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. 2001 Sep;49(3):380–386. doi: 10.1136/gut.49.3.380

Intraepithelial and lamina propria lymphocytes show distinct patterns of apoptosis whereas both populations are active in Fas based cytotoxicity in coeliac disease

S Di 1, R Ciccocioppo 1, S D'Alo 1, R Parroni 1, D Millimaggi 1, M Cifone 1, G Corazza 1
PMCID: PMC1728419  PMID: 11511560

Abstract

BACKGROUND—Lamina propria (LPLs) and intraepithelial (IELs) lymphocytes are markedly increased in coeliac mucosa, and are thought to play a crucial role in the generation of villous atrophy in coeliac disease (CD). However, the mechanisms by which they mediate the killing of enterocytes in this condition are still poorly characterised.
AIM—We investigated Fas mediated cytotoxicity and apoptosis of both LPLs and IELs, isolated from 10 untreated coeliac patients, 10 coeliac patients on a gluten free diet, and 10 biopsied controls.
METHODS—Fas and Fas ligand expression were assessed by flow cytometry and immunocytochemistry. Lymphocyte cytotoxicity against Fas expressing Jurkat cells was determined by the Jam test. The effect of the antagonist ZB4 anti-Fas antibody on apoptotic activity exerted by coeliac lymphocytes against enterocytes was analysed. Lymphocyte apoptosis was assessed by oligonucleosome ELISA.
RESULTS—LPLs and IELs showed increased apoptotic activity and higher levels of Fas ligand expression in untreated CD compared with treated CD patients and controls. Enterocyte apoptosis observed after coculturing coeliac lymphocytes and enterocytes in the presence of ZB4 antibody was reduced. In active CD, LPLs manifested increased apoptosis whereas IELs showed decreased apoptosis.
CONCLUSIONS—Our results support the involvement of the Fas/Fas ligand system in CD associated enterocyte apoptosis. Increased LPL apoptosis is likely to downregulate mucosal inflammation whereas decreased IEL apoptosis could be responsible for autoimmune and malignant complications of CD.


Keywords: apoptosis; coeliac disease; cytotoxicity assay; Fas/Fas ligand system; intraepithelial lymphocytes; lamina propria lymphocytes

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Figure 1  .

Figure 1  

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Percentages of FasL+ lamina propria lymphocytes (LPLs) in 10 untreated coeliac patients compared with those of 10 treated coeliac patients and 10 biopsied controls. Those coeliac patients on a gluten free diet and with a persistent degree of villous atrophy are indicated with a different symbol (solid square ). Horizontal bars represent median values.

Figure 2  .

Figure 2  

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Percentages of FasL+ intraepithelial lymphocytes (IELs) in 10 untreated coeliac patients compared with those of 10 treated coeliac patients and 10 biopsied controls. Those coeliac patients on a gluten free diet and with a persistent degree of villous atrophy are indicated with a different symbol (solid square ). Horizontal bars represent median values.

Figure 3  .

Figure 3  

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FasL mediated cytotoxicity by lamina propria lymphocytes (LPLs) (effectors) obtained from 10 untreated coeliac patients (A), 10 treated coeliac patients (B), and 10 biopsied controls (C) against Jurkat cells (targets). 3H labelled Jurkat cells were cultured with LPLs at various effector/target ratios (5:1; 2.5:1; 1.25:1; and 0.625:1). The percentage of DNA fragmentation of Jurkat cells was assayed after 12 hours. FasL mediated cytotoxicity by intraepithelial lymphocytes (IELs) (effectors) obtained from 10 untreated coeliac patients (D), 10 treated coeliac patients (E), and 10 biopsied controls (F) against Jurkat cells (targets). 3H labelled Jurkat cells were cultured with IELs at various effector/target ratios (3:1; 1.5:1; and 0.625:1). The percentage of DNA fragmentation of Jurkat cells was assayed after 12 hours.

Figure 4  .

Figure 4  

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Antagonist ZB4 anti-Fas antibody reduces enterocyte apoptosis induced by both lamina propria lymphocyte (LPL) and intraepithelial lymphocyte (IEL) cytotoxic activity through a Fas/FasL interaction. Untreated coeliac and control enterocytes alone, and coculture of both untreated coeliac and control LPLs and IELs with enterocytes (at an effector target ratio of 20:1 and 100:1, respectively) were incubated for four hours in the presence or absence of 2 µg/ml ZB4 antagonist antibody. The percentage of apoptotic cells was determined by TUNEL detection.

Figure 5  .

Figure 5  

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Detection of apoptosis on lamina propria lymphocytes (LPLs) (A) and intraepithelial lymphocytes (IELs) (B) by in vitro determination of cytoplasmic histone associated DNA fragments (mono and oligonucleosomes) in 10 untreated coeliac patients, 10 treated coeliac patients, and 10 biopsied controls. Quantitative results are expressed as an enrichment factor of mono and oligonucleosomes compared with controls. Horizontal bars represent median values.

Figure 6  .

Figure 6  

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Representative flow cytometric histograms of Fas expression on lamina propria lymphocytes (LPLs) isolated from duodenal biopsy specimens of an untreated coeliac patient, a treated coeliac patient, and a biopsied control.

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