1. Volume 130, Issue 7, Pages 1962-1974 (June 2006)
Implication of TNF-Related Apoptosis-Inducing Ligand in Inflammatory Intestinal Epithelial Lesions 
Bernadette Begue, Harald Wajant, Jean–Christophe Bambou, Laurent Dubuquoy, Daniela Siegmund, Jean–François Beaulieu, Danielle Canioni, Dominique Berrebi, Nicole Brousse, Pierre Desreumaux, Jacques Schmitz, Michael J. Lentze, Oliver Goulet, Nadine Cerf– Bensussan, Frank M. Ruemmele 
Gastroenterology 
Volume 130, Issue 7, Pages (June 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
TRAIL expression in intestinal epithelial cells. (A) RT-PCR analyses of the intestinal mucosa revealed transcripts for TRAIL in normal, noninflammatory duodenum, jejunum, ileum as well as colon. −, negative control; +, positive control. (B) Immunohistochemical staining of ileal sections revealed TRAIL expression (brown staining) predominantly by mature enterocytes under physiologic, noninflammatory conditions. TRAIL was merely expressed in lymphoid follicules, and its expression was markedly higher in adjacent epithelial cells. In active ileal sections of Crohn’s disease and colonic section of ulcerative colitis patients, TRAIL was markedly up-regulated in intestinal epithelial cells and extended to immature crypt cells. Some LPL stained also positive for TRAIL. (C) Isolation and purification by cell sorting (gating for ESA+ vs CD3+) of intestinal epithelial cells (IEC) from LPL derived from ileal resections allowed analysis of TRAIL gene expression in pure fractions. Under physiologic, noninflammatory conditions, IEC expressed low levels of TRAIL, which markedly increased in sections derived from inflamed ileum. LPL of inflammatory infiltrates also showed increased amounts of TRAIL transcripts, as analyzed by PCR. GAPDH was monitored as housekeeping gene. −, negative control; +, positive control. (D) Quantification by real-time PCR of whole mucosal sections or biopsy specimens from patients with active (inflammatory) Crohn’s disease (n = 17), active ulcerative colitis (n = 10), or infectious colitis (n = 10) revealed markedly increased amounts of TRAIL in inflammatory ileal and/or colonic biopsy specimens compared with normal, noninflammatory tissue sections or biopsy specimens (n = 10). In parallel, colitis induced by TNBS in Balb/c mice (n = 6) was equally accompanied by markedly increased TRAIL levels in inflammatory colonic sections compared with NaCl-ethanol-treated control animals (n = 6). *P < .01.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
TRAIL receptor profile on intestinal epithelial cells. (A) RT-PCR analysis revealed that nontransformed HIEC express transcripts for TRAIL-R1 to TRAIL-R4, whereas the tumoral-transformed enterocyte cell lines Caco-2 and HT-29 only express TRAIL-R1, -R2, and -R4. (B) Immunofluorescence analyses on HIEC confirmed the surface expression of TRAIL-R1, -R2, and -R4; however, TRAIL-R3 was undetectable.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
Expression of TRAIL by intestinal epithelial cells. (A) Using an RNA protection assay, we observed a marked up-regulation of TRAIL in HIEC in response to stimulation by IFN-γ or TNF-α alone and more pronounced in combination. In addition, TRAIL-R2 as well as FAS receptors were up-regulated after IFN/TNF treatment. (B) Cytokine-induced up-regulation of TRAIL gene expression was confirmed and quantified by real-time PCR in HIEC. IFN-γ resulted in a rapid up-regulation of TRAIL as observed after 4 hours, whereas the effect of TNF-α was delayed, and a maximal up-regulation was observed after 20 hours. TNF-α-induced TRAIL was dependent of the NF-κB pathway because the proteasome inhibitor MG132 (0.05 mmol) almost completely blocked TNF-induced TRAIL. (C) The induction of TRAIL in response to TNF-α/IFN-γ was confirmed on the protein level after immunoprecipitation of TRAIL by means of a TRAIL-R2 fusion protein. A specific double band for TRAIL at 32/34 kilodaltons was revealed by ECL staining of the Western blot. To avoid excessive cell death induced by IFN and TNF-α, HIEC were protected from apoptotic cell death using a broad-range caspase inhibitor (zVAD). One of 3 similar experiments is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Proapoptotic effects of TRAIL on intestinal epithelial cells. (A) Tumoral transformed Caco-2 cells easily responded to TRAIL stimulation with apoptosis, as analyzed by flow cytometry (AnnexinV/PI method). In contrast, nontransformed HIEC proved to be resistant, and only high doses of TRAIL (≥100 ng/mL) allowed to induce an apoptotic response. However, in the presence of low doses of the protein synthesis inhibitor cycloheximide (CHX), alone without any toxic effect, HIEC responded to TRAIL with apoptosis in a way similar to Caco-2 cells. (B) Western blot analysis of the expression of inhibitors of apoptosis proteins (IAP) revealed a high endogenous XIAP, cIAP1/2 expression in HIEC, which was not altered by