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. 2001 Mar;48(3):418–424. doi: 10.1136/gut.48.3.418

FAS engagement drives apoptosis of enterocytes of coeliac patients

L Maiuri 1, C Ciacci 1, V Raia 1, L Vacca 1, I Ricciardelli 1, F Raimondi 1, S Auricchio 1, S Quaratino 1, M Londei 1
PMCID: PMC1760155  PMID: 11171836

Abstract

BACKGROUND—Villus atrophy is the most distinctive sign of untreated coeliac disease (CD) and epithelial apoptosis is considered to be involved in this stage of the coeliac lesion. The extent of villus atrophy is, however, not homogeneous and patients with patchy or mild lesions have been described.
AIMS—To address: (a) the degree of "patchiness" in untreated CD patients; and (b) to clarify if apoptosis, and eventually which trigger drives it, causes epithelial damage.
PATIENTS—Twenty of 40 untreated, 14 treated coeliac patients, and 15 controls received five or more multiple duodenal biopsies; the remaining 20 untreated CD patients had no more than three biopsies.
METHODS—All biopsies were analysed to monitor the presence of a "flat" mucosa. Biopsies of 14 untreated, 10 treated coeliacs, and seven controls were cultured with or without gliadin. DNA fragmentation was studied by terminal deoxynucleotidyl transferase (TdT) mediated dUTP digoxigenin nick end labelling (TUNEL), and FAS and Ki67 expression by immunohistochemistry. Antiendomysium antibodies (EMA) were surveyed in biopsy culture supernatants.
RESULTS—A pattern of patchy duodenal lesions was observed in all untreated CD patients biopsied up to five times. High enterocyte FAS expression, and a high number of TUNEL+ and Ki67+ enterocytes were detected in areas with villus atrophy but not in those with a normal morphology (p<0.001). Conversely, EMA in culture supernatants and signs of immunological activation were present in all untreated CD biopsies. In vitro gliadin challenge increased the number of TUNEL+ and Ki67+ enterocytes (p<0.001 v cultures with medium alone) only in "flat" biopsies. Neutralising anti-FAS monoclonal antibodies were found to control gliadin induced enterocyte apoptosis (p>0.01) while agonist anti-FAS monoclonal antibody increased it (p<0.001).
CONCLUSIONS—Patchy lesions are observed in untreated CD mucosa and epithelial FAS engagement is a key trigger in driving villus atrophy in CD.


Keywords: apoptosis; FAS; enterocytes; coeliac disease

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Figure 1  .

Figure 1  

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DNA fragmentation and Ki67 expression in coeliac disease (CD) and control intestine. (A) DNA fragmentation in enterocytes of CD and control intestine. (B) Expression of Ki67 in crypt enterocytes of CD and control intestine (mean (SD)). Untreated CD, untreated biopsies with patchy mucosal lesions (n=20); treated CD, treated biopsies (n=14); controls, biopsies from non-coeliac patients with a normal villus architecture (n=15). ***p<0.0001 v treated CD and controls. (C) Pattern of intraepithelial DNA fragmentation in untreated CD duodenum. DNA fragmentation (TUNEL technique, red colour) is absent in the nucleus of all intraepithelial lymphocytes (CD3, green colour), while it is detected in the majority of enterocytes. Original magnification ×240. Two colour immunofluorescence: TUNEL technique, RPE labelling (red); immunohistochemistry, CD3 labelling (green) (see methods).

Figure 2  .

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FAS expression in untreated coeliac disease (CD) duodenum. A marked change in expression of FAS is observed at the border between regions with normal villus architecture (low expression; left side) and those with villus atrophy (very high expression; right side). Original magnification ×150; immunohistochemistry, peroxidase staining technique.

Figure 3  .

Figure 3  

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In vitro organ culture of untreated coeliac disease (CD) intestine with villus atrophy: DNA fragmentation and Ki67 expression after 24 hour challenge with medium or gliadin. Effect of neutralising anti-FAS M3 or agonist anti-FAS CH-11 monoclonal antibodies (mAbs) on enterocyte apoptosis. (A) DNA fragmentation and (B) Ki67 expression before and after in vitro culture. Untreated CD samples with villus atrophy before in vitro culture (n=7) and after 24 hour challenge with medium alone (n=7), gliadin (n=7), gliadin supplemented with anti-FAS M3 mAb (n=7), or medium supplemented with anti-FAS CH-11 mAb (n=4). The number of TUNEL+ enterocytes (mean (SD)) are given as percentage of both surface and crypt enterocytes and Ki67+ enterocytes (mean (SD)) as percentage of crypt enterocytes. ***p<0.001 v samples after 24 hour challenge with medium alone; †††p=0.001 v samples after 24 hour gliadin challenge. (C) Pattern of DNA fragmentation after 24 hour gliadin challenge. Note that the majority of enterocytes are TUNEL+; staining is also evident in many lamina propria mononuclear cells. (D) Pattern of DNA fragmentation after 24 hours of in vitro challenge with gliadin supplemented with neutralising anti-FAS M3 mAb. The number of TUNEL+ enterocytes is decreased compared with the pattern observed after gliadin challenge. (E) Pattern of DNA fragmentation after 24 hours of in vitro challenge with medium supplemented with agonist CH-11 anti-FAS mAb. A marked increase in the number of TUNEL+ enterocytes with derangement of epithelial architecture is evident. Original magnification: C, D and E (×180); TUNEL technique, peroxidase staining.

Figure 4  .

Figure 4  

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In vitro organ culture of untreated coeliac disease (CD) intestine with normal villus architecture. DNA fragmentation (A) and Ki67 expression (B) (mean (SD)) after 24 hour challenge with medium or gliadin and effect of agonist anti-FAS CH-11 mAb on enterocyte apoptosis

Figure 5  .

Figure 5  

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In vitro organ culture of treated coeliac disease (CD) intestine: effect of 24 hour gliadin challenge on enterocyte expression of FAS. (A) FAS expression after 24 hour challenge with medium alone. Faint staining is observed on cell membranes of some enterocytes. (B) FAS expression after 24 hour challenge with gliadin. Intense FAS expression is evident on the cell membranes of the majority of enterocytes and in some lamina propria mononuclear cells. (C) FAS expression after 24 hour challenge with gliadin: high magnification. Staining is evident on the basolateral membranes of the enterocytes. Original magnification: A, B (×160); C (×240); immunohistochemistry, peroxidase staining technique.

Selected References

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