Volume 130, Issue 2, Pages 389-397 (February 2006) A Functional Role of Flip in Conferring Resistance of Crohn’s Disease Lamina Propria Lymphocytes to FAS-Mediated Apoptosis  Ivan Monteleone, Giovanni Monteleone, Daniele Fina, Roberta Caruso, Carmelina Petruzziello, Emma Calabrese, Livia Biancone, Francesco Pallone  Gastroenterology  Volume 130, Issue 2, Pages 389-397 (February 2006) DOI: 10.1053/j.gastro.2005.10.021 Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 1 Intestinal LPLs of CD patients are resistant to FAS-mediated apoptosis. LPMCs, isolated from patients with CD and UC and normal controls (ctr), were left either unstimulated (unst) or stimulated with CH11, a human anti–FAS-activating antibody, or anti-CD3 + CD2 for 24 hours. The percentage of Annexin-V–positive CD3+ LPLs then was measured by flow cytometry. Data indicate the mean ± SD of 4 separate experiments. , Unst; □, CH11; ■, CD3 + CD2. Gastroenterology 2006 130, 389-397DOI: (10.1053/j.gastro.2005.10.021) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 2 Flip is overexpressed in CD mucosa. (A) Representative Western blot showing Flip L and S in extracts from biopsy specimens taken from 3 normal controls (ctr), 3 CD, and 3 UC patients. One of 4 blots analyzing proteins from 11 controls, 11 patients with CD, and 6 patients with UC is shown. After analysis of Flip, blots were stripped and incubated with a β-actin antibody to ascertain equivalent loading of the lanes. (B) Quantitative analysis of Flip protein in mucosal samples from 11 CTR, 11 patients with CD, and 6 UC as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units. Each point represents the Flip/β-actin ratio in mucosal samples taken from a single patient. Horizontal bars indicate mean values. (C) In CD mucosa, up-regulation of Flip occurs in sites with active inflammation. Representative Western blot showing Flip L and S in extracts form paired biopsy specimens taken from 2 healthy controls (ctr), and uninflamed (u), or inflamed (i) areas of 3 CD patients. One of 2 blots analyzing proteins from 4 CTR and 6 patients with CD is shown. Gastroenterology 2006 130, 389-397DOI: (10.1053/j.gastro.2005.10.021) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 3 Flip is overexpressed in CD3+ LPLs from CD patients. (A) Representative Western blot showing Flip L and S in total proteins extracted from CD3+ LPLs isolated from 2 controls (ctr), 2 CD, and 2 UC patients. One of 3 blots analyzing proteins from 6 CTR, 6 patients with CD, and 4 patients with UC is shown. (B) Quantitative analysis of Flip L and S protein in CD3+ LPLs from 6 CTR, 6 patients with CD, and 4 patients with UC as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units. Each point represents the Flip/β-actin ratio in mucosal samples taken from a single patient. Horizontal bars indicate mean values. (C) Western blot showing Flip L and S in proteins extracted from CD45RO+ LPLs purified from 2 normal CTR and 2 CD patients. One of 2 blots analyzing proteins from 4 CTR and 4 patients with CD is shown. Gastroenterology 2006 130, 389-397DOI: (10.1053/j.gastro.2005.10.021) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 4 Western blot showing Flip L and S isoforms (upper blot) in proteins extracted from LPMCs of 1 normal control (ctr), 1 patient with CD, and 1 with UC, and left either unstimulated (unst) or stimulated with anti-CD3 or anti-CD3 + CD2 for 24 hours. After analysis of Flip, the membrane was stripped and incubated with an anti-human IL-21 (middle blot) and then β-actin (lower blot) antibody. One of 3 separate experiments is shown. Gastroenterology 2006 130, 389-397DOI: (10.1053/j.gastro.2005.10.021) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 5 Flip is not overexpressed in celiac disease mucosa. (A) Representative Western blots showing Flip L and S (upper) and β-actin (lower) in proteins extracted from duodenal biopsy specimens of 3 untreated celiac disease patients and 3 controls (ctr). One of 2 blots analyzing proteins from 6 CTR and 6 patients with celiac disease is shown. (B) Quantitative analysis of Flip L protein in mucosal samples from 6 patients with untreated celiac disease and 6 CTR, as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units. Each point represents the Flip/β-actin ratio in mucosal samples taken from a single patient. Horizontal bars indicate mean values. Gastroenterology 2006 130, 389-397DOI: (10.1053/j.gastro.2005.10.021) Copyright © 2006 American Gastroenterological Association Terms and Conditions

Figure 6 Inhibition of Flip by antisense oligonucleotide restores the susceptibility of CD LPLs to FAS-mediated apoptosis. (A) Representative Western blot showing both FlipL and FlipS in CD LPMCs left either untreated or treated with Flip sense (s) or antisense (as) oligonucleotide for 24 hours. Right insets show the quantitative analysis of Flip L and S proteins in CD LPMCs as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units. Each point represents the Flip/β-actin ratio in LPMC samples taken from a single patient. Horizontal bars indicate mean values. (B) Flip inhibition restores the susceptibility of CD T-LPLs to FAS-mediated apoptosis. CD LPMCs were left untreated (unst) or treated with Flip sense (s) or antisense (as) oligonucleotide for 24 hours and then stimulated or not with CH11 for a further 18 hours. The percentage of Annexin-V–positive CD3+ LPLs was measured by flow cytometry. Data indicate the mean ± SD of 6 separate experiments in which LPMCs taken from 6 CD patients were used. Right inset shows a representative flow cytometric analysis of the distribution of AnnexinV+/CD3+ cells in CD LPMCs treated with sense (s) or antisense (as) oligonucleotide and then stimulated with CH11. (C, D). Treatment of (C) UC and (D) normal LPMCs with Flip antisense oligonucleotide does not enhance FAS-induced apoptosis. LPMCs were cultured and analyzed as indicated in B. Data indicate the mean ± SD of 4 separate experiments in which LPMCs taken from 2 UC patients and 2 healthy controls were used. Gastroenterology 2006 130, 389-397DOI: (10.1053/j.gastro.2005.10.021) Copyright © 2006 American Gastroenterological Association Terms and Conditions