TRAIL. However, stimulation of HIEC with TRAIL in the presence of increasing doses of CHX resulted in a marked dose-dependent down-regulation of XIAP and cIAP1/2. CHX alone had no effect on the expression of cIAP1/2 or XIAP. The inhibition of TRAIL signaling via TRAIL-R2 using an inhibitory antibody completely blocked the reduction of cIAP1/2 and XIAP. The expression of actin as housekeeping protein was analyzed in parallel; 1 of 3 similar experiments is shown. (C) Prestimulation of HIEC with low doses of TNF-α (0.1–100 ng/mL) for 6 hours, alone without any apoptotic effect, highly sensitized HIEC to TRAIL-induced apoptosis. More potent was the costimulation of HIEC by TNF-α and TRAIL, resulting in over 90% apoptotic cells after 24 hours stimulation (flow cytometric quantification, AnnexinV/PI method). Neutralizing the biologic activity of TRAIL with the 2E5 antibody clearly abolished the proapoptotic effects of TRAIL at concentrations as low as 0.25 μg/mL. Receptor studies with inhibitory antibodies selectively blocking either R1 or R2 showed that the apoptotic response to TRAIL required a functional R2. Inhibition of TRAIL-R1 did not alter the proapoptotic effect of TRAIL. Experimental conditions in these inhibitory experiments with HIEC were 6 hours prestimulation with TNF-α 1 ng/mL, followed by TRAIL (100 ng/mL for 20 hours). One of 3 similar experiments is shown. (D) Ex vivo analyses with ileal organ cultures from noninflammatory sections showed that enterocytes do not undergo apoptosis after TRAIL or TNF-α stimulation over 18 hours. However, costimulation with both cytokines (TNF-α 1 and TRAIL 100 or 500 ng/mL) resulted in a marked destruction of epithelial cells, easily seen in the occurrence of apoptotic bodies (asterisk). (E) Analysis of the effect of TNF-α on TRAIL-R expression in HIEC revealed a marked shift toward a proapoptotic constellation on mRNA and protein level. TNF-α selectively induced an over 2-fold increase in TRAIL-R2 gene expression, which resulted in an almost 2-fold up-regulation of the TRAIL-R2 membrane expression on HIEC. The expression of the other TRAIL receptors remained unaltered after TNF-α stimulation. One of 3 similar experiments is shown (flow cytometry), and the mean of 3 analyses (real-time PCR and FACS quantification) is shown.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
Signaling cascade of TRAIL-induced enterocyte apoptosis. (A) TRAIL-induced apoptosis in nontransformed HIEC (after prestimulation with TNF-α 1 ng/mL) and the tumoral-transformed Caco-2 cells required a functional caspase cascade. Inhibition with the pan-specific caspase inhibitor zVAD completely blocked the induction of apoptosis at low concentrations of 10 μmoL (HIEC) and 50 μmol (Caco-2). In parallel, selective inhibition of caspase-8 (zIETD) or caspase-3 (zDEVD) almost completely inhibited TRAIL-mediated apoptosis in both cell models. (B) Western blot analysis confirmed the activation of caspase-8 and caspase-3 in CHX-sensitized HIEC after TRAIL stimulation, seen in the appearance of the cleaved active form at 40, 36, and 23 kilodaltons (caspase-8) and 17, 12, and 8 kilodaltons (caspase-3), respectively. However, only a minimal activation was observed with high TRAIL doses (TRAIL 100 or 500 ng/mL), whereas the costimulation with CHX resulted in the potent activation of both caspases at low TRAIL concentrations. (C) Similarly, TNF (1 ng/mL)-pretreated HIEC for 6 hours responded to TRAIL stimulation with a rapid activation of caspase-3, whereas TRAIL or TNF-α alone was without or only minimal effect on caspase-3. Similarly, the costimulation of HIEC with TNF-α and TRAIL induced a rapid activation of caspase-3, seen in the occurrence of the active forms (17, 12, and 8 kilodaltons). (D) Ileal organ cultures over 20 hours showed no signs of epithelial cell apoptosis with absent staining for active caspase-3, except very few IEC (arrow) or LPL (asterisk). Cleaved and thereby activated caspase-3 stained as brown. Isolated stimulation with TNF-α or TRAIL did not activate caspase-3 in IEC. However, few apoptotic-caspase-3-positive cells were seen within the lamina propria after TRAIL stimulation. In contrast, costimulation with TNF and TRAIL resulted in marked epithelial cell apoptosis resulting in the detachment of IEC and destruction of the epithelial layer. These cells stained all positive for active caspase-3 (brown).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

7. Figure 6
Proinflammatory effects of TRAIL on intestinal epithelial cells. (A) Under noninflammatory conditions, HIEC responded with the rapid activation of NF-κB to stimulation with TRAIL as analyzed by EMSA. A maximal effect was observed after 30 minutes. (B) In response to TRAIL stimulation over 20 hours, an up-regulation of CARD15 was observed. In parallel, TRAIL-stimulated HIEC secreted increasing amounts of IL-8 in a dose-dependent manner.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions