. In the advanced stage, large areas of surface epithelium are destroyed by extensive erosions, and part of the the rarefied crypt epithelium by abscesses. However, already in the early stage of the disease there are local leaks, although extensive epithelial lesions are yet absent (Fig. 4 A,B). At the site of these leaks is apoptosis more frequent (Fig. 4 C). Thus we propose that apoptosis causes barrier defects in early ulcerative colitis. It is conceivable that these barrier defects form gates through which luminal antigens enter and elicit, or at least amplify the mucosal inflammation.

A B  C

Fig. 4 In an early stage of ulcerative colitis, leaks in the epithelial barrier are probably caused by apoptoses. A Human sigmoid colon with mild inflammation; the mucosa shows already crypt rarefaction, but visible epithelial lesions seem absent. B A map showing the spatial distribution of ion permeability indicates a local leak. C Viewed with higher magnification, the site of the leak comprises an increased rate of apoptosis in a crypt, near the surface.

 

Charité  > Departments  > Inst. Clin. Physiology  > Topics  > apoptosis:Top